Preliminary Toxicological Evaluation of Spherical Nanoparticles Containing an Imidazole Derivative (BzIm-DEA) Using the CAM Chicken Model

TL;DR

This study evaluates the safety of BzIm-DEA-loaded nanoparticles using a chicken embryo model, finding no major toxicity or developmental issues.

Contribution

The novel contribution is the preliminary toxicological assessment of BzIm-DEA-containing nanoparticles using the CAM chicken model.

Findings

No morphological abnormalities or increased mortality were observed in treated chicken embryos.

A significant increase in Apaf-1 mRNA expression was detected at a specific BzIm-DEA dose.

No dose-dependent changes in PCNA and beta-catenin expression were observed in the liver.

Abstract

Due to the increasing antibiotic resistance of microorganisms, chronic diseases, and cancer, new-generation drugs such as imidazole derivatives are being sought. Recent advances in nanotechnology enable the potential use of nanomaterials, especially nanoparticles, as drug carriers for such compounds, but also systems capable of crossing biological barriers. This study aimed to perform a preliminary toxicological assessment of nanoparticles containing BzIm-DEA ((Z)-5-benzylidene-3-[2-(diethylamino)ethyl]-2-phenyl-3,5-dihydro-4H-imidazol-4-one) embedded in chitosan films, using chicken chorioallantoic membrane (CAM) as an alternative in vivo test. Fertilized chicken eggs were treated with this therapeutic agent at various concentrations of BzIm-DEA and incubated until the 11th day of embryogenesis. No morphological abnormalities, angiogenesis-related disorders, or increased mortality were observed in any of the experimental groups. A significant increase in Apaf-1 mRNA expression was detected in CAM tissue at a dose of D3 BzIm-DEA, while no significant changes were observed for caspase-3 and catalase compared to the control group. Moreover, no changes in gene expression were observed in the liver. Immunohistochemical localization and analysis of PCNA and b-catenin expression in chicken embryonic liver did not reveal any dose-dependent changes. Within the scope of this preliminary assessment, chitosan nanoparticles loaded with BzIm-DEA did not produce gross acute embryotoxicity or major disruptions to angiogenic development under the tested conditions, providing preliminary evidence of biocompatibility as a nanoparticle carrier system.

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1. Introduction

Nanotechnology is an interdisciplinary, rapidly growing field of science [1], which has global impact and application potential in many areas of science and industries such as biotechnology, agriculture, food, cosmetics, medicine, pharmaceuticals, and beyond [2,3,4,5,6]. This enables the use of science and technology at the nanoscale (1–100 nm) by exploiting the potential of structures through precise control of their shape and size, as well as the unique properties that emerge at this scale [7]. Nanoparticles, liposomes, dendrimers, micelles, and carbon nanotubes may provide a carrier for drugs that will be able to cross the blood-brain and blood-placental barriers in the drug delivery process, giving hope for the possibility of treating neurodegenerative diseases, brain tumors, and conditions related to maternal and child health or placental dysfunctions during pregnancy in the future [8,9,10].

Carriers and stabilizers for nanostructures can be biopolymers [11]. Their biodegradability and biocompatibility [12] make them highly relevant to the medical, pharmaceutical, and cosmetic industries. Biopolymers are used as drug carriers, medical implants, wound healing dressing materials, tissue scaffolds for tissue engineering work, and are one of the base ingredients for hydrogels, whose physicochemical parameters can mimic conditions found in tissues [13,14]. An example of a biopolymer with potential for medical applications is chitosan, possessing a range of biological properties such as antimicrobial, antioxidant, immunostimulatory, anti-cancer, anti-inflammatory, biodegradability, non-toxicity, and biocompatibility [15,16]. Chitosan is formed by the deacetylation of chitin, a biopolymer widely distributed in nature that forms the exoskeleton of crustaceans, insect epidermis, fungal cell walls, and coralline algae, among others [17,18]. Both chitosan and chitin are renewable and relatively economical biopolymers [19]. According to the bibliographic data and scientific trends, chitosan is applicable for the synthesis of films, providing a matrix or carrier for nanoparticles, plant extracts, or graphene, capable of enhancing the already impressive properties of this polymer [20,21].

Imidazole, known as 1,3-diazole, is an amphoteric heterocyclic aromatic chemical compound that is a derivative of the pyrazole ring. It is constructed from a five-membered ring consisting of three carbon atoms, two nitrogen atoms (one bonded to a hydrogen atom, the other is of the pyrrole type), four hydrogen atoms, and two double bonds [22]. The imidazole ring is widely distributed in nature, being, for example, an important part of histidine and histamine molecules present in a vast number of other biomolecules [23]. Literature data indicate that imidazole derivatives are gaining interest due to their antimicrobial [24,25,26], anti-cancer [27,28] and anti-inflammatory [29] potential. This is primarily due to rapidly increasing microbial drug resistance caused by excessive, inappropriate, or unnecessary use of antibiotics in both humans and animals [30]. It is estimated that around 700,000 people die each year from this condition, while this figure is expected to increase to 10 million by 2050 if new treatments are not found [31]. In the case of cancer, despite the potential reduction in mortality in developed countries such as the USA due to reduced smoking, more effective diagnostics, and new treatments, there is still a need to search for effective and less harmful anti-cancer therapies [32,33]. Therefore, there is an opportunity to find potential anti-cancer and antimicrobial drugs among imidazole derivatives.

