Formulation and Development of Bioadhesive Oral Films Containing Usnea barbata (L.) F.H.Wigg Dry Ethanol Extract (F-UBE-HPC) with Antimicrobial and Anticancer Properties for Potential Use in Oral Cancer Complementary Therapy

Medical research explores plant extracts’ properties to obtain potential anticancer drugs. The present study aims to formulate, develop, and characterize the bioadhesive oral films containing Usnea barbata (L.) dry ethanol extract (F-UBE-HPC) and to investigate their anticancer potential for possible use in oral cancer therapy. The physicochemical and morphological properties of the bioadhesive oral films were analyzed through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), Atomic Force Microscopy (AFM), thermogravimetric analysis (TG), and X-ray diffraction techniques. Pharmacotechnical evaluation (consisting of the measurement of the specific parameters: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time) completed the bioadhesive films’ analysis. Next, oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBE-HPC in normal blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line and its influence on both cell types’ division and proliferation was evaluated. The results reveal that each F-UBE-HPC contains 0.330 mg dry extract with a usnic acid (UA) content of 0.036 mg. The bioadhesive oral films are thin (0.093 ± 0.002 mm), reveal a neutral pH (7.10 ± 0.02), a disintegration time of 118 ± 3.16 s, an ex vivo bioadhesion time of 98 ± 3.58 min, and show a swelling ratio after 6 h of 289 ± 5.82%, being suitable for application on the oral mucosa. They displayed in vitro anticancer activity on CLS-354 tumor cells. By considerably increasing cellular oxidative stress and caspase 3/7 activity, they triggered apoptotic processes in oral cancer cells, inducing high levels of nuclear condensation and lysosomal activity, cell cycle arrest in G0/G1, and blocking DNA synthesis. All these properties lead to considering the UBE-loaded bioadhesive oral films suitable for potential application as a complementary therapy in oral cancer.


. SEM Analysis
A high-resolution scanning electron microscope Quanta3D FEG (Thermo Fisher Scientific, GmbH, Dreieich, Germany) was used to evaluate both films (R and F-UBE-HPC) morphology.

Atomic Force Microscopy (AFM)
AFM investigations were performed in non-contact mode, using decoupled, flexureguided crosstalk eliminated scanners with an XE-100 microscope from Park Systems. In all AFM measurements, sharp tips were used, NCHR from Nanosensors TM , with typicallỹ 8 nm radius of curvature,~125 µm mean length, 30 µm mean width,~42 N/m force constant, and~330 kHz resonance frequency. XEI program (v 1.8.0) from Park Systems was used for image processing and roughness evaluation. Below the AFM images, presented as "enhanced contrast" view mode, representative line scans are plotted, showing in detail the surface profile of the scanned samples.

Fourier Transform Infrared Spectroscopy (FTIR)
Bioadhesive oral films were characterized by Fourier Transform Infrared (FTIR) using a Nicolet Spectrometer 6700 FTIR and a Smart DuraSamplIR HATR (Horizontal Attenuated Total Reflectance) accessory with a laminated-diamond crystal (Thermo Electron Corporation, Waltham, MA, USA). FTIR spectra were achieved in the spectral range from 4000 to 400 cm −1 , using a DTGS KBr detector, in transmittance mode. The spectra were the average of 32 scans recorded at 4 cm −1 resolution.

Powder X-ray Diffractometry
The powder X-ray diffractometry (XRD) patterns were recorded using a Rigaku Ultima IV diffractometer (Rigaku Corporation, Tokyo, Japan) in parallel beam geometry. A Cu tube (λ = 1.54056 Å) operating at 40 kV voltage and 30 mA current was used. The scanning rate employed was 2 • /min over a diffraction angle of 2θ, with a step size of 0.02 and a range of 5-60 • .

Thermogravimetric Analysis
The thermal curves of both bioadhesive oral films were recorded using a Mettler Toledo TGA/SDTA 851 e thermogravimetric analyzer (Mettler-Toledo GmbH, Greifensee, Switzerland). All experiments were conducted in a synthetic air atmosphere with a heating rate of 10 • C min −1 and a flow rate of 80 mL min −1 .

Pharmacotechnical Properties of the UBE-Loaded Bioadhesive Oral Films 2.4.1. Weight Uniformity
Twenty films from each series (F-UBE-HPC and R) were separately weighed, and the mean weight was calculated.

Thickness
Thickness was measured on 20 films of each batch using a digital micrometer (Yato Trading CO., LTD, Shanghai, China) with a 0-25 mm measurement range and 0.001 mm resolution. The average values were calculated for both films.

Folding Endurance
The films were folded and rolled repeatedly, at the same place, until they broke, or up to 300 times [17]. The folding times were recorded and reported as folding endurance values.

Tensile Strength and Elongation Ability
A digital tensile force tester for universal materials was used to determine the tensile strength and elongation behavior of a Lloyd Instruments Ltd., LR 10K Plus, West Sussex, United Kingdom digital tensile force tester for universal materials. The analysis was carried out at a speed of 30 mm/min from a distance of 30 mm. The breaking force was measured by placing the patch in a vertical position between the two bracing. The tests were performed on five patches of each batch.
The tensile strength and elongation at break were calculated using the following equations: Tensile strength kg/mm 2 = Force at breakage (kg) Film thickness (mm) × Film width (mm) (1) Elongation % = Increase in film length Inital film length × 100

Moisture Content
A thermogravimetric method was used to determine the drying loss using an HR 73 Mettler Toledo halogen humidity (Mettler-Toledo GmbH, Greifensee, Switzerland) [18]. Five films of each formulation were tested.

Surface pH
Five films of each formulation were moistened for 5 min at room temperature with 1 mL distilled water (pH 6.5 ± 0.5). The pH value was registered by touching the film surface with the electrode of a CONSORT P601 pH-meter (Consort bvba, Turnhout, Belgium).

In Vitro Disintegration Time
Using an Erweka DT 3 apparatus (Erweka ® GmbH, Langen, Germany), the time necessary for both films' disintegration was determined in a simulated saliva phosphate buffer with a pH of 6.8 at 37 ± 2 • C [19].

