Antitumor Activity of Nanoparticles Loaded with PHT-427, a Novel AKT/PDK1 Inhibitor, for the Treatment of Head and Neck Squamous Cell Carcinoma

Currently, new treatments are required to supplement the current standard of care for head and neck squamous cell carcinoma (HNSCC). The phosphatidylinositol3-kinase (PI3K) signaling pathway is commonly altered and activated in HNSCC. PHT-427 is a dual PI3K-mammalian target of the AKT/PDK1 inhibitor; however, to the best of our knowledge, the effect of the PHT-427 inhibitor on HNSCC has not been investigated. This study aims to evaluate the antitumoral effect of PHT-427-loaded polymeric nanoparticles based on α-tocopheryl succinate (α-TOS). The in vitro activity of PHT-427 was tested in hypopharynx carcinoma squamous cells (FaDu) to measure the cell viability, PI3KCA/AKT/PDK1 gene expression, and PI3KCA/AKT/PDK1 levels. Apoptosis, epidermal growth factor receptor (EGFR), and reactive oxygen species (ROS) were also measured. The presence of PHT-427 significantly enhances its antiproliferative and proapoptotic activity by inactivating the PI3K/AKT/PDK1 pathway. Nanoparticles (NPs) effectively suppress AKT/PDK1 expression. Additionally, NPs loaded with PHT-427 produce high oxidative stress levels that induce apoptosis. In conclusion, these results are promising in the use of this nanoformulation as a PHT-427 delivery system for effective HNSCC treatment.


Introduction
Of all cancer cases worldwide, head and neck squamous cell carcinomas (HNSCC) account for 5% [1]. Despite advances in diagnosis and treatment, HNSCC presents a poor prognosis, with a 5-year survival rate of 50-60% [1][2][3][4]. Therefore, novel agents that significantly enhance the effects of existing chemotherapeutic drugs with lower toxicity are needed.
In terms of genetic aberrations, a promising pathway in HNSCC is the phosphatidylinositol 3-kinase (PI3K) signaling pathway. The PI3K signaling pathway regulates cell Polymeric nanoparticles were successfully prepared using a MTOS11 polymer that was previously synthesized by free radical polymerization using N-vinyl pyrrolidone (VP) and a methacrylic derivative of α-TOS (MTOS) with a copolymer molar composition of VP: MTOS 89:11 [20,21]. These self-assembled NPs were prepared by the nanoprecipitation method. MTOS11 (10 mg/mL) and PHT-427 (10% w/w respect to the polymer) were dissolved in dioxane and incorporated drop by drop over an aqueous phase (NaCl, 100 mM) under constant magnetic stirring to obtain a polymer concentration of 2.0 mg/mL. After the nanoprecipitation, the NP suspensions were purified by dialysis for 72 h in order to remove the organic solvent and non-encapsulated PHT-427. The unloaded NPs (NP-Ø) were synthesized by the same procedure without incorporation of the drug.
To calculate the encapsulation efficiency, NP-PHT427 was freeze-dried to eliminate the aqueous phase. Afterward, PHT-427 entrapped in the NPs was quantified by absorbance spectroscopy. Specifically, NP-PHT427 (5 mg/mL) was dissolved in ethanol, and their absorbance was measured using a NanoDrop™ One/OneC. A calibration curve was previously calculated at PHT-427 concentrations ranging from 0.001 to 1 mg/mL in ethanol. The encapsulation efficiency (EE) was calculated using the formula below: where [loaded PHT-427] i is the total concentration of the PHT-427 entrapped into NPs and [loaded PHT-427] 0 is the concentration of the molecule added initially during the preparation of NPs. The particle size distribution of the unloaded and loaded NP suspensions was measured by dynamic light scattering (DLS) on a Malvern Nanosizer NanoZS Instrument (Malvern Panalytical Ltd., Malvern, UK) (laser wavelength 633 nm at 25 • C) and in square polystyrene cuvettes (SARSTEDT, Nümbrecht, Germany). Moreover, the scattering was monitored at a fixed angle of 173 • . The zeta potential was determined for NP formulations at a 0.1-mg/mL concentration containing 10 mM of NaCl and using laser Doppler electrophoresis (LDE).