Changes in societies in the second half of the twentieth century regarding ethical considerations and the increasing need to use animal models in scientific research led to the definition of the 3Rs principle (Replacement, Reduction, and Refinement) in 1959 [34,35,36]. This has resulted in the large-scale implementation of alternative methods to classical animal studies, such as, for example, computer models [37], mathematical models [38], cell and tissue cultures [39,40], lower vertebrates such as Danio rerio [41], invertebrates [42,43], and microorganisms [44,45]. An alternative model that fits the 3Rs principle, is low-cost and can replace mammalian models in preliminary studies prior to proper clinical trials, is the chicken chorioallantoic membrane (CAM) model [46]. Other bird species, such as quail, ducks, and turkeys, in which the embryogenesis period is longer or shorter than in the chicken, which may be crucial for some analyses, can also be used [47]. The chorioallantoic membrane in the chicken embryo develops very dynamically from about embryonic day 3.5 and is an extraembryonic structure formed by the fusion of the chorion and the allantois. This is accompanied by the process of angiogenesis, as a result of which a network of undifferentiated blood vessels develops very dynamically in this bilayered membrane, which on the eighth day of embryogenesis gives rise to a plexus of capillaries whose dynamic proliferation continues until the 11th day of embryogenesis. In the following days of embryonic development, until day 18, the formation of the definitive blood system for survival outside the egg takes place [48,49]. The main functions of the CAM during embryogenesis are to support embryonic development through gas exchange, calcium transport, acid-base balance, and ion and water reabsorption [50]. Literature data suggest the use of the chorioallantoic membrane (CAM) model due to its specificity in research in oncology [51,52], tissue engineering [53,54,55], microbiology [56], and pharmacology [57].

Biologically active substances may affect homeostasis, induce cell death, or generate oxidative stress in the cell. Apaf-1 (apoptotic peptidase activating factor 1), upon binding cytochrome c and dATP, forms an oligomeric apoptosome. The apoptosome binds and cleaves procaspase-9, releasing its activated form. Activated caspase-9 stimulates the subsequent caspase cascade that commits the cell to apoptosis. Caspase-3 is a crucial executioner protease in the apoptotic pathway [58]. Apoptosis is a key cell death process during organogenesis, tissue homeostasis, and also in the etiology of multiple physiological disorders. Exogenic factors, drugs, and toxicants may affect the expression of apoptosis regulators in many species, including chicken embryos [59] as well as in older birds [60]. Environmental factors and biologically active compounds may also affect oxidative status in the organism and generate oxidative radicals. Key enzymatic cellular antioxidants are superoxide dismutases (SODs) and catalases (CATs). SOD enzymes catalyze the dismutation of superoxide (O_2_^•−^), generating hydrogen peroxide (H_2_O_2_). CAT, as well as glutathione peroxidases (GPXs), convert H_2_O_2_ into water. Their activity increases in the event of stress associated with the production of free oxygen radicals. In our previous study, we analyzed the effect of environmental toxicants and drugs on mRNA expression and immunolocalization of catalase and superoxide dismutase (SOD) in avian embryos [61] and laying hen [62].

While the CAM model has been previously utilized for evaluating nanoparticle toxicity in oncology [51,52,63], tissue engineering [53,54,55], and pharmacology, the specific combination of (1) chitosan-based spherical nanoparticles, (2) loaded with the imidazole derivative BzIm-DEA, and (3) evaluated through integrated morphological, molecular, and histological analyses, represents a novel contribution. This study directly addresses a gap in knowledge regarding the toxicological profile of BzIm-DEA in a developmental context. Specifically, this work combines: development and optimization of a chitosan film-based delivery system for BzIm-DEA nanoparticles; comprehensive preliminary toxicological evaluation using the ethically advantaged CAM model (3Rs compliance); integration of morphological observations, gene expression profiling, and immunohistochemical analysis to assess acute embryotoxicity, angiogenic response, and apoptotic/oxidative stress pathways.

Given the lack of prior in vivo toxicological data for BzIm-DEA, the aim of this study was to develop and optimize methods of application and analyze the biological activity and potential to affect angiogenesis, apoptosis and oxidative stress regulators of (Z)-5-benzylidene-3-(2-(diethylamino)ethyl)-2-phenyl-3,5-dihydro-4H-imidazole-4-one (C_22_H_25_N_3_O) using a preliminary screening approach with a logarithmic dose range. This approach allows for the establishment of basic biocompatibility and identification of potential dose–response relationships, while explicitly acknowledging that further pharmacokinetic and dose–response characterization will be required before translation toward clinical applications.

2. Results

2.1. Physicochemical Characterization of Chitosan Films Containing BzIm-DEA Nanoparticles

The thickness of the prepared chitosan films containing BzIm-DEA nanoparticles ranged from 0.123 ± 0.002 mm (D3) to 0.220 ± 0.004 mm (D1), with statistically significant differences observed among formulations (p < 0.05, Table 1). The successful formation of spherical nanostructures was confirmed by scanning electron microscopy (SEM). Representative SEM micrographs of sample D1 at magnifications of 5000× and 25,000× (Figure 1) demonstrate uniformly shaped spherical particles with well-defined morphology distributed on the chitosan film surface. High magnification imaging revealed core–shell architecture with entrapped oil droplets within the chitosan matrix, confirming successful nanoparticle encapsulation. Stability considerations: the chitosan films were stored at ambient conditions (20–25 °C, relative humidity ~60%) before application. Visual assessment of nanoparticle dispersity via SEM suggested uniform distribution across prepared films. Batch-to-batch reproducibility was assumed based on consistent preparation procedures and consistent film thickness measurements (Table 1), though formal inter-batch comparisons and stability testing over extended storage periods were not performed in this preliminary study. These aspects represent important areas for future optimization to ensure consistent formulation quality.