Swelling Ratio
Six films of each series were placed in Petri plates on a 1.5% agar gel and incubated at 37 ± 1 • C. The films were weighed every 30 min for 6 h. The swelling ratio is calculated through the following equation: Wt is the patch weight at time t after the incubation, and Wi is the initial weight [20][21][22].

Ex Vivo Bioadhesion Time
This determination was assessed using the technique described by Gupta et al. [23] on a detached porcine oral mucosa. The fat layer and any tissue residue were removed from the membrane surface; then, it was fixed on a glass plate after being rinsed with distilled water and a phosphate buffer pH of 6.8 at 37 • C. Each film was hydrated in the center with 15 µL phosphate buffer and pressed onto the mucosa surface for 30 s. The glass plate was Pharmaceutics 2022, 14, 1808 6 of 34 submerged in 200 mL phosphate buffer (pH 6.8) and kept at 37 • C for 2 min. A paddle set at a 28 rpm stirring rate simulated the oral cavity conditions, and the time requested for each film to detach from the oral mucosa was measured. This registered time is known as residence time or bioadhesion time. All tests were carried out in triplicate.

Inoculum Preparation
The direct colony suspension method (CLSI) was used to prepare the bacterial inoculum. 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 achieved 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-UBE-HPC was dissolved in 1 mL of diluted phosphate buffer. As standards, Ceftriaxone (Cefort 1g Antibiotice SA, Ias , i, Romania) solutions 30 mg/mL and 150 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 S.R.L., Otopeni, România) was selected as standard for Candida sp.

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. [24].
All 96-well plates were incubated for 24 h at 37 • C for bacteria and 35 • C for yeasts in My Temp mini Z763322 Digital Incubator (Benchmark Scientific Inc., Sayreville, NJ, USA).

Reading and Interpreting
The 96-well plates were examined with a free eye to see the color differences between standard and samples after 24 h incubation [25]. 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 [26,27].

Cytotoxic Activity of UBE-Loaded Bioadhesive Oral Films on A. salina Larvae
F-UBE-HPC was placed in 1 mL of diluted buffer and left to incubate for 15 min at 37 • C; after this time, its homogenous dispersion in the buffer solution was used as a treatment for A. salina larvae.
Brine shrimp larvae were achieved by placing the A. salina cysts in a saline solution of 0.35%, for 24-48 h, under continuous aeration and light, at room temperature. (20-22 • C). At the first larval stage, they were separated and introduced into experimental well plates (with a volume of 1 mL) in 0.3% saline solutions [28]. The prescreen was compared with a blank (untreated brine shrimp nauplii) to obtain accurate results regarding the F3 patch cytotoxic effect. During the test period, A. salina larvae were not fed to not interfere with the tested extracts. Their evolution was investigated after 24 h and 48 h, during which the larvae had embryonic energy reserves as lipids [29].

Fluorescent Microscopy
The brine shrimp larvae were stained with 3% acridine orange (Merck Millipore, Burlington, MA, USA) for 5 min. The samples were dried in darkness for 15 min and placed on the microscope slides. Fluorescent microscopy (FM) 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 (λex = 510-550 nm; λem = 590 nm). These images were obtained at 100×, 200×, and 400× magnification and processed with Optikam Pro 3 Software (OPTIKA S.R.L., Ponteranica, BG, Italy) [30]. All observations were performed in triplicate. The study platform for in vitro cytotoxicity analysis was the Attune Acoustic focusing cytometer (Applied Biosystems, Bedford, MA, USA). Before cell analysis, the flow cytometer was first set using fluorescent beads (Attune performance tracking beads, labeling, and detection, Life Technologies, Europe BV, Bleiswijk, Netherlands [31], with standard size (four intensity levels of beads population). The cell quantity was established by countering the cells below 1 µm. Over 10,000 cells per sample were gated by Forward Scatter (FSC) and Side Scatter (SSC) for each analysis.

Human Blood Cells Cultures
Blood samples were collected into heparinized vacutainers and used throughout the experiment. The blood (1.0 mL) was introduced in 6.0 mL of Dulbecco's phosphate buffered saline (with MgCl 2 and CaCl 2 ) medium, supplemented with 10% bovine fetal serum, L-glutamine, and antibiotics mix solution. The mixture was placed in untreated Nunclon Vita Cell culture 6-well plates (Kisker Biotech GmbH & Co.KG, Steinfurt, Germany) were incubated in a Steri-Cycle™ i160 CO 2 Incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA), with 5% CO 2 , at 37 • C for 72 h. Next, blood cell cultures were treated with the samples and controls and incubated for 24 h in the same conditions. All the flow-cytometry analyses were performed after this incubation time.

CLS-354 Cell Line, Cell Culture
The CLS-354 cells were cultured for 7 days in DMEM High Glucose with 10% FBS supplemented with antibiotic mix solution in a 5% CO 2 humidified atmosphere at 37 • C, according to https://www.clsgmbh.de/pdf/cls-354.pdf (accessed on 5 March 2022). Then, the cells were dissociated with Trypsin-EDTA and centrifugated at 3000 rpm for 10 min in a Fisher Scientific GT1 Centrifuge (Thermo Fisher Scintific Inc., Waltham, MA, USA). Then, the cells were distributed in Millicell™ 24-Well Cell Culture Microplates (Termo Fisher Scientific Inc., Waltham, MA, USA). After treatment, the cells 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-UBE-HPC was dispersed in 1 mL of suitable culture media for both types of cells with 1% DMSO and incubated at 37 • C for 24 h. Usnic acid of 125 µg/mL in 1% DMSO was selected as positive control; 1%DMSO was also the negative control. The cells were placed in flow-cytometry tubes (with 2 µL Annexin V-FITC and 2 µL PI of 20 µg/mL) and incubated at room temperature, in darkness, for 30 min. Then, 1 mL of FCB was added, and viable cells, early apoptotic cells, late apoptotic cells, and necrotic cells were examined with 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
A Magic Red ® Caspase-3/7 Assay Kit containing Hoechst 33,342 stain (200 µg/mL) and acridine orange (AO, 1.0 µM) was used. Hoechst 33,342 is a cell-permeant nuclear stain; when it is linked to double chain DNA, it emits blue fluorescence, highlighting condensed nuclei in apoptotic cells.
Acridine orange is a chelating dye that can be used to reveal lysosomal activityhttps://www.abcam.com/caspase-37-assay-kit-magic-red-ab270771.html (accessed on 3 April 2022). Therefore, 300 µL of cell culture was introduced in flow-cytometry tubes; then, 2 µL of Hoechst 33,342 stain was added, and the cells were well-mixed. Then, 50 µL of acridine orange 1.0 µM was added, and the cells were incubated in darkness, at room temperature, for 30 min. Finally, 1 mL FCB was added, permitting the examination of the cells at the flow cytometer. UV excitation and blue emission for Hoechst 33,342 (VL2) at 488 nm and green emission acridine orange (BL1 channel) were used for examination.

