Treatment of Maxillofacial Cancers by Zein Nanoparticles Loaded with Anticancer Peptide Pistacia Zardin1: Enhanced Cytotoxicity and Apoptosis Induction in Head and Neck Squamous Cell Carcinoma (HNSCC)

TL;DR

Researchers developed zein nanoparticles loaded with an anticancer peptide to treat head and neck cancers, showing improved effectiveness and cancer cell targeting.

Contribution

A novel pH-responsive nanoparticle delivery system for the anticancer peptide PZ1, enhancing its cytotoxicity and apoptosis in HNSCC.

Findings

PZ1-Zein NPs reduced IC50 values in HNSCC cell lines compared to free peptide, showing higher efficacy.

The nanoparticles increased caspase-3/7 activity, indicating enhanced apoptosis in cancer cells.

PZ1-Zein NPs demonstrated improved selectivity for cancer cells over normal cells.

Abstract

Head and neck squamous cell carcinomas (HNSCCs) are considered the most common histological type of head and neck cancer. This study aims to develop a drug delivery system based on zein protein nanoparticles (Zein NPs) to enhance the therapeutic effect of the anticancer peptide, Pistacia zardin1 (PZ1), for the treatment of maxillofacial cancers. PZ1-Zein NPs were synthesized by the desolvation method. These spherical nanoparticles (size: 162.8 nm, PDI: 0.27) showed high encapsulation efficiency (89%) and pH-responsive release (with higher drug release in the acidic tumor microenvironment). In vitro cytotoxicity assays showed that PZ1-Zein NPs significantly reduced IC50 values in HNSCC cell lines (e.g., SCC-25: 7.5 µM vs. 19.3 µM for free peptide, p < 0.001) while exhibiting improved selectivity for cancer cells over normal HaCaT cells. Mechanistic investigations confirmed that PZ1-Zein NPs significantly increased apoptosis, as shown by increased caspase-3/7 activity (5.8-fold vs. 2.6-fold). These findings highlight PZ1-Zein NPs as a promising nanomedicine strategy and a candidate functional component for future dual-functional scaffolds aimed at targeted hard tissue engineering and surgery in HNSCC management.

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1. Introduction

Head and neck squamous cell carcinoma (HNSCC), particularly in the maxillofacial region, presents a complex clinical challenge that extends beyond oncological control. HNSCC is the sixth most common cancer worldwide [1,2]. The standard treatment for HNSCC is typically aggressive surgical resection. This treatment leads to significant hard tissue defects. Although conventional therapies (radiotherapy and chemotherapy) are commonly used alongside surgery, the 5-year survival rate remains low due to locally advanced disease, high recurrence rates, and the development of therapeutic resistance [3,4,5]. Furthermore, the systemic toxicity of current chemotherapies can worsen the patient’s quality of life and is associated with many side effects [6]. Therefore, developing new cancer treatment strategies is essential to both curing cancer and avoiding the side effects of existing treatments [7,8].

In the field of maxillofacial cancers, the development of biocompatible therapeutic models with the ability to slowly release anticancer agents can be effective and beneficial in improving the treatment of these cancers [9,10]. Anticancer peptides (ACPs) are one of the most effective and newest alternatives to chemotherapy drugs that can prevent the unwanted side effects of traditional drugs. These peptides have high specificity towards cancer cells, low systemic toxicity, and the ability to modulate multiple signaling pathways involved in proliferation, metastasis, and apoptosis [11]. Pistacia zardin1, a bioactive peptide derived from the hydrolysis of pistachio proteins, has shown effective anticancer efficacy. This peptide was obtained in our previous study and has been patented. However, Pistacia zardin1, similar to other bioactive peptides, has limitations such as inherent instability, rapid enzymatic degradation, and a short half-life, which make its use as a therapeutic agent problematic [12].