2.2. Evaluation of Survival Rate and Morphology of Embryos

No morphological changes were observed in the embryos of both groups in which a fragment of chitosan film was applied with nanoparticles containing the compound BzIm-DEA at different concentrations (D1, D2, D3, D4, D5) and in the case of the sample without the test compound (D0). Representative images of chicken eggs and embryos on the 11th day of embryogenesis after different BzIm-DEA doses are shown in Figure 2. Visual inspection of CAM tissue revealed extensive vascular development in all experimental and control groups. Morphological assessment indicated a well-branched network of blood vessels with active angiogenesis evident in the vicinity of the applied chitosan films. However, quantitative image-based analysis of vascular characteristics, such as vessel density (vessels per unit area), branching points (branch points per unit length), or fractal dimension of vascular networks, was not performed in this preliminary study. Such quantitative metrics would strengthen conclusions regarding angiogenic responses and are recommended for future investigations. The intensification of angiogenesis and the high survival rate of embryos may have been influenced by chitosan, which is the main component of the film, which is a biopolymer used in the medical industry due to, among other things, its non-toxicity, its properties to mimic tissue-specific conditions, its bactericidal, bacteriostatic, and antifungal properties [64]. Due to the high similarity between the samples, it is difficult to determine whether compound BzIm-DEA also exerted a similar effect or enhanced the effect of chitosan properties, as compounds containing an imidazole ring or its derivatives in their structures show potential for anti-inflammatory, anti-cancer, antioxidant, antibacterial, and antifungal properties.

Embryonic survival outcomes: A total of 4 embryonic deaths were recorded across experimental groups (n = 60 total eggs; D0: 2/6, D1: 1/6, D3: 1/6; overall survival rate 93.3%). These losses were attributable to serious shell damage and compromised membrane integrity during the window preparation procedure (see Section 4.4.2), rather than treatment-related effects. Thus, the survival data indicate that the selected doses of BzIm-DEA did not produce treatment-related embryonic lethality under these conditions. The death of embryos was caused by serious damage to the egg and, consequently, by the lack of continuity of the shell, membranes, and the protective slices and parafilm. If the study continues, the range of doses of the test compound can be increased, and a different type of oil in the emulsion, biopolymer, adhesive acid, or its content can be introduced as a variable. The indisputable advantage of using the CAM model over classical injections is the very small chance of losing a significant number of eggs due to imprecision, so it is an ideal model for preliminary studies.

In addition to static photographic documentation, video recording of exposed embryos and the CAM region was performed and is provided as Supplementary Material (Video S1). This Supplementary Video offers visual documentation complementary to static images: (1) vascular perfusion visualization; the video demonstrates active blood circulation within CAM vessels in the region surrounding the applied chitosan film, providing dynamic evidence of vascular integrity and adequate perfusion to embryonic tissues. While static images depict vessel morphology, video documentation reveals the functional aspect of ongoing circulation—a critical indicator of normal embryonic development and absence of acute vascular toxicity. (2) Embryonic viability confirmation: active blood flow, normal tissue coloration, and the absence of excessive hemorrhage or vascular disruption provide dynamic confirmation of acute embryonic health and the non-lethal nature of BzIm-DEA exposure under tested conditions. (3) Technical feasibility documentation: video documentation of the shell removal and film application procedures demonstrates the practical feasibility and reproducibility of the CAM model for treatment application and monitoring.

2.3. Immunohistochemical Analysis of PCNA and β-Catenin Expression in Embryonic Liver

Expression of mRNA encoding the apoptosis-regulating protein Apaf-1, as well as caspase-3, was significantly higher in the CAM tissue (chorioallantoic membrane) than in the liver tissue. A statistically significant increase in Apaf-1 mRNA expression was observed specifically in CAM tissue from the D3 dose group compared to controls (Figure 3A), with a mean fold-change of 2.0-fold, p < 0.05. Notably, this upregulation was not accompanied by corresponding increases in caspase-3 mRNA (Figure 3B), and no dose-dependent expression patterns were observed at lower (D1, D2) or higher (D4, D5) doses. The caspase-3 and catalase (CAT) mRNA expression levels did not differ significantly among any experimental groups and remained similar to control values (Figure 3B,C). The lack of dose-dependent changes across the concentration range and the dissociation between Apaf-1 upregulation and caspase-3 expression warrant cautious interpretation and further mechanistic investigation.