Cell Viability Assay
The cell viability was measured using Alamar Blue assay (AB) (ThermoFisher Scientific, Waltham, MA, United States). In brief, the FaDu cells were seeded at 10 4 cells/well in 96-well plates. At 24 h of incubation, the medium was replaced with the corresponding NP treatments for 24 and 48 h. Then, AB solution (10% (v/v) solution of AB dye) was added to each well, and fluorescence was measured at 3 h of incubation with a FLUOstar Omega (BMG Labtech, Ortenberg, Germany) plate reader at an excitation wavelength of 571 nm and an emission of 601 nm. NP treatment at 0 mg/mL was taken as 100% viability. The results were averaged over 3 different independent experiments, with 16 replicates for each experimental condition per experiment.

Reactive Oxygen Species Detection
ROS generation was determined using red fluorescent probe dihydroethidium (DHE) (Calbiochem, San Diego, CA, United States). In brief, the FaDu cells were seeded at 1 × 10 5 cells/well in 24-well plates. After 24 h of incubation, the cells were treated and the plates were incubated for 24 h. After fixation with 4% PFA, the cells were incubated with DHE (4 µmol/L) at 37 • C for 90 min. Next, the cells were stained with 300 nM of DAPI (Sigma-Aldrich, San Luis, CA, United States) for 5 min at 37 • C and were scored in an Olympus BX51 fluorescence microscope. ImageJ was used for quantitative analysis of the image.

Evaluating Reference Gene Expression
A web-based tool RefFinder (www.leonxie.com/referencegene.php, accessed on 9 August 2021) was used to determine the reference genes' stability. RefFinder is a webbased tool developed for evaluating and screening reference genes from experimental datasets. It integrates major computational programs including BestKeeper, geNorm, Normfinder, and the comparative Delta-Ct method to rank and compare the candidate reference genes. Based on the rankings from each program, it assigned an appropriate weight to an individual gene and calculated the geometric mean of their weights for the overall final ranking [22].

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction
The total RNA derived from the FaDu cells was extracted using miRNeasy Tissue/Cells Advanced Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. RNA recovery was determined spectrophotometrically in NanoDrop One (Thermo Scientific, Waltham, MA, United States). The total RNA (1 µg) was reverse transcribed using a StaRT kit (AnyGenes, Paris, France) according to the manufacturer's instructions, using Veriti Thermal Cycler (Applied Biosystems, Waltham, MA, United States).
A quantitative real-time reverse transcription-polymerase chain reaction was performed to study gene expression using the 7500 Fast real-time PCR detection system (Applied Biosystems, Waltham, MA, United States). Complementary DNA (cDNA) templates (2 µL) were added to 8 µL of Perfect Master Mix SYBRG (AnyGenes, Paris, France). The final volume was 10 µL. Polymerase chain reactions were carried out according to the procedures provided by the manufacturer in triplicate. The transcript levels were normalized to GUSB and RPLPO (used as reference genes). Determination of the relative expression levels was performed using the comparative "Ct" method. The following human primers were used in this study: PI3KCA, AKT1, AKT2, and PDK1 (AnyGenes, Paris, France).

Apoptosis Quantification
Apoptosis was measured by the Human Cleaved Caspase-3 (Asp175) ELISA Kit (Abcam) in cell culture extracts from each treatment group according to the manufacturer's instructions. Absorbance was measured using a FLUOstar Omega (BMG Labtech Ortenberg, Germany) microplate reader at 450 nm.

Statistical Analysis
The results are expressed as mean ± standard deviation. Statistical significance (p < 0.05) was evaluated using the analysis of variance (ANOVA, Tukey test) by Origin 9.

Nanoparticle Characterization: PHT-427 Encapsulation
PHT-427 was successfully entrapped in the inner core of self-assembled nanoparticles based on the MTOS11 copolymer. This copolymer was previously synthesized by free radical polymerization using VP as hydrophilic monomer and a methacrylic derivative of α-TOS (MTOS) with a molar composition of VP:MTOS 89:11. The structure of this copolymer is shown in Figure 1. Additionally, the most relevant properties of this copolymer are summarized in Figure S1 (see the Supplementary Materials).
formed to study gene expression using the 7500 Fast real-time PCR detection system (Applied Biosystems, Waltham, MA, United States). Complementary DNA (cDNA) templates (2 µL) were added to 8 µL of Perfect Master Mix SYBRG (AnyGenes, Paris, France). The final volume was 10 µL. Polymerase chain reactions were carried out according to the procedures provided by the manufacturer in triplicate. The transcript levels were normalized to GUSB and RPLPO (used as reference genes). Determination of the relative expression levels was performed using the comparative "Ct" method.