In order to overcome these limitations, the use of nanoparticles as carriers and delivery systems can be useful [13]. Nanocarriers can protect therapeutic peptides from degradation, increase their solubility, and facilitate their sustained release directly at the tumor site. Furthermore, when incorporated into biomaterial scaffolds, these nanoparticles can serve as the active component of a scaffold-based drug delivery system [14]. Recently, the field of nanomedicine has advanced significantly towards smart, multifunctional platforms. For instance, sophisticated nanoprobes have been engineered to combine targeted therapy, deep-tissue near-infrared imaging, and real-time monitoring of the tumor microenvironment (e.g., highly sensitive temperature sensing and controlled photothermal therapy) [15], as well as real-time visual detection of biological markers in complex organoid and in vivo models [16]. Alongside these inorganic and theranostic systems, researchers are also focusing on organic and natural protein-based nanoparticles. Among them, zein protein has attracted widespread attention as a highly biocompatible carrier. Protein-based nanoparticles, such as zein protein, have attracted attention as a biocompatible carrier. In addition to being a carrier system, zein protein nanoparticles have excellent properties for forming porous scaffolds suitable for bone regeneration [17]. These nanoparticles have the ability to load and deliver a variety of hydrophobic and amphiphilic drugs with high efficiency. For environmentally sensitive drugs, such as peptides, these nanostructures provide a protective barrier against enzymatic degradation and enhance their cellular uptake. These properties make zein nanoparticles a multifunctional biomaterial for the development of functional scaffolds that combine bone conduction with local anticancer activity [18].

This study was designed to synthesize zein nanoparticles loaded with Pistacia zardin1 (PZ-Zein NPs) and evaluate their effect on HNSCC cell lines as a first step towards their later incorporation into dual-functional scaffolds for simultaneous tumor therapy and tissue repair.

2. Materials and Methods

2.1. Cell Lines and Culture Conditions

Human head and neck squamous cell carcinoma (HNSCC) and normal cell lines (HaCaT) were obtained from the Pasteur Institute of Iran. HNSCC and HaCaT cells were cultured in DMEM (enriched with 0% (v/v) heat-inactivated FBS and 1% (v/v) penicillin-streptomycin).

2.2. Preparation of Pistacia Zardin1-Loaded Zein Nanoparticles (PZ-Zein NPs) as a Scaffold-Based Delivery System

Pistacia zardin1-loaded zein nanoparticles were prepared using a modified anti-solvent precipitation method. Briefly, 50 mg of zein protein was dissolved in 5 mL of 90% (v/v) ethanol containing 0.1% (w/v) glacial acetic acid to create the organic phase. Pistacia zardin1 peptide was dissolved separately in deionized water at a concentration of 2 mg/mL. For peptide loading, a specified amount of Pistacia zardin1 solution was added to the zein solution, and the mixture was stirred for 30 min.

The organic phase (zein and peptide solution) was slowly added dropwise (0.5 mL/min) into 20 mL of deionized water (aqueous phase) under constant magnetic stirring at 800 rpm. The acquired suspension was then sonicated using a probe sonicator (50 W for 5 min) for reduction in nanoparticle size. Ethanol and unencapsulated peptide were removed by dialysis against deionized water using a dialysis membrane (MWCO 3.5 kDa) for 24 h. The purified PZ-Zein NP suspension was centrifuged at 10,000 rpm for 15 min for nanoparticle collection. The PZ-Zein NP suspension was centrifuged at 10,000 rpm for 15 min for nanoparticle collection. The pellet was then resuspended in 5 mL of deionized water containing 5% (w/v) mannitol (as a cryoprotectant). To achieve lyophilization, the suspension was pre-frozen at −60 °C for 12 h, followed by drying under a vacuum of 0.1 mBar at −20 °C for 24 h. The acquired powder was collected and stored at 4 °C. Empty Zein nanoparticles (empty Zein NPs) were prepared using the same procedure without the addition of Pistacia zardin1.

2.3. Characterization of PZ-Zein Nanoparticles

The synthesized nanoparticles were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Encapsulation efficiency (EE%) and loading capacity (LC%) were determined by high-performance liquid chromatography (HPLC).

Briefly, the supernatant collected after centrifugation of the nanoparticle suspension was analyzed for unencapsulated peptide using HPLC. A C18 reverse-phase column was used with a mobile phase consisting of acetonitrile and 0.1% trifluoroacetic acid in water at a flow rate of 1 mL/min and detection at 220 nm. A standard curve for Pistacia zardin1 was prepared at various known concentrations.