2.4. Analysis of mRNA Expression of Selected Genes Key to Apoptosis and Oxidative Stress in Embryonic Liver and Chorioallantoic Membrane

Expression of catalase, SOD2, PCNA, and b-catenin was detected in all embryonic liver samples. No significant differences in hepatic gene expression were observed among experimental groups. Quantitative analysis of PCNA and β-catenin expression via immunohistochemistry was performed qualitatively. Immunofluorescence patterns are presented in Section 4.4.3; quantitative morphometric analysis of proliferation index (PCNA-positive nuclei count) and β-catenin subcellular localization distribution was not undertaken in this preliminary study. Limitations of hepatic gene expression assessment: The restricted panel of molecular markers examined (Apaf-1, caspase-3, catalase, SOD2) represents a significant limitation of this preliminary study. The liver, as the primary organ for xenobiotic metabolism and detoxification, represents a critical target for potential acute and chronic toxicological effects. More comprehensive assessment would require evaluation of: phase I/II/III xenobiotic metabolism genes (cytochrome P450 family members, glutathione S-transferases, UDP-glucuronosyltransferases)-Inflammatory mediators (TNF-α, IL-6, IL-1β); extended apoptotic pathway genes (Bcl-2 family members, caspase-8, Fas/FasL); stress response elements (nuclear factor-erythroid 2-related factor 2 [Nrf2] and target genes, heat shock proteins). The absence of changes in the limited markers analyzed does not exclude potential hepatotoxicological effects that might be revealed through more comprehensive transcriptomic or proteomic approaches. The expression of PCNA localized in the cell nucleus has been observed in control and experimental groups. β-catenin, a protein with dual roles in cell–cell adhesion (via adherent junctions) and canonical Wnt signaling pathway regulation, typically presents distinct subcellular localization patterns depending on its functional context. When serving structural roles in cell adhesion, it localizes to the cell membrane; when serving signaling roles, it translocates to the nucleus [65]. In all examined embryonic liver samples, β-catenin was predominantly observed at the hepatocyte cell membrane (Figure 4), consistent with its structural role in maintaining cell–cell junctions. No dose-dependent alterations in β-catenin subcellular localization pattern (membrane vs. cytoplasmic/nuclear accumulation) were visually apparent, suggesting no activation of canonical Wnt signaling under these acute exposure conditions. However, qualitative visual assessment without quantitative image analysis may lack sensitivity to detect subtle changes.

3. Discussion

Comparative context and uniqueness of findings: This study represents one of the first in vivo toxicological evaluations of the compound BzIm-DEA and, to the authors’ knowledge, the first application of this compound in a developmental embryonic model. Consequently, direct comparison of the present findings with existing toxicological literature is not feasible, as prior in vivo toxicological data for BzIm-DEA are not available. This gap represents both a limitation of the current analysis and an opportunity for preliminary characterization and hypothesis generation to guide future, more comprehensive toxicological investigations.

Apaf-1 (apoptotic peptidase activating Factor 1) represents a critical initiator of the intrinsic apoptotic pathway, forming the apoptosome complex that activates caspase-9 and initiates the executioner caspase cascade in response to mitochondrial cytochrome c release. The observed statistically significant increase in Apaf-1 mRNA expression in CAM tissue at the D3 dose (Figure 3A) represents potential activation of early apoptotic signaling in response to BzIm-DEA exposure.

However, several important caveats warrant cautious interpretation of this finding:

• (1)Incomplete apoptotic cascade activation: the absence of corresponding increases in caspase-3 mRNA (the key executioner protease downstream of Apaf-1-mediated caspase-9 activation) suggests incomplete progression to terminal apoptotic events. This pattern could reflect: activation of apoptotic machinery subsequently blocked by anti-apoptotic protective signals; developmental stage-specific resistance to apoptotic progression in embryonic tissues; reversible stress response that does not culminate in cell death.
• (2)Non-linear, non-dose-dependent response: the upregulation at D3 but not at lower (D1, D2) or higher (D4, D5) doses is atypical of classical dose–response relationships and may indicate; a threshold effect with a narrow window of sensitivity, non-monotonic dose–response curves, increasingly recognized in toxicology but requiring mechanistic clarification, potential biological variability requiring confirmation with larger sample sizes.
• (3)Limited mechanistic context: whole-tissue mRNA quantification does not identify which cell populations express elevated Apaf-1, nor does it assess downstream apoptotic events (caspase activation, mitochondrial depolarization, DNA fragmentation). Spatial transcriptomics or immunofluorescence for active caspase-3 would clarify the cellular distribution and significance of this signal.
• (4)Temporal dynamics: single-timepoint assessment at embryonic day 11 provides a snapshot of only one developmental stage. Earlier or later timepoints might reveal additional apoptotic responses not captured in this study.

The Apaf-1 upregulation at D3 warrants further investigation but should not be interpreted as definitive evidence of apoptotic toxicity. Recommended confirmatory approaches include Western blotting for protein levels and caspase activation, flow cytometry with Annexin V/PI staining for quantification of apoptotic cells, TUNEL staining for apoptotic body detection, and expanded qPCR assessment of pro- and anti-apoptotic gene networks.