Apoptosis Quantification
Apoptosis was measured by the Human Cleaved Caspase-3 (Asp175) ELISA Kit (Abcam) in cell culture extracts from each treatment group according to the manufacturer's instructions. Absorbance was measured using a FLUOstar Omega (BMG Labtech Ortenberg, Germany) microplate reader at 450 nm.

Statistical Analysis
The results are expressed as mean ± standard deviation. Statistical significance (p < 0.05) was evaluated using the analysis of variance (ANOVA, Tukey test) by Origin 9.

Nanoparticle Characterization: PHT-427 Encapsulation
PHT-427 was successfully entrapped in the inner core of self-assembled nanoparticles based on the MTOS11 copolymer. This copolymer was previously synthesized by free radical polymerization using VP as hydrophilic monomer and a methacrylic derivative of α-TOS (MTOS) with a molar composition of VP:MTOS 89:11. The structure of this copolymer is shown in Figure 1. Additionally, the most relevant properties of this copolymer are summarized in Figure S1 (see the Supplementary Materials). The appropriate hydrophilic and hydrophobic balance in the polymer chains of MTOS 11 allowed for the formation of nanoparticles, where the PHT-427 was entrapped in their inner core through inter-and intramolecular hydrophobic interactions. The appropriate hydrophilic and hydrophobic balance in the polymer chains of MTOS 11 allowed for the formation of nanoparticles, where the PHT-427 was entrapped in their inner core through inter-and intramolecular hydrophobic interactions.
Unloaded (NP-Ø) and PHT-427-loaded NPs (NP-427) were successfully prepared and characterized. The results are summarized in Table 1. Both NP suspensions exhibited unimodal size distributions with an appropriate hydrodynamic diameter that slightly increased for NP-427 (132 nm and 155 nm for NP-Ø and NP-427, respectively). Additionally, PHT-427 was efficiently entrapped during the nanoprecipitation process, with an EE of 75.4% as a result of its hydrophobicity and low water solubility. In the nanoprecipitation process, the hydrophobic interactions between PHT-427 and the MTOS nanodomains in the core of the nanoparticles regulates the encapsulation of this drug. Moreover, the hydrophilic shell that was rich in VP stabilized them due to the optimal hydrophilic-hydrophobic balance in the particles. Table 1. Most relevant characteristics of NP-Ø and NP-427: hydrodynamic diameter (Dh), polydispersity index (PDI), zeta potential (ξ), encapsulation efficiency (EE), loading capacity (LC), and encapsulated drug concentration. Values represent mean ± SD.

NP Sample
PHT-427 (%w/w) The NPs exhibited slightly negative zeta potentials, indicating an almost neutral charge of the surface. These values are typical of α-TOS-based NPs, as recently reported by our group [15,16].