Encapsulation efficiency (EE%) and loading capacity (LC%) were calculated using the following formulas (Equations (1) and (2)):

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2.4. In Vitro Drug Release Study

The in vitro release profile of Pistacia zardin1 from PZ-Zein NPs was assessed in PBS (pH 7.4) and acetate buffer (pH 5.5, for simulation of the acidic tumor microenvironment) at 37 °C under constant shaking. Briefly, PZ-Zein NPs (2 mg) were dispersed in 2 mL of each release medium and placed in dialysis bags (MWCO 3.5 kDa). The bags were then immersed in 20 mL of the respective release medium. At determined time intervals (0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h), 1 mL aliquots of the release medium were collected and replaced with fresh medium to maintain sink conditions. The concentration of released Pistacia zardin1 in the aliquots was quantified using HPLC. All experiments were performed in triplicate.

For evaluation of the release kinetics of Pistacia zardin1 from PZ-Zein NPs, the obtained release data were fitted to various mathematical models (zero-order, first-order, Higuchi, and Korsmeyer–Peppas models). The correlation coefficient (R^2^) was used to determine the best-fitting model for each pH condition.

2.5. In Vitro Cytotoxicity Assay

The cytotoxic effects of free Pistacia zardin1, Zein NPs, and PZ-Zein NPs on HNSCC cell lines and the non-malignant cell line (HaCaT) were evaluated using the MTT assay. Cells were cultured into 96-well plates (5 × 10^3^ cells/well in 100 μL of growth medium) and incubated overnight. After 24 h, the cells were treated with various concentrations of free Pistacia zardin1, Zein NPs, and PZ-Zein NPs (equivalent peptide concentrations ranging from 0.1 to 100 μM) for 24, 48, and 72 h. Untreated cells were used as a negative control, and cells treated with 0.1% DMSO were also included. Viable cells were evaluated by MTT assay. Cell viability was calculated as a percentage relative to untreated control cells. The half-maximal inhibitory concentration (IC50) values were determined using non-linear regression analysis (e.g., GraphPad Prism 9.0).

2.6. Apoptosis Induction Assays

To investigate the mechanism of cell death induced by designed nanostructures, apoptosis was assessed using the evaluation of caspase activity. The activation of effector caspases (caspase-3 and caspase-7) was measured using a fluorometric assay kit according to the manufacturer’s protocol. HNSCC cells (SCC-25) and non-malignant HaCaT cells were seeded in opaque 96-well plates at a density of 1 × 10^4^ cells/well and treated with free Pistacia zardin1, empty Zein NPs, and PZ-Zein NPs (at IC50 concentrations) for 24 h. After treatment, 100 μL of Caspase-Glo^®^ 3/7 Reagent was added to each well, and the plates were incubated at room temperature for 1 h. Luminescence was measured using a microplate reader. Caspase activity was expressed as a fold increase relative to untreated control cells.

2.7. Statistical Analysis

All experiments were performed in three independent replicates. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software. Differences between groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. A p-value less than 0.05 (p < 0.05) was considered statistically significant.

3. Results

3.1. Characterization of Pistacia Zardin1-Loaded Zein Nanoparticles (PZ-Zein NPs)

The successful fabrication of Pistacia zardin1-loaded Zein nanoparticles (PZ-Zein NPs), intended as a core component for a scaffold-based drug delivery system, was confirmed using various physicochemical characterization techniques. Dynamic light scattering (DLS) analysis showed that the empty Zein NPs had a mean hydrodynamic diameter of 135.2 ± 4.1 nm with a polydispersity index (PDI) of 0.21 ± 0.03, indicating a relatively narrow size distribution. After loading with Pistacia zardin1, the PZ-Zein NPs displayed a slightly increased mean diameter of 162.8 ± 5.5 nm and a PDI of 0.27 ± 0.04. Both empty Zein NPs and PZ-Zein NPs exhibited a positive zeta potential of +27.5 ± 1.0 mV and +24.1 ± 0.8 mV, respectively. This positive surface charge contributes significantly to colloidal stability and promotes interaction with negatively charged cell membranes, a desirable feature for enhancing cellular uptake in both cancerous and hard tissue-associated cells. These DLS results are summarized in Figure 1 and Table 1.