While this study evaluated mRNA expression of key molecular mediators of apoptosis (Apaf-1, caspase-3) and oxidative stress (catalase), the absence of significant changes in caspase-3 and catalase does NOT definitively exclude apoptotic or oxidative stress responses occurring at the cellular level. This represents an important methodological limitation:

• (1)Single developmental timepoint: assessment at embryonic day 11 captures only acute early-stage exposure effects. Apoptotic and oxidative stress responses are inherently dynamic, with gene expression changes potentially occurring at earlier developmental stages (rapid early response), in different tissue compartments, or at later timepoints not captured in this single-timepoint study.
• (2)Limited gene panel: analysis of only three apoptotic/oxidative stress markers (Apaf-1, caspase-3, catalase) represents a very narrow window into complex signaling networks. Comprehensive assessment would require evaluation of: extended apoptotic markers: caspase-9, Bcl-2 family members (Bcl-xL, Bcl-w, Bax, Bad, Bim, Bok), SMAC/DIABLO, IAPs (cIAP1, XIAP); extended oxidative stress regulators: SOD1, SOD3, glutathione peroxidases (GPx1, GPx2, GPx3), thioredoxins, catalases (CATs), peroxiredoxins, and the nuclear factor-erythroid 2-related factor 2 (Nrf2); stress response elements: heat shock proteins (Hsp70, Hsp90), unfolded protein response markers (BiP, CHOP, eIF2α phosphorylation, XBP1 splicing); additional defense systems: autophagy markers (LC3-II/LC3-I ratio, Beclin-1, ATG genes), and ferroptosis markers.
• (3)mRNA vs. protein vs. activity: mRNA expression quantification does not assess protein abundance, post-translational modifications, subcellular localization, or enzymatic activity. Examples: catalase activity is regulated not only by transcription but also by heme cofactor availability, protein quaternary structure, and age-related modifications. Caspase-3 activity depends on proteolytic activation of procaspase-3 by upstream caspases, which may not correlate with steady-state mRNA levels. Apaf-1 function requires assembly with cytochrome c and dATP into the functional apoptosome—a biochemical event not detectable by mRNA quantification alone.
• (4)Tissue-level analysis masking localized responses: whole-tissue homogenates represent averages across multiple cell types with potentially divergent responses. Localized stress responses in specific cell populations (endothelial cells remodeling vasculature, hepatocytes undergoing metabolic stress, immune cells) would be diluted by non-responding neighboring cells and may escape detection.
• (5)Developmental and species context: the embryonic chicken at day 11 represents a unique developmental state with heightened metabolic activity, ongoing organogenesis, and stress response mechanisms that may differ substantially from adult tissues and non-avian species.

Recommendations for improved assessment in future studies: RNA-sequencing or quantitative nanostring for high-throughput gene expression profiling-multi-timepoint sampling (days 7, 9, 11, 14 post-treatment) to capture temporal dynamics; spatial transcriptomics, laser microdissection, or flow cytometry sorting for cell-type-specific expression patterns; western blotting and immunofluorescence for protein-level validation; functional assays: flow cytometry for apoptosis/necrosis detection, enzymatic assays for catalase/SOD activity; investigation of alternative stress pathways (autophagy markers, ferroptosis indicators, necrosis markers).

The mRNA expression of Apaf-1, CASP3, and CAT genes was demonstrated in both the chorioallantoic membrane and liver. The results obtained suggest that, apart from a significant increase in Apaf-1 expression in the chorioallantoic membrane in the D3 group, there is no correlation between the concentration of the compound BzIm-DEA in the film and changes in the expression of genes both key to apoptosis and oxidative stress. However, in order to confirm the results obtained, the size of the groups should be increased, more samples should be considered, and expression analyses of a larger group of genes should be carried out, but the expression of other genes key to apoptosis, oxidative stress phenomena, and other relevant physiological processes and cell signaling pathways should be expanded. Probably the best solution would be to use cell cultures to test the effect of the analyzed compound on other cellular processes, such as the cell cycle or differentiation pathways. The properties of the biopolymer used and the potential properties of the BzIm-DEA compound, such as non-toxicity, bactericidal, bacteriostatic, antifungal properties, and the ability to mimic tissue-specific conditions, offer the potential application of the obtained films in the production of dressings and packaging for the storage of medical instruments. The CAM model, compared to other frequently used models in toxicological research, is characterized by the possibility of maximizing the use of the animal for research purposes. Due to the developmental state of the embryo, the pain and suffering that commonly accompanies standard animal models is minimized, and it is possible to minimize waste in the form of plastics, infectious material, and reagents required for cell or tissue culture. It can be concluded that the prepared BzIm-DEA compound carrier was stable and did not interfere with biological processes in the chicken yak during incubation. The selected doses of compound BzIm-DEA, as well as the method of application, were not toxic to the chicken embryos. Assessment of CAM morphology and vascular development: under the specific experimental conditions evaluated (embryonic exposure until day 11), BzIm-DEA-loaded chitosan nanoparticles did not produce gross morphological abnormalities, embryonic lethality directly attributable to treatment, or visual evidence of vascular structural injury (hemorrhaging, necrosis, disrupted branching pattern). However, this assessment was limited to qualitative morphological observation. Comprehensive evaluation of CAM angiogenesis would require quantitative assessment of vascular parameters, including:

• -vessel density (vessels per unit area)
• -branching point analysis (branch points per unit length of main vessel)
• -fractal dimension analysis of vascular networks
• -measures of vascular tortuosity and branching angles
• -quantification of avascular zones or regions of reduced perfusion.

Without such quantitative metrics, we cannot definitively exclude subtle, dose-dependent effects on angiogenic processes. Visual inspection provides valuable preliminary information but lacks the objectivity and sensitivity of automated vessel segmentation and morphometric analysis. Recommendations for future studies include high-resolution digital imaging of CAM with automated vessel segmentation software to quantify potential angiogenic effects.