Evaluation of Total ROS Activity
A total of 100 µL of stain solution from ROS Assay was added to each 1 mL of cell culture in flow-cytometry tubes and well-mixed. After incubation at 37 • C, in 5% CO 2 , for 60 min, the cells were examined with a flow cytometer using a 488 nm excitation and green emission for ROS (BL1 channel).

Cell Cycle Analysis
A total of 1 mL of cell culture was washed in FCB and fixed for 10 min with 50 µL ethanol. Next, the cells were treated with PI (20 µg/mL) and RNase A (30 µg/mL) and incubated at room temperature, in darkness, for 30 min. Then, 1 mL FCB was added, and the cell cycle distribution was examined by flow cytometry using a 488 nm excitation and orange emission for PI (BL2 channel).

Evaluation of Cell Proliferation
Volumes of 1 mL of cell cultures were incubated with 50 µM EdU (500 µL) at 37 • C for 2 h. Then, the cells were fixed (with 100 µL 4% paraformaldehyde in PBS) and permeabilized (with 100 µL Triton X-100). After washing in 3% buffer sodium azide (BSA) and centrifuging at 300 rpm for 5 min, at 4 • C, the cells were incubated with a reaction mix (500 µL) for 30 min at room temperature, in darkness. Then, they were washed in permeabilization buffer and centrifuged at 300 rpm for 5 min, at 4 • C. After these procedures, 1 mL FCB was added, and the cells were examined by flow cytometry using a 488 nm excitation and green emission for EdU-iFluor 488 (BL1).

Data Analysis
All analyses were performed in triplicate, and the results were presented as means values ± standard deviation (SD). The results are presented as percent (%) of cell and nuclear apoptosis, caspase 3/7 activity, autophagy, cell cycle, DNA synthesis, and count (×10 4 ) of oxidative cellular stress after flow-cytometry analyses were performed with SPSS v. 23 software, IBM, Armonk, NY, USA, 2015. The Levene test was analyzed for homogeneity of variances of samples. At the same time, a paired t-test was used to evidence the differences between sample and controls; p < 0.05 was established as statistically significant. The Principal Component Analysis was achieved with XLSTAT v. 2022.2.1. by Addinsoft (New York, NY, USA).

Development of the Bioadhesive Oral Films
Both films are transparent, flexible, and homogenous with smooth surfaces ( Figure S1, Supplementary Material). F-UBE-HPC ( Figure S1a) has a yellowish-green, faint-brown color, while R is colorless. (Figure S1b). The patches peeling and cutting processes occurred without forming air bubbles, cracks, or other defects.
Each UBE-loaded bioadhesive oral film has 0.330 mg UBE, with a total phenolic content (TPC) of 573.234 mg/g (one film has a TPC of 0.189 mg) and 108.742 mg/g usnic acid (one film contains 0.036 mg UA).

AFM Analysis
The AFM 2D images at the scale of (8 × 8) µm 2 in the so-calle view mode are presented in Figure 2a

AFM Analysis
The AFM 2D images at the scale of (8 × 8) µm 2 in the so-called "enhanced contrast" view mode are presented in Figure 2a

AFM Analysis
The AFM 2D images at the scale of (8 × 8) µm 2 in the so-called "enhanced contrast" view mode are presented in Figure 2a  The first row shows the Reference (R) (Figure 2a). Some differences in the morphology of the parental patch can be noticed as the sample R appears more compact with local accumulations of material (as indicated with a few arrows in Figure 2a). Some large pits (surface cavities) are also observed on the R surface. The arbitrary lines plotted below the AFM images of R suggest a similar z-scale (level oscillations) of about 120 nm (the vertical scale of the plotted line scans in Figure 2a).
The morphology of F-UBE-HPC (Figure 2b) is much more uniform compared with The first row shows the Reference (R) (Figure 2a). Some differences in the morphology of the parental patch can be noticed as the sample R appears more compact with local accumulations of material (as indicated with a few arrows in Figure 2a). Some large pits (surface cavities) are also observed on the R surface. The arbitrary lines plotted below the AFM images of R suggest a similar z-scale (level oscillations) of about 120 nm (the vertical scale of the plotted line scans in Figure 2a).
The morphology of F-UBE-HPC ( Figure 2b) is much more uniform compared with the parental sample, exhibiting small protuberances. There is a decrease in roughness (Rq), as also observed in the line scan from Figure 2b, where a vertical scale of~50 nm can be observed (from −30 to +20 nm). Moreover, the peak-to-valley (Rpv) parameter decreases after "functionalization" (from R to F-UBE-HPC), as visible in Figure 2c. The histograms of the roughness (Rq) and peak-to-valley (Rpv) (Figure 2c) were plotted for all the line scans, indicated by red horizontal lines in the AFM images. When the corrugation parameters are calculated for the whole scanned area (i.e., (8 × 8) µm 2 ), as displayed in Figure 2d, it was observed that the R and F-UBE-HPC samples are roughed.
Relevant images of both films' morphology are presented in Figure 2e,f at the scale of (3 × 3) µm 2 , choosing some areas as flat as possible. In this case, it is evident that locally, meaning in small areas, the films are relatively uniform regarding the morphological characteristics but maintain the same trend regarding their roughness.