Scanning electron microscopy (SEM) micrographs (Figure 2) confirmed the nanoscale morphology of the zein-based formulations. Both empty Zein NPs and PZ-Zein NPs appeared as nearly spherical particles forming a densely packed but non-fused layer. Individual nanoparticles could be distinguished with diameters in the range of approximately 100–200 nm, which is in good agreement with the hydrodynamic sizes obtained from DLS. No large micron-sized aggregates or collapsed structures were observed, indicating that the preparation and drying procedures preserved the integrity of the nanocarriers. Importantly, loading of Pistacia zardin1 did not produce any obvious changes in particle shape or surface texture, suggesting that peptide encapsulation does not compromise the structural stability of the zein nanoparticles, which is essential for their potential incorporation into tissue engineering scaffolds.

The encapsulation efficiency (EE%) and loading capacity (LC%) of Pistacia zardin1 within the zein nanoparticles were determined by HPLC analysis. The PZ-Zein NPs achieved an encapsulation efficiency of 88.7 ± 2.9% and a loading capacity of 10.2 ± 0.7%, demonstrating efficient incorporation of the peptide into the zein matrix.

3.2. In Vitro Drug Release Study

The in vitro release kinetics of Pistacia zardin1 from PZ-Zein NPs were evaluated in PBS (pH 7.4, simulating physiological conditions) and acetate buffer (pH 5.5, mimicking the acidic tumor microenvironment often associated with resorbed bone defects and tumor sites) over 72 h at 37 °C. As shown in Figure 3, PZ-Zein NPs exhibited a sustained release profile under both pH conditions, avoiding the rapid burst release often associated with free peptide delivery, which is advantageous for maintaining long-term therapeutic levels in a scaffold.

In PBS (pH 7.4), Pistacia zardin1 was released gradually, with approximately 18.5 ± 2.1% of the loaded peptide released within the first 8 h, followed by a more sustained release, reaching about 58.2 ± 3.4% cumulative release over 72 h. In contrast, at pH 5.5, a moderately faster release rate was observed, with approximately 28.1 ± 2.5% of the peptide released within the initial 8 h and a cumulative release of about 71.5 ± 4.1% after 72 h. This pH-responsive release behavior, with enhanced release in acidic conditions, suggests that the zein nanoparticles could potentially deliver a higher concentration of the therapeutic peptide specifically at the tumor site within a maxillofacial defect while minimizing systemic exposure.

As shown in Table 2, the Korsmeyer–Peppas model exhibited the highest correlation coefficient (R^2^ = 0.9918 for pH 7.4 and R^2^ = 0.9936 for pH 5.5). Thus, this model completely describes the release kinetics of Pistacia zardin1 from PZ-Zein NPs under both pH conditions. The release exponent (n) values were 0.432 and 0.458 for pH 7.4 and pH 5.5, respectively. These values show a combination of diffusion-controlled and polymer matrix degradation/swelling processes. The higher value at pH 5.5 suggests that acidic conditions may promote greater polymer swelling or degradation. This condition leads to the accelerated release observed at this pH. The Higuchi model also showed good correlation (R^2^ > 0.986), supporting the predominance of diffusion-controlled release. These findings indicate that the release of Pistacia zardin1 from PZ-Zein NPs is primarily controlled by a combination of peptide diffusion through the zein matrix and pH-responsive matrix degradation.

3.3. Cytotoxicity of PZ-Zein NPs on HNSCC Cell Lines

The in vitro cytotoxicity of free Pistacia zardin1, empty zein nanoparticles (NPs), and PZ-Zein NPs was assessed on three HNSCC cell lines (SCC-25) and a non-malignant control cell line (HaCaT) using the MTT assay after 24, 48, and 72 h of treatment.

As shown in Table 3, empty Zein NPs demonstrated negligible cytotoxicity towards all tested cell lines, with IC50 values greater than 200 µM. This confirms the excellent biocompatibility of the zein carrier for biomedical applications. Free Pistacia zardin1 showed dose- and time-dependent cytotoxicity against the HNSCC cell line.

PZ-Zein NPs significantly enhanced the cytotoxic effects of Pistacia zardin1 on the HNSCC cell line compared to the free peptide. After 48 h of treatment, the IC50 value for PZ-Zein NPs in SCC-25 cells was significantly lower than that for free Pistacia zardin1 (7.5 ± 0.5 µM vs. 19.3 ± 1.3 µM, p < 0.001). The enhanced cytotoxicity of PZ-Zein NPs was consistent at all time points, with prolonged exposure generally resulting in lower IC50 values.