No dose-dependency was shown for mRNA expression among both genes key to apoptosis (except for an increase in Apaf-1 expression at the D3 dose) and oxidative stress. WNT4 is involved in various developmental processes, including axis formation, tissue and organ development, and stem cell maintenance. Dysregulation of β-catenin signaling can lead to developmental abnormalities and is also implicated in cancer development, and in this experimental model, expression of this protein in the liver tissue was not affected by the BzIm-DEA compound. To summarize, the CAM model may be used in the preliminary study in the process of dose optimization, delivery method, and angiogenesis, as well as an embryotoxicity study. Further complex analysis is needed to test the biological potential of the BzIm-DEA compound.

The present study used chitosan film without BzIm-DEA (D0) as the primary negative control to isolate the effect of the active compound. However, the inclusion of a completely intact, non-windowed embryo group would provide additional baseline data for gene expression and developmental parameters and is recommended for future comprehensive studies.

4. Materials and Methods

4.1. Reagents

Chitosan (deacetylation degree ≥ 85%, Pol-Aura, Morąg, Poland), lactic acid (purity ≥ 85%, Sigma-Aldrich, St. Louis, MO, USA), glycerol (purity ≥ 99%, Merck, Darmstadt, Germany), olive oil (extra virgin, local supplier), and compound BzIm-DEA (synthesized as per the method in Section 4.2). All reagents were used without further purification.

4.2. Synthesis of Compound (Z)-5-Benzylidene-3-[2-(diethylamino)ethyl]-2-phenyl-3,5-dihydro-4H-imidazol-4-one (BzIm-DEA) and Calculations

The compound (4Z)-4-benzylidene-1-[2-(diethylamino)ethyl]-2-phenyl-4,5-dihydro-1(H)-imidazol-5-one (denoted as 13 in the source literature and referred to herein as BzIm-DEA) was synthesized according to the method described by Topuzyan et al. [66]. The synthesis involved cyclodehydration of the corresponding N-benzoyl-α,β-dehydroamino acid amide using trimethylchlorosilane (Me_3_SiCl) in DMF under microwave irradiation (360 W, 4.5 min), yielding 74.4% of the target product. The structure was confirmed by ^1^H and ^13^C NMR spectroscopy (Mercury-300 Varian, 300 MHz, DMSO-d_6_) and FT-IR spectroscopy (Nicolet Avatar 330). The structure of BzIm-DEA was confirmed by ^1^H NMR, ^13^C NMR, and FTIR spectroscopy. The spectra are provided in Supplementary Materials (Figures S1–S3) together with characteristic peak assignments. The obtained spectral data (^1^H NMR, ^13^C NMR, FT-IR) were identical to those reported in the cited work [66], confirming the Z-configuration and purity of the compound.

Conformational analysis and electronic structure calculations were performed using Gaussian 09W (Rev. A.02) [67]. Geometry optimizations and frequency calculations were carried out at the DFT/B3LYP/6-31G(d) level, including the polarizable continuum model (PCM) for modeling molecules in water solution at 298 K. Gibbs free energies were corrected by a scaling factor of 0.9804 for zero-point energies [68]. The energy barriers of transitions between conformers were found by scanning the corresponding dihedral angles (dh1 and dh2, Figure 5). The found transition states were saddle-points of the first order.

The X-ray structure is most closely related to the cf1 conformation, as the two key dihedral angles, dh1 and dh2, correspond to the same conformer. Differences are observed only within the amino group, where the spatial orientation varies; evidently, in solution, this group adopts a different arrangement from that observed in the crystal. The authors of the crystallographic study [66] did not comment on the fact that the crystal contained only one of the enantiomeric conformers. In principle, every second crystal should contain molecules adopting the opposite conformation. The well-defined crystal structure clearly demonstrates that intermolecular interactions with neighboring molecules are capable of suppressing conformational transitions. The PCM used in the theoretical calculations represents the solvent in an idealized manner—as a continuous medium with a given polarizability—and therefore does not take into account specific interactions such as substrate–solvent hydrogen bonding, which may restrict or modify the molecular geometry.

In solution, the cf2 and cf2* conformations were found to be energetically more favorable. The energy differences between these conformers are small, and the reversal of their energetic order relative to the solid state is therefore not unexpected. The calculated energy barriers for the conformational transitions amount to 13.3–13.8 kJ·mol^−1^ for rotation about dh1 and 24.7–25.4 kJ·mol^−1^ for rotation about dh2. The observed hysteresis is typical of molecules whose spatial structure is constrained by steric hindrance; during rotation, certain molecular fragments undergo deformations induced by steric interactions.

The calculated dihedral angles, relative Gibbs free energies (ΔG), and Boltzmann populations of the BzIm-DEA conformers are summarized in Table 2. The cf1 conformation shows the closest correspondence to the crystallographic structure, particularly in terms of the dh1 and dh2 dihedral angles, which define the relative orientation of rings B and C. Only minor deviations are observed for the aminoethyl side chain, confirming that this fragment adopts a different spatial orientation in solution than in the solid state. The nearly identical free energies of cf1 and cf1* (and of cf2 and cf2*) reflect the presence of mirror-image conformers with comparable stability.

The computed activation barriers and rate constants for conformer interconversion in solution at 298 K are listed in Table 3. Rotations about dh1 (13.3–13.8 kJ·mol^−1^) are energetically more accessible than those involving dh2 (24.7–25.4 kJ·mol^−1^), indicating limited flexibility of the aminoethyl fragment. The rate constants derived from the Eyring–Polanyi equation confirm that transitions around dh1 are substantially faster, consistent with the observed hysteresis typical for sterically constrained molecular systems.