NPs Loaded with PHT-427 Reduce FaDu Cell Viability
The cell viability studies were assessed using an Alamar Blue assay. Figure 2 shows the effects of NP-Ø and NP-427 after 24 (A) and 48 h (B) and demonstrates that the relative FaDu viability decreased in a dose and time-dependent manner, and was lower for NP-427. Particularly, cell viability was reduced below 70%, being toxic for cancer cells (a cell viability lower than 70% is cytotoxic according to ISO 10993-5: 2009) when NP-427 was administered at concentrations higher than 0.5 mg/mL for 24 and 48 h (Figure 2A,B). The effect of NP-Ø was observed after 24 h, and this effect did not increase over time (no significant differences between 24 and 48 h were observed). The activity of NP-427 was significantly higher after 48 h; this means that the NPs were taken up by the cells at 24 h, but that the release of the PHT-427 inhibitor took longer. For instance, NP-427 at 0.5 mg/mL reduced the cell viability of FaDu from 61.5% at 24 h to 45% at 48 h.
NP-427 at concentrations of 0.5 and 0.75 mg/mL were used in the following experiments, as these concentrations were the concentrations of the NPs loaded with the incorporated highest concentration of the PHT-427 inhibitor that presented selectivity against cells at the lowest treatment time, i.e., 24 h. Moreover, NP-Ø at the concentrations of 0.5 and 0.75 mg/mL were considered to analyze the intrinsic biological activity of the polymeric nanovehicles.
The results clearly showed that NP-427 was cytotoxic for FaDu cells with an IC50 of 0.629 mg/mL at 24 h and an IC50 of 0.538 mg/mL at 48 h (see Table S1, Supplementary Materials). The total cytotoxicity (100%) was reached at a concentration of 1 mg/mL at 48 h. For NP-Ø, the IC50 is 0.738 mg/mL at 24 h, the IC50 is 0.735 mg/mL at 48 h, and the total cytotoxicity (100%) is not reached even at a concentration of 1 mg/mL. Therefore, an antiproliferative effect is seen in cancer cells. NP-427 has the lowest IC50 and, therefore, the highest efficiency.
In Figure S2 (see the Supplementary Materials), the FaDu viability when in contact with free or encapsulated PHT-427 is compared against drug concentration after 24 and 48 h of treatment. For the same drug concentration, free PHT-427 leads to a lower cell viability in contrast with the encapsulated one. After 24 h, the cell viability values were 0.047 and 0.016 mg/mL for encapsulated and free PHT-427, respectively. Therefore, NP-PHT-427 represents an excellent vehicle that allows us to encapsulate high concentrations of hydrophobic PHT-427, enhancing their bioavailability and dosage on the targeted side. In spite of the reduction in PHT-427 in vitro toxicity, NP-PHT427 allows for a sustained released of the highly encapsulated drug concentration from the inner core of the particles on the tumor area. Figure 2C shows images obtained by optical microscopy of the FaDu culture treated with NP-Ø and NP-427 at 24 h. NP-treated groups were affected, particularly in the case of NP-427. In these groups, the cell morphology was not polygonal but rounded together with a qualitative decrease in the number of cells when compared with the control.
PHT-427 represents an excellent vehicle that allows us to encapsulate high concentrations of hydrophobic PHT-427, enhancing their bioavailability and dosage on the targeted side. In spite of the reduction in PHT-427 in vitro toxicity, NP-PHT427 allows for a sustained released of the highly encapsulated drug concentration from the inner core of the particles on the tumor area. Figure 2C shows images obtained by optical microscopy of the FaDu culture treated with NP-Ø and NP-427 at 24 h. NP-treated groups were affected, particularly in the case of NP-427. In these groups, the cell morphology was not polygonal but rounded together with a qualitative decrease in the number of cells when compared with the control.

NPs Loaded with PHT-427 Induce FaDu Cell Apoptosis
Apoptosis induction by NPs in FaDu cells was quantitatively studied by measuring the levels of caspase-3 (apoptosis effector protein) by ELISA in cell lysates. The FaDu cells had significantly higher levels of caspase-3 in the NP treatment groups compared with the control group. Statistically significant differences between the levels of caspase-3 for NP-Ø and NP-427 groups were found, with the caspase-3 activity being the highest for

NPs Loaded with PHT-427 Induce FaDu Cell Apoptosis
Apoptosis induction by NPs in FaDu cells was quantitatively studied by measuring the levels of caspase-3 (apoptosis effector protein) by ELISA in cell lysates. The FaDu cells had significantly higher levels of caspase-3 in the NP treatment groups compared with the control group. Statistically significant differences between the levels of caspase-3 for NP-Ø and NP-427 groups were found, with the caspase-3 activity being the highest for NP-427 ( Figure 3A). Apoptosis was also qualitatively analyzed by immunofluorescence using Annexin V staining cells. The fluorescence micrographs revealed an increase in red fluorescence in FaDu cells incubated with both NPs groups, being higher in the NP-427 group, indicating an increase in apoptosis ( Figure 3B). NP-427 ( Figure 3A). Apoptosis was also qualitatively analyzed by immunofluorescence using Annexin V staining cells. The fluorescence micrographs revealed an increase in red fluorescence in FaDu cells incubated with both NPs groups, being higher in the NP-427 group, indicating an increase in apoptosis ( Figure 3B).