FTIR Analysis
FTIR spectroscopy was used to study the formation of bioadhesive films. The FTIR spectra of both films (R and F-UBE-HPC) are shown in Figure 3.  In Figure 3, for the peak's optimal visualization, evidencing the differences be both films, the spectral range was divided into two parts: 4000-2000 cm −1 (a) and 200 cm −1 (b); moreover, R was marked with black color and F-UBE-HPC with blue.
The FTIR spectrum of pure HPC shows absorption bands at 3431 cm −1 due to th group from the pyranose unit, at 2967 cm −1 and 2932 cm −1 due to CH2 and CH stre vibrations, at 1533 cm −1 due to C=C stretching vibration, and at 1079 cm −1 due to stretching vibration [32]. In Figure 3, for the peak's optimal visualization, evidencing the differences between both films, the spectral range was divided into two parts: 4000-2000 cm −1 (a) and 2000-400 cm −1 (b); moreover, R was marked with black color and F-UBE-HPC with blue.
The FTIR spectrum of pure HPC shows absorption bands at 3431 cm −1 due to the OH group from the pyranose unit, at 2967 cm −1 and 2932 cm −1 due to CH2 and CH stretching vibrations, at 1533 cm −1 due to C=C stretching vibration, and at 1079 cm −1 due to C-O-C stretching vibration [32].
The FTIR spectrum of PEG shows a broad band around 3500 cm −1 due to the OH group forming hydrogen bonds with other polymers. The characteristic bands appearing at 1724 cm −1 and 1633 cm −1 are attributed to the carbonyl group (C=O) and stretching vibrations of CH, CH2, and CH3 groups. The bands at 1464 and 1343 cm −1 are associated with the C-H bending. The stretching vibrations of the C-C-O group appear at 1280 and 1100 cm −1 . At around 950 and 840 cm −1 , the harmonic bands of the C-C-O group appear [33].
The FTIR spectra of R and F-UBE-HPC bioadhesive films show almost the same peaks as in the pure polymers but with some displacement. This aspect proves that between the two polymers exists a strong interaction. The UBE presence reduces the FTIR peak intensity compared with R ( Figure 3-blue line), evidencing its complete dispersion in the polymer matrix. Figure 4a shows the XRD patterns of the reference R and F-UBE-HPC films. According to Ishii et al. [34], the XRD pattern of HPC showed an amorphous characteristic, with a broad peak at 2θ • = 9 • and 20 • . UBE is loaded, but the peak intensities are reduced, thus proving that UBE was successfully dispersed in the polymer matrix.

TG Analysis
Thermogravimetric and differential thermal analyses were performed to assess the bioadhesive films' thermal behavior and stability. Reference (R) and F-UBE-HPC exhibited similar behavior when a heating program from 25 to 600 °C (Figure 4b) was performed.
In the first step (between 25 and 100 °C), the films' mass loss (of 2.4% for R and 2.3% for F-UBE-HPC) can be associated with the loss of residual solvent and physisorbed water. The decomposition process of the organic compounds occurs in two distinct steps, between 190-380 °C and 380-550 °C. The mass losses and maximum decomposition temperatures obtained from DTA curves associated with the three steps are presented in Table 2. Table 2. Thermal data of the studied samples.

Film
1st Step (%) 2nd Step (%) 3rd Step (  In the XRD pattern of R, the amorphous character was observed with the two broader peaks characteristic of the HPC compound. The amorphous aspect is maintained when UBE is loaded, but the peak intensities are reduced, thus proving that UBE was successfully dispersed in the polymer matrix.

TG Analysis
Thermogravimetric and differential thermal analyses were performed to assess the bioadhesive films' thermal behavior and stability. Reference (R) and F-UBE-HPC exhibited similar behavior when a heating program from 25 to 600 • C (Figure 4b) was performed.
In the first step (between 25 and 100 • C), the films' mass loss (of 2.4% for R and 2.3% for F-UBE-HPC) can be associated with the loss of residual solvent and physisorbed water. The decomposition process of the organic compounds occurs in two distinct steps, between 190-380 • C and 380-550 • C. The mass losses and maximum decomposition temperatures obtained from DTA curves associated with the three steps are presented in Table 2. Table 2. Thermal data of the studied samples.
The swelling rate over the 6 h of study is presented in Figure 5. Table 3 and Figure 5 show that F-UBE-HPC and R have the same swelling performance after 6 h (289 ± 5.82 vs. 288 ± 6.13, p > 0.05).
The swelling rate over the 6 h of study is presented in Figure 5. Table 3 and Figure 5 show that F-UBE-HPC and R have the same swelling performance after 6 h (289 ± 5.82 vs. 288 ± 6.13, p > 0.05).

Antimicrobial Activity
The standard drugs and F-UBE-HPC microdilutions used in antimicrobial activity evaluation are registered in Table 4.

Antimicrobial Activity
The standard drugs and F-UBE-HPC microdilutions used in antimicrobial activity evaluation are registered in Table 4. F-UBE-HPC inhibitory activity on both bacterial and fungal strains was dose-dependent, and the obtained data are displayed in Table 5 and Figure 6. Table 5 shows that F-UBE-HPC of [5.5-0.085] mg/mL concentration act on S. aureus similar to CTR of [1.5-0.024] mg/mL (Table 5). On P. aeruginosa, their inhibitory activity is higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate.  Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate. higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate. higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate. higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate. higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate. higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate. higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate. higher: in the range of [5.5-0.685] mg/mL and act similarly to CTR of [2.498-0.187] mg/mL. The final F-UBE-HPC concentration range of [0.342-0.085] mg/mL inhibits bacterial colonies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate.  nies proliferation such as a CTR of [0.093-0.024] mg/mL. Figure 6 shows that F-UBE-HPC of [5.5-2.75] mg/mL induces C. albicans partial death and a low proliferation of C. parapsilosis. At the following concentration range [1.375-0.171] mg/mL, it similarly acts on both Candida sp., determining a moderate proliferation. The final concentration (0.085 mg/mL) leads to C. albicans fast proliferation and C. parapsilosis moderate.

Cytotoxic Activity on A. salina Larvae
After 24 h, the larvae were alive, swimming, and showing normally visible movements. We investigated them under a microscope to observe the changes after 24 and 48 h of exposure. All these microscopic images are presented in Figures 7 and 8.