4.3. Synthesis of Chitosan Film with Nanoparticles Containing BzIm-DEA Compound from the Imidazoles Group

Dose selection rationale: as BzIm-DEA represents a novel compound with no prior in vivo toxicological data, dose selection employed the following approach:

• (1)Structural analogy and reference compound: reference was made to clotrimazole (Canesten^®^), a well-established imidazole antifungal with extensively documented in vivo biological activity, safety profiles, and clinical applications [69]. The structural similarity of BzIm-DEA to clotrimazole provided a reference framework for initial dose estimation and helped inform the concentration range selected.
• (2)Experimental design strategy: five dose levels (D1–D5) were designed as a logarithmic dilution series spanning approximately four orders of magnitude (from 0.0000000694 g to 0.000694 g BzIm-DEA per 100 g chitosan film, Table 1). This wide concentration window allows for preliminary identification of potential dose–response relationships and helps identify any concentration-dependent effects across a broad range.
• (3)Preliminary nature of dose selection: the selected doses are NOT intended to represent anticipated therapeutic or toxic exposure levels in clinical or veterinary applications. Rather, they constitute a preliminary screening tier designed to: assess basic biocompatibility of the chitosan nanoparticle delivery system; identify gross toxicological red flags under acute embryonic exposure conditions; generate preliminary data to guide dose selection in future pharmacokinetic and dose–response characterization studies.
• (4)Pre-screening safety margin: all selected doses were pre-screened in preliminary cell culture experiments to ensure they would not produce catastrophic embryotoxicity or 100% embryonic lethality in vivo.
• (5)Future dose characterization requirements: determination of dose–response relationships and clinical relevance of the current findings will require: quantitative pharmacokinetic assessment of BzIm-DEA absorption, distribution, metabolism, and clearance; comparison with published acute reference doses (ARfD) for structurally similar imidazole compounds; dose escalation studies to establish no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect-level (LOAEL) thresholds; understanding of species-specific metabolism and toxicological sensitivity differences.

Nanoparticles were synthesized according to the general procedure reported by [70,71,72]. A 2% w/w chitosan gel was prepared by dissolving 20.0 g chitosan in 965.0 g distilled water containing 5.0 g lactic acid and 10.0 g glycerol, stirred at 70 °C (700 rpm) until homogeneous. Emulsions (oil:water 1:1) containing BzIm-DEA (0.1 mM stock in olive oil) were prepared by ultrasonication (20 kHz, 5 min) and blended with the chitosan gel according to Table 1. Films were cast (25.0 g portions), dried (5 days, ambient conditions), and characterized for thickness (micrometer, eight points per film). Prepared chitosan films containing BzIm-DEA nanoparticles were stored at ambient conditions (temperature 20–25 °C, relative humidity approximately 60%) in sealed containers before application to eggs. Films were visually inspected immediately before application to confirm continued integrity and absence of obvious degradation. Stability testing over extended storage periods and assessment of physicochemical parameter changes (thickness, particle size distribution, encapsulation efficiency) during storage were not performed in this preliminary study, representing an area for future development.

Detailed composition and thickness measurements for all formulations are presented in Table 1. Notably, Entry 4 (D3 film) showed the smallest thickness (0.123 ± 0.002 mm), while Entry 2 (D1 film) exhibited the largest thickness (0.220 ± 0.004 mm), suggesting that the concentration of BzIm-DEA in the emulsion inversely affected film thickness.

The addition of lactic acid is intended to dissolve the chitosan [73], while the addition of glycerin has the effect of increasing the flexibility of the resulting film [74]. Another organic acid, such as acetic acid, could have been used instead of lactic acid, while lactic acid was chosen because of its widespread use in the cosmetic industry [75].

In all figures, D0–D5 denote the control film without BzIm-DEA (D0) and increasing BzIm-DEA doses (D1–D5) as specified numerically in Table 1.

The successful formation of spherical nanostructures was confirmed by scanning electron microscopy (JEOL 7550 scanning electron microscope, Akishima, Tokyo, Japan). Representative SEM micrographs of sample D1 are shown in Figure 1, illustrating uniformly shaped particles with well-defined morphology distributed on the chitosan film surface. However, capturing sharp images of the spherical particles proved challenging, as during measurement, we observed the spherical structures rupturing in real-time under high vacuum conditions and electron beam bombardment. Under these conditions, some capsules ruptured, revealing the internal oil droplet entrapped within the spherical chitosan structure, which corroborates their core–shell architecture.

4.4. Animals

4.4.1. CAM Model

Studies on the CAM (chicken embryonic chorioallantoic membrane) model were conducted on the chorioallantoic membrane of domestic chicken embryos. Fertilized eggs (Leghorn, n = 60) were purchased from a local breeder (Tarnów, Poland). Eggs were selected based on their shape, size, and shell structure. The selected eggs were disinfected by washing with 70% ethanol [76], and then thoroughly wiped and dried. The eggs were placed in an incubator (Brinsea 190 Advance, Brinsea, Titusville, FL, USA) arranged on dedicated trays. The incubator used had an automatic egg turning function. The temperature was set at 37.6 °C (the optimal temperature during incubation of chicken eggs is 37.5–37.8 °C [77]), while the humidity was set at 60% [78]. From the first day of incubation until the day of the end of the experiment, water (distilled) was replenished at least once a day in the incubator tanks to ensure humidity.