Reference Gene Analysis
For the selection of genes that could be used as normalizers in the qPCR analysis of the expression of target genes for FaDu cells, we analyzed eight reference gene expressions for the different experimental conditions: C (control), NP-Ø (NP without loading, 0.5 and 0.75 mg/mL), and NP-PHT427 (NP loaded with PHT427, 0.5 and 0.75 mg/mL) at 24 h.
First, the expression of the reference genes was analyzed by qPCR. Afterward, the data obtained were analyzed using the algorithm of the Red Finder software. Table 2 showed the ranking order of eight reference genes analyzed by the computational programs Normfinder, BestKeeper, Genorm, and comparative Delta-Ct method algorithms separately and recommended comprehensive ranking by the RedFinder program.

Reference Gene Analysis
For the selection of genes that could be used as normalizers in the qPCR analysis of the expression of target genes for FaDu cells, we analyzed eight reference gene expressions for the different experimental conditions: C (control), NP-Ø (NP without loading, 0.5 and 0.75 mg/mL), and NP-PHT427 (NP loaded with PHT427, 0.5 and 0.75 mg/mL) at 24 h.
First, the expression of the reference genes was analyzed by qPCR. Afterward, the data obtained were analyzed using the algorithm of the Red Finder software. Table 2 showed the ranking order of eight reference genes analyzed by the computational programs Normfinder, BestKeeper, Genorm, and comparative Delta-Ct method algorithms separately and recommended comprehensive ranking by the RedFinder program.
Calculations based on the algorithm of RedFinder found GUSB and RPLP0 to be the most stable genes for the NP-427 group, followed by the TFRC, PPIA, B2M, TBP, ACTB, and HPRT1 genes (Figure 4). For a good quantification of gene expression, the use of more than one reference gene for the efficient normalization of gene expression data is suggested [23]. Thus, we combined the use of the two most stable reference genes for normalization. Calculations based on the algorithm of RedFinder found GUSB and RPLP0 most stable genes for the NP-427 group, followed by the TFRC, PPIA, B2M, TBP and HPRT1 genes (Figure 4). For a good quantification of gene expression, the use than one reference gene for the efficient normalization of gene expression data is su [23]. Thus, we combined the use of the two most stable reference genes for normaliza

PHT-427-Loaded NPs Reduced Gene Expression of the PI3K Pathway
Quantitative statistical analysis through a histogram is used to show the r PCR results for PI3K pathway proteins ( Figure 5). The expressions of almost every involved in the pathway (PI3K, AKT1, AKT2, and PDK1) were significantly (p < duced in cells treated with NP-427 in the 0.5 and 0.75 mg/mL concentrations co with the control and NP-Ø groups.

PHT-427-Loaded NPs Reduced Gene Expression of the PI3K Pathway
Quantitative statistical analysis through a histogram is used to show the real-time PCR results for PI3K pathway proteins ( Figure 5). The expressions of almost every protein involved in the pathway (PI3K, AKT1, AKT2, and PDK1) were significantly (p < 0.05) reduced in cells treated with NP-427 in the 0.5 and 0.75 mg/mL concentrations compared with the control and NP-Ø groups.

Levels of PI3K Pathway Components Decreased with PHT427-Loaded NPs
The immunofluorescence identification of PI3KCA protein (green color) at 24 h with concentrations of 0.5 mg/mL and 0.75 mg/mL of NP-427 shows an objective decrease in intracellular PI3KCA ( Figure 6A,B) congruent with a decrease in results in the Western blot analyses ( Figure 6C) compared with untreated and NP-treated unloaded cells. This cellular decline is statistically significant (p < 0.05). PDK1 identification in immunofluorescence has a similar pattern to that of PI3KCA ( Figure 7A). In FaDu cultures, green fluorescence decreased in the group with NP-427, in a dose-dependent manner, compared with the control and unloaded NP groups.
The reduction in fluorescence was quantified using Image J software, and the differences found were statistically significant (p < 0.05) ( Figure 7B). The Western blot shows the same results, with a significant decrease in PDK1 levels when the concentration of PHT427-loaded nanoparticles increased ( Figure 7C). NP-427 at 0.5 and 0.75 mg/mL exhibits a statistically significant decrease in PDK1 concentration (p < 0.05).
In the case of AKT1/2, immunofluorescence is reduced following the augmentation of NP-427 concentrations ( Figure 8A-D). Similar to the anterior cases, the Western blot showed a decrease in phospho-Thr308-Akt, phospho-Ser473-Akt, and total AKT at 24 h in cells treated with NP-427 at 0.5 and 0.75 mg/mL (p < 0.05) ( Figure 8E). Pharmaceutics 2021, 13, x 10 of 19