Cytotoxic Activity on A. salina Larvae
After 24 h, the larvae were alive, swimming, and showing normally visible movements. We investigated them under a microscope to observe the changes after 24 and 48 h of exposure. All these microscopic images are presented in Figures 7 and 8.  . albicans (a,b) and C. parapsilosis (c,d). The results were compared with Terbinafine (a,c) as an antifungal drug. (e) Results interpretation adapted from Bitacura et al. [27], as follows: blue-cells are dead; violet-blue-cells are partially dead; violet-cells are alive, no proliferation; light-violet-low proliferation; dark pink-moderate proliferation; pink-fast proliferation; light pink-very fast proliferation. F-UBE-HPC-bioadhesive oral film loaded with U. barbata dry ethanol extract.

Cytotoxic Activity on A. salina Larvae
After 24 h, the larvae were alive, swimming, and showing normally visible movements. We investigated them under a microscope to observe the changes after 24 and 48 h of exposure. All these microscopic images are presented in Figures 7 and 8.
Pharmaceutics 2022, 14, 1808 17 of 35 After 24 h of exposure, A. salina larvae showed an early digestive blockage, with contraction of the digestive tube, a low amount of food in the upper digestive part, and a low detachment of cuticle from peripheral tissues (Figure 7e-h). After 48 h, 35.82% of larvae were alive, and 8.95% were in a sublethal stage. The registered mortality was 55.22% due detachment of cuticle from peripheral tissues (Figure 7e-h). After 48 h, 35.82% o were alive, and 8.95% were in a sublethal stage. The registered mortality was 55.2 to massive digestive blockage and tissue destruction (Figure 7m-p).
The FM images show A. salina larvae after 48 h exposure at F-UBE-HPC (Fig  f) compared with blank (Figure 8a-c). Figure 8e,f shows intracellular lysosomes ac in the cell death process, evidenced by red fluorescence. After 24 h of exposure, A. salina larvae showed an early digestive blockage, with contraction of the digestive tube, a low amount of food in the upper digestive part, and a low detachment of cuticle from peripheral tissues (Figure 7e-h). After 48 h, 35.82% of larvae were alive, and 8.95% were in a sublethal stage. The registered mortality was 55.22% due to massive digestive blockage and tissue destruction (Figure 7m-p).
The FM images show A. salina larvae after 48 h exposure at F-UBE-HPC (Figure 8d-f) compared with blank (Figure 8a-c). Figure 8e,f shows intracellular lysosomes activated in the cell death process, evidenced by red fluorescence.

In Vitro Analysis of the Biological Effects of UBE-Loaded Mucoadhesive Oral Films on Human Blood Cell Cultures and CLS-354 Cancer Cell Line
The effects of F-UBE-HPC on blood cell cultures and CLS-354 tumor cell line were studied by highlighting the biological mechanisms implied in caspases 3/7 activity, nuclear shrinkage, autophagy, oxidative stress, cell cycle, apoptosis, and DNA synthesis and fragmentation, following previous preliminary studies with U. barbata dry ethanol extract [35,36].

Caspase 3/7 Activity
To prove that F-UBE-HPC causes cell apoptosis in normal blood cells and OSCC tumor cells, the effector caspase 3/7 intracellular activity was determined by flow cytometry. The results are illustrated in Figure 9.
cytometry. The results are illustrated in Figure 9.

Nuclear Condensation and Lysosomal Activity
After 24 h of F-UBE-HPC treatment in normal blood cells and CLS-354 tumor cells, the presence of pyknotic nuclei (stained with Hoechst 33342) and the lysosomal activity (evidenced by acridine orange) are displayed in Figure 10.

Nuclear Condensation and Lysosomal Activity
After 24 h of F-UBE-HPC treatment in normal blood cells and CLS-354 tumor cells, the presence of pyknotic nuclei (stained with Hoechst 33342) and the lysosomal activity (evidenced by acridine orange) are displayed in Figure 10.

ROS Levels
In blood cell cultures and CLS-354 tumor cell line, F-UBE-HPC induced powerful oxidative stress expressed as total ROS level measured through flow cytometry. The obtained results are indicated in Figure 11. ROS levels substantially increased in the blood cells treated with F-UBE-HPC compared with the 1% DMSO negative control and positive control of 125 µg/mL of UA (2833.33 × 104 ± 152.75; vs. 242.00 × 104 ± 2.00; 846.66 × 104 ± 5.77; p < 0.01, Figure 11A-C,G,I).

Cell Cycle Analysis
Human blood consists of leukocytes (white blood cells, WBC), thrombocytes (platelets), and erythrocytes (red blood cells, RBW). Only leukocytes contain a cell nucleus, essential when estimating the amount of DNA in human blood cell cultures [37].
DNA content was performed by propidium iodide/RNase stain to explore the effects of F-UBE-HPC in normal blood cells and CLS-354 tumor cells (Figure 12).

Cell Cycle Analysis
Human blood consists of leukocytes (white blood cells, WBC), thrombocytes (platelets), and erythrocytes (red blood cells, RBW). Only leukocytes contain a cell nucleus, essential when estimating the amount of DNA in human blood cell cultures [37].
DNA content was performed by propidium iodide/RNase stain to explore the effects of F-UBE-HPC in normal blood cells and CLS-354 tumor cells ( Figure 12).

Apoptosis
The effects of F-UBE-HPC treatment in normal blood cells and CLS-354 tumor cells were achieved by examining morphology and cell membrane integrity with annexin V- As shown in Figure 12A,B,G,I, in normal blood cell cultures, F-UBE-HPC induces a higher cell cycle arrest in G1/G0 phase (93.91 ± 1.41) than the negative control (88.52 ± 0.54, p < 0.05). DNA synthesis phase of the cell cycle presented significantly more decreased values than both controls: 0.99 ± 0.24 vs. C1: 4.76 ± 0.68, C2UA: 2.86 ± 0.23; p < 0.01 (Figure 12A-C,G,I).

