THC, an isolated major metabolite of curcumin, is a polyphenolic compound that exhibits improved bioavailability over curcumin [11]. It possesses exemplary chemo-preventive and anticancer potential [14]. Despite its advantages over curcumin in terms of chemical stability [20]. THC shows low aqueous solubility, limiting its bio-accessibility [21]. Therefore, several drug delivery systems have been created to enhance its efficiency and solubility.

Phytosomes (phyto-phospholipid complexes) are lipid-based organic delivery systems that can increase the absorption and stability of poorly soluble phytochemicals [29]. Having a high affinity for polyphenols, they can create hydrogen bonds between their polar heads and the polar part of the active ingredients they carry [50]. These bonds enhance the efficiency of drug entrapment, water solubility, and minimizes drug leakage. This, in turn, improves drug stability, increases membrane absorption, conclusively maximizing bioavailability [31]. There are several phytosomal preparations of a wide range of phytochemicals. Those with the most positive clinical evidence are curcumin and silybin phytosomes, having been the most commonly prepared [51]. Most phytosomal curcuminoids behave more superiorly than free curcuminoids, showing enhanced cytotoxicity and increased cellular uptake [52]. In another context, the nanoformulation of polyvinyl pyrrolidone-stabilized curcumin has previously shown a preferential uptake to SCC4 over normal cell lineage [9].

Hence in our present work, we aim to develop and characterize THC-phytosomes as an efficient nanoformulation. We also aim to ascertain its cytotoxic efficacy and selectivity against SCC4 cell line compared to its free solution counterpart and the conventional chemotherapeutic drug, cisplatin.

Several studies attest to the dose and time-dependent cytotoxic effect of THC on treatment-resistant cancers [17, 53]. The decrease in IC50 over time could insinuate the increased cellular uptake of the nanoparticles by endocytosis, evading phagocytosis and being more penetrative to cancer cells [54]. Meanwhile, THC-solution was inconsistent in its cytotoxic profile, depicting a difficulty in cellular uptake over time, an expected finding owing to the decreased water solubility of THC. Despite the shooting increase in the sensitivity of oral cancer cells to THC-solution compared with the phytosomes, we should not neglect the toxic effect of DMSO. Regarding the nanoformulation and the standard chemotherapeutic, as the dose increased and time elapsed, the cell viability decreased drastically. Therefore, THC-phytosomes proved to have both dose and time-dependent cytotoxic responses similar to cisplatin.

Evaluating the selectivity index of each drug, our results established the THC-phytosomes to be the most selective to cancer cells, preserving the viability of normal oral keratinocytes and gingival fibroblasts at all concentrations. However high the selectivity of the solution to cancer cells was, it showed a drastic decrease in normal cell viability when dosages increased. Of all the three medications, cisplatin showed the least selectivity to cancerous cells and was the most toxic to normal cells, highlighting this as a significant side effect of cisplatin.

Studying the ability of cells to migrate and proliferate is an integral component of cancer studies. Herein, we implored the scratch assay in cell culture after treatment with the corresponding IC50s of the three medications over 72 h. The THC-phytosomes group revealed the widest wound gap, designating that the treatment was the most effective in impeding proliferation and caused the lowest percentage of migration. As for THC-solution and cisplatin, they did not inhibit the proliferation as effectively as the nanoformula, with a faster rate of closure and a comparable wound gap. Similarly, combining THC-solution with radiation on C6 cells has revealed its potency as an anti-migratory over THC alone [37].

Clonogenic survival analysis represents a metric by which we can measure cellular proliferative loss in response to treatment or injury [55]. Asserting the anti-proliferative efficacy, the nanopreparation of THC proves to have the upper hand in inhibitory potential to colony survival, producing the lowest number of spheres and recording the lowest survival fraction among all groups. THC-solution and cisplatin again show comparable pro-proliferative clonogenic responses. These anti-proliferative findings display how effective the phytosomes strategy is for allowing a successful uptake and diffusion through the cell membrane. This is attributed to the enhanced solubility and P_o/w of THC-phytosomes, which could be assigned to the amphiphilic nature of the developed THC-SPC complex [32, 45], in contrast to the lipophilic nature of pure THC [31]. Further supporting these results is the drug release study, which showed obvious improvement in the hydrophilicity and aqueous solubility of THC-phytosomes formulations, enhancing both the rate and the extent of their release compared to pure THC [31].