Levels of PI3K Pathway Components Decreased with PHT427-Loaded NPs
The immunofluorescence identification of PI3KCA protein (green color) at 24 h with concentrations of 0.5 mg/mL and 0.75 mg/mL of NP-427 shows an objective decrease in intracellular PI3KCA ( Figure 6A,B) congruent with a decrease in results in the Western blot analyses ( Figure 6C) compared with untreated and NP-treated unloaded cells. This cellular decline is statistically significant (p < 0.05).

PHT427-Loaded NPs Increase Levels of Superoxide Anions
Fluorescence for the oxidized dihydroethidium (DHE) probe was used to detect superoxide anion (O 2− ) by visualizing the increased intensity of red fluorescence in the nuclei of cells. FaDu cell cultures in the NP-427 group ( Figure 9A) show an increase in fluorescence in the cell nucleus regarding control and NP-Ø groups. Statistical significance was obtained by analyzing fluorescence using ImageJ software; thus, NP-427 showed an DHE intensity of 21.6 ± 0.6 (at 0.5 mg/mL) and 23.8 ± 0.5 (at 0.75 mg/mL) arbitrary units (AU) (p < 0.05) in comparison with the control group, which showed 16.7 ± 0.4, and with the NP-Ø group, which showed 17.1 ± 0.5 AU ( Figure 9B).

EGFR Decreases in the Presence of NPs Loaded with PHT-427
The EGFR levels were measured by Western blot. The results show that EGFR levels in FaDu cells after the treatment at 24 h were lower in the case of NP-Ø and NP-427 when compared with the control (Figure 10). The EGFR levels were significantly (p < 0.05) decreased using NP-427 concerning the control and NP-Ø. Particularly, the EGFR expression decreased by 1981 ± 12 and 1211 ± 18 AU at concentrations of 0.5 mg/mL and 0.75 mg/mL of NP-427, respectively, concerning the control (3610 ± 11 AU). PDK1 identification in immunofluorescence has a similar pattern to that of PI3KCA ( Figure 7A). In FaDu cultures, green fluorescence decreased in the group with NP-427, in a dose-dependent manner, compared with the control and unloaded NP groups. The reduction in fluorescence was quantified using Image J software, and the differences found were statistically significant (p < 0.05) ( Figure 7B). The Western blot shows the same results, with a significant decrease in PDK1 levels when the concentration of PHT427-loaded nanoparticles increased ( Figure 7C). NP-427 at 0.5 and 0.75 mg/mL exhib its a statistically significant decrease in PDK1 concentration (p < 0.05).
In the case of AKT1/2, immunofluorescence is reduced following the augmentation of NP-427 concentrations ( Figure 8A-D). Similar to the anterior cases, the Western blo showed a decrease in phospho-Thr308-Akt, phospho-Ser473-Akt, and total AKT at 24 h in cells treated with NP-427 at 0.5 and 0.75 mg/mL (p < 0.05) ( Figure 8E).  Fluorescence for the oxidized dihydroethidium (DHE) probe was used to detect superoxide anion (O 2− ) by visualizing the increased intensity of red fluorescence in the nuclei of cells. FaDu cell cultures in the NP-427 group ( Figure 9A) show an increase in fluorescence in the cell nucleus regarding control and NP-Ø groups. Statistical significance was obtained by analyzing fluorescence using ImageJ software; thus, NP-427 showed an DHE intensity of 21.6 ± 0.6 (at 0.5 mg/mL) and 23.8 ± 0.5 (at 0.75 mg/mL) arbitrary units (AU) (p < 0.05) in comparison with the control group, which showed 16.7 ± 0.4, and with the NP-Ø group, which showed 17.1 ± 0.5 AU ( Figure 9B).