Apoptosis
The effects of F-UBE-HPC treatment in normal blood cells and CLS-354 tumor cells were achieved by examining morphology and cell membrane integrity with annexin V-FITC/PI stain ( Figure 13). .* p < 0.05 and ** p < 0.01 represent significant statistical differences between controls and sample made by paired samples t-test; PI-propidium iodide; C1-negative control with 1% dimethyl sulfoxide (DMSO); C2UA-positive control with 125 µg/mL usnic acid (UA); F-UBE-HPC-bioadhesive oral film loaded with U. barbata dry ethanol extract; AP-apoptotic cell fraction (subG0/G1); S-synthesis of cell cycle phases.

Apoptosis
The effects of F-UBE-HPC treatment in normal blood cells and CLS-354 tumor cells were achieved by examining morphology and cell membrane integrity with annexin V-FITC/PI stain ( Figure 13).

Principal Component Analysis
The Principal Component Analysis (PCA) was performed for F-UBE-HPMC oral mucoadhesive patches and both controls (C1-DMSO and C2UA) and variable parameters determined in both cell types (normal blood cells and CLS-354 tumor cells) according to the correlation matrix and PCA-Correlation circle from the Supplementary Material. The results are displayed in Figure 15.  The two principal components explained the total data variance, with 51.43% a tributed to the first (PC1) and 48.57% to the second (PC2). The PC1 was associated wit both controls (C1-DMSO and C2UA), viability, early and late apoptosis, nuclear conden sation, and DNA fragmentation (subG0/G1) in both cell types and cell cycle arrest i G0/G1 and necrosis in CLS-354 tumor cells. The PC2 was related to F-UBE-HPC, ROS, an caspase 3/7 activity, DNA synthesis and autophagy in both cell types, and cell cycle arres in G0/G1 and necrosis in normal blood cells. The two principal components explained the total data variance, with 51.43% attributed to the first (PC1) and 48.57% to the second (PC2). The PC1 was associated with both controls (C1-DMSO and C2UA), viability, early and late apoptosis, nuclear condensation, and DNA fragmentation (subG0/G1) in both cell types and cell cycle arrest in G0/G1 and necrosis in CLS-354 tumor cells. The PC2 was related to F-UBE-HPC, ROS, and caspase 3/7 activity, DNA synthesis and autophagy in both cell types, and cell cycle arrest in G0/G1 and necrosis in normal blood cells.
By correlating and interpreting these data, the places of F-UBE-HPC patches and both controls (C1-DMSO and C2UA) in the PCA-correlation biplot ( Figure 15) were explained, highlighting the corresponding processes triggered in CLS-354 cancer cells and normal blood cells and their complementary mechanisms.