Several reports account for the ability of numerous anti-cancerous agents to halt the cell cycle at different checkpoints, eliciting cellular apoptosis [56]. Per the considerable variation among results in the literature in the cell cycle assay, THC has resulted in the accumulation of cells in the G0/G1 phase in MCF-7 and 4T1 breast cancer cell lines [17]. In another study on glioma cells, it caused a G2/M phase arrest [57]. Our results displayed an S-phase cell population arrest by THC-solution in SCC4 cells at 24 and 48 h. This is consistent with our previous results on the hamster precancerous model, where THC decreased the immune expression of cyclin D1, a cell cycle checkpoint regulator [28]. On the other hand, cisplatin showed a G0/G1 phase arrest at 24 h, similar to that described previously on leukemic cells [58]. Then, at 48 h, the cell populations shifted substantially to cause a stark arrest at the M-phase. A previous study on gastric cancer also confirms the G2/M phase arrest after 48 h of treatment by cisplatin [59].

Our study manifested that treatment with THC-phytosomes led to a significant escalation of cell percentages in the S-phase of the cell cycle. That escalation was both increasing and consistent throughout 48 h, unlike the THC-solution, whose escalation of cell percentages in the S-phase seemed somewhat decreasing over time, and unlike cisplatin with the inconsistent responses at 24 and 48 h. Halting the cell cycle at the S-phase is a character seen in numerous researched anticancer drugs [60, 61]. This S-phase arrest would prevent DNA replication, consequently preventing cell division and growth, essentially freezing the cells. That constitutes cellular stress that might induce the cell to undergo apoptosis.

In cancer management, apoptotic pathway targeting may result in cancer death, reversing drug resistance or enhancing drug sensitivity [62]. THC-phytosomes predominated the apoptotic flow cytometric analysis over 48 h of treatment. The high count of apoptotic cells complemented by the undermined necrotic response could suggest that the nanosized preparation of THC, owing to the chemical bond between the drug and the phospholipid, allows the medication to be uptaken more readily by the cells, eliciting a more intense apoptotic response with the formation of apoptotic bodies, which can then be safely phagocytosed, Moreover, this nanosized formulation would enhance the absorption and bioactivity of the free counterpart of the drug, as it has been established with different cancer drugs when compared to their respective nanoformulations that not only did the nanosized loaded-drug provide an enhanced response but also had reduced adverse effects [63].

Backing up our Annexin V-FITC cytometric results, Hoechst nuclear and H&E cytological visualizations in the phytosomes group exhibited the hallmark features of apoptosis, namely intense chromatin fragmentation, apoptotic bodies, and nuclear membrane blebbing. For instance, in our previous oral in vivo model, the nanoformulated THC mucoadhesive composite was able to regress the evolving proliferative leukoplakic lesions that showed severe dysplasia into simple white patches with minimal atypia [28].

As for THC-solution, it showed the weakest apoptosis-inducing effect among the groups possibly because THC has a higher molecular weight in addition to a decreased aqueous solubility, which makes its passive diffusion into the cells much harder. Similarly, THC-solution has induced apoptosis in glioma cells only through combination treatment with radiation, albeit effectively enhancing their radiosensitivity [37]. Hence the microscopic features of apoptosis in the THC-solution group were less in abundance than the phytosomes group.

The favorable result of cisplatin described in the flow cytometric analysis is not entirely without consequences since cisplatin shows the highest number of necrotic cells among all groups at all time points, which seems to be a commonly described feature of the drug in literature. Evidence has shown that cisplatin-induced necroptosis (programmed cell necrosis) could be the culprit behind its routinely presented nephrotoxicity [64]. Our microscopic visualization results indeed showed that cisplatin-treated cells exhibited distinctive features of necrosis, ranging from chromatin fading (karyolysis) to cloudy degeneration and ghosted cells. Accordingly, the cost versus benefit provided by cisplatin could be arguable.