PZ-Zein NPs showed improved therapeutic selectivity for cancer cells over healthy tissues. While free Pistacia zardin1 exhibited moderate toxicity towards non-malignant HaCaT cells (IC50 of 58.2 ± 4.5 µM at 48 h), the IC50 of PZ-Zein NPs in these cells was significantly higher (95.5 ± 7.1 µM, p < 0.01), indicating a better therapeutic window and reduced off-target effects for the nanoparticle formulation. This selectivity is crucial for preserving the viability of surrounding healthy hard and soft tissues during maxillofacial reconstruction. Representative dose–response curves for SCC-25 cells after 48 h are shown in Figure 4.

3.4. PZ-Zein NPs Induce Apoptosis in HNSCC Cells

To further elucidate the mechanism by which PZ-Zein NPs exert their enhanced cytotoxicity, apoptosis induction was investigated using a caspase-3/7 activity assay. Confirmation of apoptosis induction was obtained by measuring the activity of effector caspases (caspase-3 and caspase-7). As shown in Figure 5A, treatment of SCC-25 cells with PZ-Zein NPs for 24 h led to a significant increase in caspase-3/7 activity compared to untreated control cells and empty Zein NPs. Specifically, PZ-Zein NPs induced a 5.8 ± 0.6-fold increase in caspase-3/7 activity in SCC-25 cells. These activations were significantly higher than the 2.6 ± 0.3-fold increases observed with free Pistacia zardin1 (p < 0.001). These findings strongly suggest that the enhanced pro-apoptotic effects of PZ-Zein NPs are mediated, at least in part, through the activation of caspase cascades. PZ-Zein NPs did not significantly induce apoptosis in non-malignant HaCaT cells. As shown in Figure 5B, the caspase-3/7 activity in HaCaT cells treated with PZ-Zein NPs was only 1.2 ± 0.2-fold relative to the untreated control, which was not significantly different from empty Zein NPs (1.1 ± 0.1-fold) or free Pistacia zardin1 (1.3 ± 0.2-fold). This finding is consistent with the cytotoxicity results and further supports the improved therapeutic selectivity of the nanoparticle formulation for cancer cells over normal tissues.

4. Discussion

The therapeutic approach to HNSCC in the maxillofacial region faces two major challenges: eliminating invasive tumor cells and reconstructing hard tissue defects resulting from surgical resection [19]. Current treatments are largely both systemic toxic and harmful to surrounding healthy tissues [20]. Given these challenges, the development of scaffolds with dual functions (inhibition of cancer cell growth and bone regeneration) is of great importance [21]. In this study, zein protein nanoparticles loaded with the anticancer peptide Pistacia zardin1 (PZ-Zein NPs) were synthesized, and their potential as a therapeutic approach in this field was evaluated. The findings of this study showed that encapsulation of Pistacia zardin1 in a zein matrix significantly enhanced its physicochemical stability, cytotoxic efficacy, and ability to induce apoptosis in HNSCC cells.

PZ-Zein nanoparticles were synthesized with a mean hydrodynamic diameter of approximately 163 nm and a narrow size distribution (PDI 0.27). In the field of hard tissue engineering, particle size is a key and pivotal parameter. Studies show that nanoparticles in the range of 100–200 nm are suitable for passive tumor targeting through the enhanced permeability and persistence (EPR) effect, allowing preferential accumulation in leaky tumor vasculature [22]. This size range also facilitates deep penetration into the porous structure of biomaterial scaffolds and ensures uniform drug distribution when incorporated into a tissue engineering construct [23]. The observed positive zeta potential (+24.1 mV) is also noteworthy. Although zein is generally hydrophobic, its surface charge allows electrostatic interaction with the negative charge of the cancer cell membrane, thereby enhancing cellular uptake [24]. This finding is consistent with previous studies on zein-based drug delivery systems, such as those containing curcumin or paclitaxel, in which a positive surface charge was associated with improved drug entry into cancer cells [25,26,27].