Discussion
Until the preparation of UBE-loaded bioadhesive oral films analyzed in the present work for potential applications in oral cancer treatment, U. barbata lichen was thoroughly studied for almost six years.
First, it is essential to mention that this lichen was harvested from a coniferous forest in the same area (47 • 28 N, 25 • 12 E, and 900 m altitude), belonging to the highest Romanian volcanic mountains (Calimani mountains) [30,38]. Coniferous forest soil is adjacent to the Tinovul Mare Poiana Stampei peat bog with a natural origin, its accumulation beginning in the post-glacial period [39]. The specific conditions of the U. barbata native zone consist of seasonal water-level fluctuations with thermic variations between −1 • C and 14 • C, with a precipitation range of 600-800 mL [39]. The soil has pH values between 4.09 and 5.89 and contains several trace/heavy metals under the alert threshold from national protocols [39].
A detailed elemental analysis was performed on dried lichen harvested in 2020 [30,38]. Twenty-three metals were investigated by inductively coupled plasma mass spectrometry (ICP-MS) [38]. The heavy metal concentrations were associated with those reported by Cazacu et al. [39] in the forest soil, and the results reveal that autochthonous U. barbata contains all, in lower amounts than the permissible limits for medicinal plants. However, the mercury content (0.671 ± 0.020 µg/g) was lower than the WHO's and FDA's approvable limit (1 µg/g) and over the one mentioned in the European Pharmacopoeia (0.1 µg/g) [38]. The metal concentrations were compared with those reported in the scientific literature regarding U. barbata from unpolluted zones (Mountain Natural Park in Bulgaria [40], Sri Lanka rain forest [41] and central and southern Tierra del Fuego, Patagonia, Argentina [42]). Thus, the data obtained confirmed the autochthonous lichen's suitability for pharmaceutical use [30], according to regulatory agencies [43], which restrict the harvesting area for accurate results in human-safe formulations.
The loss of drying of the dried lichen was determined [44], and bioactive secondary metabolites were identified and quantified [45]. The research on U. barbata from the Calimani mountains continued with the preparation of various extracts in different solvents through low-cost and easy-to-use conventional processes [38,44,46], investigating the suitable solvents and extraction conditions to obtain U. barbata extracts with pharmacological potential. All extracts were comparatively analyzed, quantifying their metabolites through HPLC-DAD [45,47] and evaluating their antioxidant, antimicrobial, and cytotoxic potential. The cytotoxicity prescreen was performed using A. salina as an animal model [44], continuing with complex studies on normal and tumor cells [35,36].
This previously described interdisciplinary research on U. barbata from the Calimani mountains led to selecting the most appropriate extracts for bioadhesive oral film formulation, including the dry ethanol extract. UBE contains usnic acid and other various phenolic compounds [48][49][50], including common polyphenols (ellagic acid and gallic acid) with high pharmacological potential [51]. Thus, U. barbata ethanol extract shows a dose-dependent inhibitory activity on the most known bacteria responsible for oral infectious disease [52] in immunocompromised patients: S. aureus, S. pneumoniae, S. pyogenes, Enterococcus sp., other Gram-positive bacteria isolated from oral cavity and pharynx (S. epidermidis, S. oralis, S. intermedius), and P. aeruginosa [38,51,53,54]. It also reported inhibitory effects on C. albicans [38]. Phenolic compounds extracted in ethanol have antibacterial and antifungal properties and could act synergistically in UBE. Moreover, inhibitory activity on bacterial strains varies in direct proportion to antioxidant potential [55], and UBE exhibits an intense antioxidant activity [51]. In normal cells, it shows antioxidant and protective effects, neutralizing a part of the free radicals generated by 0.2%DMSO, thus reducing the lipids peroxidation [56] and activity of antioxidant enzymes Superoxide dismutase (SOD) and Catalase (CAT) induced by cellular oxidative stress [36]. Contrarily, UBE demonstrated an in vitro anticancer effect on CAL-27 tumor cell line, triggering cell death processes. It acts as a prooxidant, increasing the oxidative stress induced by 0.2% DMSO and thus stimulating cellular antioxidant defense-Glutathione peroxidase (GPx) and SOD-and augmenting the malondialdehyde (MDA) levels [36,57].
Knowing the UBE bioactive metabolites' content and its pharmacological potential, the present work aimed to study a pharmaceutical formulation containing this U. barbata dry extract. Thus, UBE-loaded bioadhesive oral films were prepared and analyzed, their properties being compared with References (films without UBE). As a film-forming polymer, HPC was selected due to its non-ionic character and water solubility. In addition, it proved to be physiologically inert, non-irritating, bioadhesive, and biodegradable [58][59][60][61]. PEG 400 was used as a plasticizer in a 5% (w/w) amount to improve the film flexibility and obtain suitable mechanical properties [62,63]. Both films are transparent, flexible, and homogenous. The yellowish-green, faint-brown color of F-UBE-HPC is due to lichen dry ethanol extract, and its morphology shows a smooth surface.
The physical and chemical investigation of the bioadhesive oral films containing Usnea barbata dry ethanol extract demonstrated the successful incorporation of the UBE in the polymer matrix. The pharmacotechnical analysis of F-UBE-HPC was realized according to other previously published studies on bioadhesive films containing plant extracts [64,65].
Therefore, both films' thickness values show that UBE does not influence their dimensions. Because the films' thickness is directly connected to active ingredient concentration and the polymer's mucoadhesive function, uniformity is required. The selected plasticizer type and amount also influence the film thickness. Generally, the suitable thickness for bioadhesive oral films is 0.05-1.0 mm [66][67][68], and the UBE-loaded formulation belongs to this. The film's considerable folding endurance highlights appreciable flexibility and mechanical resistance provided by PEG 400 (used as a plasticizer in a 5% amount). The plasticizer's role is to modify the viscoelastic properties of the polymer, which will subsequently influence the adhesion ability and release behavior. Moreover, the UBE load did not change the films' mechanical properties.
The bioadhesive films' elongation evidenced their remarkable flexibility. The tensile strength indicates the films' high resistance, suitable to withstand the mechanical stress that occurs during unpacking and appliance on the oral mucosa. The films' hardness depends on the type and amount of polymer and plasticizer used. The obtained results prove the accurate selection of the excipients appropriate to incorporate UBE as an active ingredient in achieving high-quality bioadhesive films. Moreover, the films' flexibility is essential in aiding the penetration of active ingredients and attachment to the oral mucosa.
Bharkatiya et al. [69] stated that low tensile strength and elongation characterize a soft and weak polymer. In contrast, moderate tensile strength and low elongation are typical for a hard and brittle polymer. Furthermore, high tensile strength and elongation describe a soft and tough polymer, as in the studied formulations.
The flexibility of the chains diminishes as the crosslinking density in water-soluble polymers increases, thus protecting against overhydration. The bound water can build H-links with the HPC's accessible chains, allowing them to move more freely [70,71]. Repka et al. [72] stated that HPC has less water affinity. The obtained values also indicate a certain amount of moisture content in the films' structure, but it is well-known that humidity is needed to ensure bioadhesion. Moreover, bioadhesion is improved by functional groups that can create hydrogen bonds.
Both films' neutral pH ensures their tolerability and biocompatibility with oral mucosa. This property confirms that UBE inclusion in the polymer matrix does not affect the base films' pharmacotechnical properties; they exclusively depend on the selected excipients. In addition, pH value can influence the polymer's bioadhesive properties, and a neutral value is optimal for mucoadhesion [73,74]. HPC used as polymer film-forming is responsible for rapid disintegration, and the plasticizer also significantly influences this property. Another essential factor is the manner of active ingredient dispersion in the matrix base. In the studied formulation, UBE was solubilized and mixed with the polymer matrix, thus diminishing the disintegration time.