EGFR Decreases in the Presence of NPs Loaded with PHT-427
The EGFR levels were measured by Western blot. The results show that EGFR levels in FaDu cells after the treatment at 24 h were lower in the case of NP-Ø and NP-427 when compared with the control (Figure 10). The EGFR levels were significantly (p < 0.05) decreased using NP-427 concerning the control and NP-Ø. Particularly, the EGFR expression decreased by 1981 ± 12 and 1211 ± 18 AU at concentrations of 0.5 mg/mL and 0.75 mg/mL of NP-427, respectively, concerning the control (3610 ± 11 AU).

Discussion
Targeted therapy using inhibitors has led to a new paradigm in the treatment of cancer. Overexpression or mutation of the PI3K signaling pathway has been identified as one of the key regulators in several malignancies, including HNSCC [5][6][7][8]. PHT-427 is a mol-

Discussion
Targeted therapy using inhibitors has led to a new paradigm in the treatment of cancer. Overexpression or mutation of the PI3K signaling pathway has been identified as one of the key regulators in several malignancies, including HNSCC [5][6][7][8]. PHT-427 is a molecule inhibitor-targeting PI3K/AKT pathway with promising antitumor activity in various cancers but is not studied at HNSCC [11,12]. Therefore, this is the first study to investigate the effect of PHT-427 therapy on FaDu (HNSCC) cells.
In our study, PHT-427 was chosen as a competitive inhibitor derived from sulphonamides that acts specifically at two sites, AKT and PDK1, in the PI3K pathway at the same time [12,24]. These two sites are related to a PH domain involved in the PIP3-AKT joint and phosphorylation of Threonine 308 from AKT by PDK1. This inhibitor is used in research in different tumors such as in the pancreas [25], prostate [26], or skin [27], but as a hydrophobic drug and insoluble in an aqueous medium, its bioavailability is limited to oral formulation. Therefore, more efficient methods are required to improve the delivery of PHT-427 to cancer cells Polymeric nanoparticles are promising vehicles to transport chemotherapeutic drugs due to their high versatility, easy surface functionalization, and modulation of physicochemical properties by changing the polymer composition and microstructure [14][15][16][17][18]. In the present preclinical study, we investigated the employment of the poly(VP-co-MTOS) nanoparticle drug delivery system, developed by our group, to improve the therapeutic effect of PHT-427 for the treatment of HNSCC. Previously, we successfully used this delivery system to transport α-TOS (an antitumor drug) in the treatment of HNSCC [20,21]. The present study shows the efficacy of PHT-427 loaded in poly(VP-co-MTOS) nanoparticles in HNSCC, demonstrating antitumoral activity.
Treatment with NP-427 was confirmed to significantly decrease the viability and to increase apoptosis of FaDu cells. NP-427 concentrations between 1.0 mg/mL and 0.5 mg/mL inhibited the FaDu viability below 70% during the first 24-48 h (Figures 2 and 3). The data we obtained on cell viability are related to previous studies of Lucero-Acuña et al. [19]. They encapsulated the inhibitor PHT-427 in poly (lactic-co-glycolic acid) nanoparticles (PLGA) to treat BxPC3 and MiaPaCa-2 cells (pancreatic cancer cells). In cell viability tests that assumed the biphasic release behavior of PHT-427, the results showed that encapsulating in PLGA decreased the cell viability of MiaPaCa-2 and BxPC3 cells relative to treatment with PHT-427 alone. However, when they considered an alternative assumption that the PHT-427-PGLA rapidly delivered its entire contents to the cytosol soon after cell internalization (not biphasic release), the results showed that the encapsulation did not improve the therapeutic efficacy against the BxPC3 and MiaPaCa-2 cells. Moreover, these effects on cell viability were studied in periods of 1 to 7 days against our tests, where we saw effects from 24 h. Previous studies of Tian et al. with the inhibitor BEZ235 (dual inhibitor of PI3K-mTOR) loaded in delivery systems, such as liposomes, significantly reduced the cell viability of the HN5 head and neck squamous cell carcinoma cell line compared with the control and inhibitor alone at 72 h [28]. The increase in PI3K/Akt overexpression of up to 50% of all HNSCC cases suggests that inhibition of the PI3K/Akt/PDK1 signaling pathway is an effective HNSCC cancer therapy [29,30]. Changes in gene expression, protein levels, and immunofluorescence