One of the most critical attributes of an effective scaffold-based delivery system is its release kinetics [28]. The PZ-Zein NPs demonstrated a sustained release profile over 72 h, avoiding the initial “burst release” often associated with nanoparticle formulations. More importantly, the release was pH-responsive, with a significantly higher cumulative release at pH 5.5 (mimicking the tumor microenvironment) compared to physiological pH 7.4. The tumor microenvironment in maxillofacial malignancies is characteristically acidic due to the Warburg effect (aerobic glycolysis) [29]. This pH-sensitive behavior suggests that the zein matrix remains relatively stable in healthy tissues (pH 7.4) but degrades or swells more rapidly in the acidic tumor site, triggering targeted drug release. This “smart” release mechanism minimizes off-target toxicity—a crucial advantage for preserving the viability of osteoblasts and fibroblasts essential for bone healing and tissue integration. Similar pH-responsive behaviors have been reported in zein nanoparticles loaded with chemotherapeutics, attributed to the protonation of amino acid residues in zein and subsequent protein swelling in acidic conditions [30,31].

In vitro cytotoxicity assays revealed that PZ-Zein NPs significantly enhanced the anticancer potency of Pistacia zardin1. The IC50 values decreased substantially (e.g., from 19.3 µM to 7.5 µM in SCC-25 cells) compared to the free peptide. This 2.5-fold enhancement can be attributed to several factors. First, the hydrophobic core of the zein nanoparticle protects the peptide from enzymatic degradation by proteases present in the culture medium and in vivo, thereby extending its half-life. Second, improved cellular uptake via endocytosis, driven by the nanoparticle’s size and charge, ensures a higher intracellular concentration of the peptide [32]. Crucially, PZ-Zein NPs demonstrated improved selectivity, exhibiting significantly lower toxicity to non-malignant HaCaT keratinocytes compared to the free peptide. This broader therapeutic window is highly desirable for maxillofacial applications, as it indicates that the formulation can target cancer cells while sparing the surrounding healthy mucosal and stromal tissues necessary for successful reconstruction.

The results showed that the main mechanism of cell death induced by PZ-Zein nanoparticles is apoptosis. This was confirmed by a significant increase in caspase-3/7 activity (5.8-fold increase). These findings suggest that zein nanoparticles not only deliver the peptide but also can enhance its interaction with intracellular apoptotic pathways by facilitating endosomal escape or mitochondrial targeting. Compared with synthetic polymer carriers such as PLGA, which can generate acidic degradation products and lead to disruption of cellular metabolism, natural protein carriers such as zein appear to provide a more biocompatible substrate that effectively activates the caspase cascade [33,34].

Various studies have shown that positively charged bivalent peptides that have anticancer effects often exert their effects by disrupting mitochondrial membrane integrity, generating reactive oxygen species, and modulating pro-survival signaling pathways such as PI3K/Akt and MAPK. Some peptides also act by activating caspase-8. Based on the data from our study, it can be considered that any of the mechanisms presented in the above studies could play a role in the case of the peptide in this study. In order to confirm the exact mechanism, therefore, measurement of initiator caspases 8 and 9, assessment of mitochondrial membrane potential, analysis of cytochrome c release and Bcl-2 family protein expression, as well as Western blotting of key nodes in the PI3K/Akt and MAPK cascades are needed to determine the exact apoptotic signaling cascade involved by PZ-Zein nanoparticles.

Comparing the results of this study with similar studies in the field of peptide delivery reveals several key distinctions. Liposomal formulations of anticancer peptides often suffer from stability and rapid clearance issues that limit their efficacy [35,36]. In contrast, zein is a material with superior stability and ease of fabrication. Although zein nanoparticles have been extensively studied for hydrophobic small molecules (e.g., 5-fluorouracil, paclitaxel), their use for peptide delivery in HNSCC has not been investigated [37,38]. Our study shows that the encapsulation efficiency of zein for peptides (approximately 89%) is comparable to or even better than many synthetic polymers. Furthermore, the natural origin of zein makes it an ideal candidate for scaffold-based drug delivery systems for hard tissue engineering, as it can be mixed with ceramics such as hydroxyapatite without causing significant immune reactions [39].

According to the presented results, the development of novel scaffolds in the field of maxillofacial surgery and hard tissue engineering based on peptide-loaded zein nanoparticles appears feasible. In the present study, we did not yet fabricate such scaffolds; instead, we propose, at a conceptual level, that by incorporating PZ-Zein nanoparticles into a zein/ceramic scaffold via 3D printing, a “functional scaffold” can be created that provides mechanical support for bone defects and simultaneously releases Pistacia zardin1 to eliminate tumor cells and residual microscopic disease after tumor resection. Experimental validation of this dual-functional scaffold concept, including scaffold fabrication, mechanical characterization, and evaluation of osteogenic performance, will be addressed in future work. This approach addresses the common clinical problem of local tumor recurrence, which often impairs tissue repair. The sustained release profile observed in vitro suggests that such a scaffold can maintain therapeutic drug levels for weeks or months, covering the critical period of postoperative recovery.