Attempting to elucidate the rationale behind the enhanced apoptotic effect of THC-phytosomes, we had to determine the pathway tackled by apoptosis. Consequently, we stained the SCC4 cells with MitoTracker Red, whose intensity and distribution have been previously used to describe the morphology and integrity of the mitochondrial structure of HeLa cells undergoing apoptosis [49]. Herein, oral cancer cells treated with THC-phytosomes displayed a remarkably intense fluorescence that marks increased mitochondrial activity and swelling [49, 65]. A previous study on aclarubicin, a fluorescent anticancer drug, showed that its fluorescence merged predominantly with the Mitotracker Deep Red staining of the mitochondria and was relevant to its increased cytotoxicity [66]. Our results from using Mitotracker Red provide insight into the mitochondria-related signaling pathway of apoptosis.

Caspase-3 is an apoptotic executioner protein, whose high levels correlate with cells displaying morphologic features of apoptosis [67]. Immunohistochemically, we found the highest detectable positivity in the phytosomes group with cytoplasmic homing similar to that previously described in paclitaxel-treated HT29, MDA-MB231, and KB cells [68]. This finding is in agreement with Liu et al. and Han et al., who concluded that THC was effective in inducing apoptosis in hepatocellular carcinoma and breast cancer cells, respectively, by increasing caspase-3 and caspase-9 protein expression [69, 70]. Since our results have found that the fluorescence of Mitotracker Red positively and significantly correlated with caspase-3 protein expression, we can hence assume that mitochondrial signaling was implicated in inducing apoptosis of SCC4 cells by THC-phytosomes. Further validating this is the increased number of apoptotic cells and the abundance of morphological apoptotic nuclear features in the THC-phytosomes group coupled with the completely low fluorescence emitted by the proliferating untreated cancer cells. This can be attributed to the fact that the water-soluble THC-phytosomes were quite easily engulfed by the cells owing to their nanoscale, causing the cells to be saturated with the drug, hence producing an elevated response.

Tracing the apoptotic pathway back to the initiator protein, caspase-8 gene expression was extremely high in the phytosomes-treated group. Interestingly, a similar elevated expression was attained by FLLL32, a synthetic curcumin analog, previously tested on oral cancer cell lines SCC9 and HSC-3 [71]. Widely regarded as the initiator protein responsible for the extrinsic pathway of apoptosis, caspase-8 can also trigger the intrinsic mitochondria-related pathway, suggesting an interplay between both mechanisms [72].

Bax is considerably the main pro-apoptotic protein of the Bcl-2 family. In the cancer milieu, overexpression of Bax decelerates tumor enlargement [73]. A PCR result retrieved from the H22-induced mice model, where THC-treated tumors revealed overexpression of Bax, was related to reduced malignant ascites volume, indicating the possible functional role of THC in triggering the mitochondrial pathway of apoptosis [70]. Mimicking a similar Bax genetic profile, our results showed an enormously high expression of Bax in the phytosomes group compared with the other groups. This would later cause mitochondrial membrane permeabilization, releasing small pro-apoptotic molecules such as cytochrome C [74].

Tackling another arm of cellular functional phenotyping, we aimed to verify the protective antioxidant capacity of THC by measuring the released ROS, crucial players in carcinogenesis [75, 76]. Here, we assessed the oxidative stress index by calculating the ratio of MDA, a frequent byproduct of (LPO) to the total antioxidant capacity (TAC). We chose MDA as an index of LPO because of its high levels in patients with oral and oropharyngeal cancer inversely combined with the low plasma levels of antioxidants [77]. Our results mirror the evidence in the literature about the potent and superior antioxidant effect THC possesses owing to the lack of conjugated bonds in its central chain [11, 78]. Both THC formulas showed promising results and the lack of significance between both is a true affirmation of the innate antioxidant nature of the herb itself. Cisplatin shows the highest MDA/TAC ratio, indicating the highest oxidant potential of the groups, an expected finding since most chemotherapeutic agents generate ROS to induce apoptotic damage in cancer cells [79]. Precisely because of that, this strategy poses dangerously toxic side effects to normal cells, which do not escape this therapeutic approach [80]. In contrast, an antioxidant agent can promote cellular function and immunity, while reducing the side effects of ROS-producing drugs, such as cisplatin. For example, a study has prescribed antioxidant supplements to reduce cisplatin-mediated nephrotoxicity [81], which sheds light on the possible use of including THC in anti-cancer regimens.

Finally, it is important to note that verifying our results on additional cell lines and profiling cell cycle proteins such as cyclin A1 and PCNA would be beneficial. Additionally, coupling these in vitro investigations with in vivo studies would be highly recommended to help transfer our hypothesis to the translational field.