The swelling index plays an essential role in regulating the release rate of the active ingredient. The diffusion of water molecules into the hydrated decreases the number of hydrogen bonds and increases the strength between the polymers. Panomsuk et al. [75] proved that a polymer chain with a low ability to make hydrogen bonds cannot build a strong matrix structure, and water penetration would be difficult. The polymeric matrix swelling ability depends on its resistance to the migration of the water molecules, and the hydroxyl group is essential for hydrophilic cellulose polymers' matrix integrity [37]. Moreover, the polymer's substituted groups significantly limit the active ingredients' influence on the swelling properties. Hence, the similar swelling ratio of F-UBE-HPC and R demonstrates the impact of the active ingredient dispersion on the swelling degree because UBE incorporated as a solution in F-UBE-HPC did not change the polymer properties.
The swelling behavior also has an impact on the film's bioadhesive performance. The increased residence time is the result of the elastic behavior induced by the used plasticizer. The interaction between free polymer chains and mucin is essential; the active ingredients loaded into the matrix could occupy the chains [76], proving that their dispersion directly impacts the adhesion performance. The obtained data show that both oral films have the same bioadhesion time, demonstrating that UBE load did not influence the matrix adhesivity.
The F-UBE-HPC rapid disintegration and neutral pH were essential for accurately evaluating their pharmacological potential because the afferent studies were conducted in vitro (using bacterial, blood, and tumor cell cultures) and in vivo (on A. salina larvae used as an animal model) [77]. These previously mentioned analyses were performed with homogenous dispersions of UBE-loaded films in different culture media and, respectively, in phosphate buffer. On the other hand, the tested cultured cells and A. salina larvae are strongly affected by pH variation; thus, the films' neutral pH was requested to obtain accurate results.
For optimal evaluation, the studies used for the UBE-loaded films had a similar trend to previous ones for UBE investigation. Thus, the F-UBE-HPC antimicrobial effects were examined on similar bacterial and fungal species, and a cytotoxicity prescreen was performed on A. salina larvae. Anticancer activity was evaluated on a similar OSCC cell line (CLS-354, mouth epithelial squamous cell carcinoma). As normal cells, blood cell cultures were used from a single donor (the same as previously described in UBE's biological studies) because buccal mucosa has a substantial blood supply and is permeable for most blood cells. Therefore, the most common types of WBC in blood-lymphocytes (with T cells) and segmented cells (polymorphonuclear, including neutrophils and eosinophils)-are the predominant leukocytes mixed with various endothelial cell types in the oral mucosa. No significant differences in the proportions of these cells between healthy children and adults indicate that both innate and adaptive immune systems function in the oral cavity [78]. All selected studies reproduced, in a certain measure, the conditions of the previous UBE pharmacological evaluation, aiming to know whether the UBE-loaded films' activities are similar to the UBE ones.
The results show that F-UBE-HPC displayed dose-dependent inhibitory effects against S. aureus, P. aeruginosa, and both Candida sp. through various mechanisms belonging to UBE's phenolic secondary metabolites: inhibiting protein, DNA, and cell wall synthesis, permeabilizing the cell wall and chelating various microbial micronutrients and reducing their disponibility for cell growth [79]. These microbial species are frequently implied in immunocompromised patients' oral cavity infections. UBE-loaded bioadhesive films could protect them, facilitate their recovery after radiotherapy, and generally increase their life quality. Moreover, the significant inhibitory action against C. albicans could also be helpful in oral cancer prevention [80,81].
The BSL assay was used as cytotoxicity prescreen because it is a low-cost, fast, and effective analysis due to A. salina larvae sensitivity. It can be adapted to various applications [82][83][84]. The brine shrimp larvae used as biotester have a rapid growth rate and, in 24 h, pass to another developing stage (i.e., nauplii). Their mortality could be associated with blocking DNA synthesis or cell cycle arrest [29]. The movements of larvae and different morphological changes (visualized due to the larvae's transparency) could be associated with various cytotoxic mechanisms [28,82,85]. Previous studies consider that BSL assay results could predict the antitumor activity of tested products [86,87]. Thus, morphological changes induced by F-UBE-HPC on A. salina larvae and intracellular lysosomes activated in cell death processes (evidenced in FM images) anticipate the previously described in vitro anticancer effects on oral epithelial squamous cell carcinoma [86]. The brine shrimp larvae mortality could be associated with the blockage of DNA synthesis and cell cycle arrest in G0/G1 and intracellular lysosomes from FM images with autophagy.
All previously mentioned factors implied in oral cancer development produce oxidative stress (an imbalance between ROS generation and antioxidant defense, leading to high intracellular ROS levels) [88]. Excessive ROS causes oxidative damage to cellular constituents (lipids, proteins, and DNA), hardly affecting normal cells and then contributing to carcinogenesis [89,90]. ROS are also essential in cell signaling, modulating the most significant cell death pathways (mediated by mitochondria, endoplasmic reticulum, and death receptors) [91]. ROS in high concentration could induce caspase 3/7 activation, triggering cellular processes associated with cell death (DNA damage with cell cycle arrest in different phases [92], nuclear condensation [93], blockage of DNA synthesis, and autophagy [88]).
Generally, cancer cells (due to mitochondrial abnormalities) have higher ROS levels compared with normal cells [94]. Therefore, an overproduction of ROS in cancer cells may offer a cancer-specific therapy [95]. F-UBE-HPC acts on CLS-354 cancer cells, considerably increasing cellular oxidative stress and caspase 3/7 activity, triggering nuclear condensation and autophagy, inducing cell cycle arrest in G0/G1, and blocking DNA synthesis. Compared with UA 125 µg/mL, F-UBE-HPC generates an ROS level 3 × higher; the caspase activity level is double that of UA. Moreover, DNA synthesis is completely stopped, and lysosomal activity is substantially increased compared with UA. Even though CLS-354 tumor cell viability is slowly diminished after 24 h, all intracellular and molecular processes leading to tumor cell death are triggered at a significant level.
On the other hand, in normal blood cells, the basal ROS level is lower than the one in the cancer cell line. A moderate ROS level is essential to promote cell proliferation and survival. In blood cell cultures, oxidative stress induced by UBE-loaded films is lower than in the CLS-354 tumor cell line; their viability after 24 h is over 98%. Compared with cancer cells, it could be considered that F-UBE-HPC displays a protective role in blood cell cultures, remarkably diminishing caspase 3/7 activity, nuclear condensation, and lysosomal activity triggered by 1% DMSO.
If any concerns regarding the F-UBE-HPC safety for human health [96] in relationship with usnic acid hepatotoxicity still exist, the following data could be significant. LipoKinetix (Syntrax, Cape Girardeau, MO), the fat-burning diet supplement [97], was a multi-ingredient product, including 100 mg usnic acid per capsule, together with 25 mg of norephedrine hydrochloride, 100 µg of 3,5-diiodothyronine, 3 mg of yohimbine hydrochloride, and 100 mg of caffeine [98]. The recommended daily dose associated with severe liver failure was 3-6 capsules, corresponding to 300-600 mg of usnic acid [99]. One UBE-loaded bioadhesive oral film contains 0.330 mg UBE, corresponding to 0.036 mg UA; hence, over 2700 films should be necessary to ensure the usnic acid content of 100 mg from a single capsule of LipoKinetix.

Conclusions
In the present study, bioadhesive oral films loaded with U. barbata dry ethanol extract were formulated and manufactured using HPC as a polymer matrix and PEG 400 as a plasticizer.
F-UBE-HPCs and References (the same formulation without UBE) were investigated through physicochemical and pharmacotechnical analyses, and the obtained data reported no significant differences, proving the active ingredient's consistent incorporation.
The F-UBE-HPC properties are optimal for accurately maintaining the lichen extract's pharmacological potential. The results prove it, recommending UBE-loaded bioadhesive oral films for possible application in oral cancer treatment and prevention. Additional research will optimize the bioadhesive oral film formulation, performing drug release in vivo and clinical studies, aiming to complete this interdisciplinary work and confirm their therapeutical benefits.