Despite the highly promising formulation and baseline efficacy results, several critical limitations of the current study must be acknowledged to provide a realistic perspective on its clinical translation. First, the in vitro anticancer efficacy was validated using a single representative HNSCC cell line (SCC-25). Future studies must incorporate a broader panel of representative HNSCC cell lines (e.g., CAL-27, HN6) to confirm the cell line generality of PZ-Zein NPs’ anticancer efficacy. Second, the current biological evaluations rely entirely on 2D monolayer culture models. These static models fail to simulate the 3D stereostructure of actual tumor tissues and the crucial interactions between cells in the tumor microenvironment (e.g., the complex crosstalk between tumor cells, osteoblasts, and immune cells). This simplification has limited relevance to actual clinical tumor scenarios and may overestimate the in vivo therapeutic effect of the nanoparticles. Furthermore, while the study confirms that PZ-Zein NPs induce apoptosis by activating executioner caspases (caspase-3/7), it does not clarify the upstream molecular mechanisms (e.g., involvement of the mitochondrial apoptotic pathway, death receptor pathway, or regulation of survival signaling pathways such as PI3K/Akt and MAPK). Additionally, the specific cellular uptake mechanisms of the nanoparticles (e.g., clathrin-mediated vs. caveolae-mediated endocytosis) and the precise intracellular localization and targets of the released PZ1 peptide remain unexplored.

Another significant limitation is the absence of a lateral comparison. We did not compare the anticancer activity and biocompatibility of PZ-Zein NPs with clinically used chemotherapeutic drugs (e.g., paclitaxel or 5-fluorouracil) or mainstream nanocarriers (e.g., PLGA nanoparticles or liposomes), making it challenging to definitively highlight its clinical application advantages over current standards of care. Moreover, for subsequent practical applications and large-scale production, comprehensive stability profiling is mandatory. In this initial study, the long-term storage stability of the nanoparticles (e.g., changes in particle size and loading efficiency under different temperature and humidity conditions) and performance attenuation after repeated freeze–thaw cycles were not evaluated. Crucially, the influence of the physiological serum environment on the stability and release behavior of the nanoparticles was not explored, which is a vital missing link for systemic or localized in vivo applications.

Most importantly, while a core conceptual objective of this research direction is the eventual development of a “tumor therapy–tissue repair” dual-functional scaffold, this remains a conceptual proposal in the current manuscript. The present study is strictly limited to the synthesis and validation of the nanoparticulate active component. We did not fabricate a macroscopic tissue engineering scaffold (e.g., a zein/hydroxyapatite composite scaffold) loaded with PZ-Zein NPs. Consequently, key properties of the scaffold (mechanical strength, porosity, uniform dispersion, and sustained release of NPs) were not tested. Furthermore, the actual impact of such a composite scaffold on the proliferation and differentiation of osteoblasts is not verified here. The absence of sophisticated animal models of maxillofacial cancer (such as nude mouse subcutaneous xenograft models or orthotopic implantation models) means that the true in vivo tumor-suppressive effect, biodistribution, metabolic pathways, long-term biocompatibility, and systemic toxicity of PZ-Zein NPs remain unverified. Addressing these gaps—specifically benchmarking against clinical drugs, conducting stability and serum assays, fabricating the composite scaffold, and testing the complete system in appropriate animal models for bone regeneration and scaffold integration—will be the absolute focus of our future research pipeline.

5. Conclusions

In conclusion, this study successfully synthesized and characterized Pistacia zardin1-loaded zein nanoparticles as a potent anticancer formulation with significant potential for application in hard tissue engineering. The PZ-Zein NPs exhibited favorable physicochemical properties, pH-responsive release, enhanced cytotoxicity, and induction of apoptosis in HNSCC cells. These results support the integration of PZ-Zein NPs into functional scaffolds, offering a novel, dual-functional therapeutic strategy for the comprehensive management of maxillofacial cancers—simultaneously eradicating the tumor and providing a foundation for tissue reconstruction.