patterns illustrate the inhibition to the PI3K/AKT/PDK1 pathway implicated in cell growth and survival (Figures 5-8). A gene expression study is crucial to understand how the inhibitor affects cell growth and promotes apoptosis. Our in vitro results demonstrated that NP-427, compared with the control and unloaded-nanoparticle groups, significantly suppress the gene expressions of PI3KCA, PDK1, and AKT ( Figure 5). At the protein level, immunofluorescence patterns and Western blot data illustrate the decrease in markers such as PI3K, p-PDK1-Ser241, Total AKT, p-AKT-Ser473, and p-AKT-Thr308 (Figures 6-8). This decrease in markers coincides with a decrease in the cell population, gene expression, and the induction of apoptosis in FaDu cells (Figures 2, 3 and 5).
The tensin homolog (PTEN), a tumor suppressor and phosphatase in normal physiology, regulates PI3K. However, the loss or inactivation of PTEN is a frequent alteration in cancer, leading to hyperactivity of the PI3K pathway [6]. Several inhibitors have been developed to inhibit this pathway for HNSCC, in combination or not with chemotherapy, immunotherapy, or radiation [10,31,32]. BYL719, an inhibitor of the PI3K pathway, concomitant tested with Dacomitinib [33], demonstrates synergic action against HNSCC cells with PI3CA mutation. In another study, adenosine induces intrinsic apoptosis of HNSCC cells via the PI3CA/AKT/mTOR signaling pathway, decreasing the phosphorylation levels of PI3K, AKT, and mTOR [34]. Herzog and colleagues [35] studied double inhibition on PI3K/AKT/mTOR by oral intake of PF-04691502. This tumor suppressor acts as a competitive inhibitor of ATP in PI3K and mTOR. To date, there are no other studies that analyze the PHT-427 inhibitor in HNSCC. Finally, PHT-427 has also been studied as a stabilizer of AKT dysregulation in skin cancer promoted for Rapamicine [27]. Our results are in agreement with the antitumor effect observed in the study by Kobes et al. [25]. This research sought to improve the treatment of pancreatic cancer via the drug delivery of inhibitor PHT-427 from PLGA nanoparticles. PLGA-PHT-427 showed the elimination of primary pancreatic tumor in 68% of mice and a six-fold to four-fold reduction in tumor volume relative to untreated tumors.
One extensively studied receptor tyrosine kinase upstream of PI3K/Akt signaling is EGFR. Overexpression of EGFR has been identified in many cancers from epithelial origins, including HNSCC, where overexpression of EGFR is found in over 95% of all tumors [25,26] and has been associated with a more aggressive malignant phenotype, including increased resistance to treatment and poorer clinical outcome. The findings of the current study show that PHT-427-loaded nanoparticle decrease EGFR levels in the FaDu cell line ( Figure 10). This is comparable with a previous report of BYL719 (a specific inhibitor for PI3K p110α) in human head and neck cancer cell lines.
Oxidation in tumoral cells is important to maintain homeostasis. Normally, tumoral cells tend to endure high levels of oxidation to maintain a considerable division rate. For this reason, mitochondrial activity is increased and transmembrane proteins are capable of ejecting the overflow of superoxide anion out of the cell. In cases with an overload of superoxide anion in cells, homeostasis mechanisms may not be enough and apoptosis could be triggered, even in tumoral cells [36]. DHE, for superoxide anion detection, shows an increase in oxidation in cells treated with NPs (red color intensified) most clearly seen in cells with 0.75 mg/mL (Figure 9). The MTOS included in the NP structure promotes an elevation in cell oxidation and may help the apoptotic properties of PHT-427 itself, as our group demonstrated before [20,21].

Conclusions
The inhibitor PHT-427 is a promising targeted antitumor agent that has shown strong effects against several cancers but has not been tested in HNSCC. Our research in vitro shows the possibility to use this AKT/PDK1 pathway in new treatments of HNSCC, especially with the PHT-427 inhibitor. By utilizing NPs, we can resolve the problem of the administration and hydrophobic properties of PHT-427. However, an anticancer evaluation of the PHT-427-loaded MTOS-nanoparticles in animal models is still needed. Further preclinical and clinical studies of PHT-427-loaded nanoparticles alone or in combination with chemotherapeutic drugs could be beneficial to cancer patients.