4.4.2. Day 3 of Embryogenesis

On the third day, the eggs were again disinfected in order to cut an oval-shaped hole in the shell. To determine the location of the air chamber and its surface, the egg was candled with an ovoscope and marked. The egg with the marked location of the air chamber was placed on a dish, and then a small puncture was gently made with a syringe needle on the opposite side. Through the hole made, 3 mL of egg white was taken from the egg with a syringe and then disposed of. The puncture site was carefully protected with parafilm and then sealed with a piece of plaster. The reduced volume of egg white in the egg made it possible to make a puncture in one of the sides of the egg with surgical scissors and then cut out part of the shell. The cut hole (about 2 × 2 cm) was secured with parafilm, over which a patch was applied. Eggs that were unfertilized or in which the embryos died in the early stages of embryogenesis were disposed of. Eggs with the hole made and secured were placed in an incubator with the automatic rotation of eggs function turned off from then on.

4.4.3. Day 8 of Embryogenesis

On the eighth day of incubation, eggs were signed and divided into six groups, with six eggs included in each group due to the exclusion of unfertilized eggs and accounting for losses during shell perforation or embryo death. Eggs were marked according to the coding system (D0–D5) used for both the samples and the chitosan films (D0–D5 films) synthesized with nanoparticles containing the BzIm-DEA compound. Before applying the prepared films, they were treated with UV light to eliminate the possibility of contamination and cut into small squares of 0.5 cm × 0.5 cm with sterile scissors. The cut piece of foil was placed with tweezers on the branching of the main blood vessels (Figure 6) after uncovering the protected opening in the eggshell, then secured with parafilm and plasters and placed back in the incubator. Technical considerations and photographic documentation: due to technical limitations in optical access through the smaller observation window created in the eggshell, high-quality photographic documentation of embryonic tissue with the applied chitosan film could not be obtained from all specimens using standard imaging protocols without further manipulation of the preparation. To overcome this constraint and capture adequate visual documentation of CAM morphology, vascular integrity, and the precise positioning and appearance of the chitosan carrier, the eggshell was carefully removed from selected embryos to expose the chitosan layer positioned on the chorioallantoic membrane. This approach enabled improved visualization and photographic documentation (Figure 6) while maintaining embryonic viability throughout the observation and imaging period. Following the removal of eggshell protective barriers and parafilm, video recording of the exposed embryo and CAM region was performed to document CAM integrity, embryonic viability, and vascular perfusion in the region of chitosan film application. Video files were retained as supporting documentation of treatment application methodology, embryonic health status, and absence of acute vascular dysfunction or hemorrhaging. Videos were not subjected to quantitative analysis of hemodynamic parameters (e.g., blood flow velocity, perfusion rate measurements); recording served primarily as qualitative visual confirmation of CAM vascular integrity and embryonic viability (Video S1, Supplementary Materials).

4.4.4. Proceedings on Day 11 of Embryogenesis

The experiment was completed on the 11th day of incubation. From each egg removed from the incubator, a safety slice and parafilm were pulled off the opening, after which, fragments of the chorioallantoic membrane were cut out, and then the embryo was removed and placed on the dish. The embryo was immediately decapitated with surgical scissors, and then the liver was isolated from it. Both the fragment of the liver and the membrane were washed with saline solution (on ice) and placed in tubes with StayRNA reagent (A&A Biotechnology, Gdynia, Poland). The tubes were placed in the refrigerator and then in the freezer at −20 °C until RNA isolation. Two lobes of each liver were fixed in 10% (v:v) buffered formalin, pH = 6.9 (Leica, Wetzlar, Germany), then processed and embedded in paraffin for immunodetection of SOD2, catalase, PCNA, and β-catenin.

4.4.5. RNA Isolation, Reverse Transcription Reaction, qPCR

RNA isolation and reverse transcription were carried out according to the manufacturer’s instructions and a previously published protocol [60]. Analysis of mRNA expression by qPCR was performed according to the kit manufacturer’s instructions (Solis BioDyne, Vilnius, Lithuania). Total RNA (2 μg from each tissue extracted with TRI Reagent) was reverse transcribed using a high-throughput cDNA Reverse Transcription Kit (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific, Waltham, MA, USA) containing random primers. Samples were incubated in a thermocycler (Mastercycler Gradient; Eppendorf, Hamburg, Germany) according to the following thermal profile: 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. The resulting cDNA was used in real-time qPCR for catalase (CAT), APAF-1, caspase-3 (CASP3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Amplifications were performed in 96-well plates in a thermocycler (StepOnePlus; Applied Biosystems, Waltham, MA, USA) according to the recommended cycling program: 15 min at 95 °C, 40 cycles of 15 s at 95 °C, 20 s at 62 °C, and 20 s at 72 °C. The primers (Table 4) were synthesized according to previously used and tested sequences [69,79]. Single real-time qPCR reactions for the genes tested were carried out in a volume of 10 μL containing 2 μL of 5× HOT FIREPol EvaGreen qPCR Mix Plus, 0.12 μL of each primer (10 pmol/μL), 1 μL cDNA (10× diluted sample after RT reaction), and sterile water was added to 10 μL. Each sample was analyzed in duplicate. A negative control without cDNA was included during each analysis. Relative expression of the genes tested was calculated after normalization with GAPDH transcript using the 2^−ΔΔCT^ method.

4.4.6. Immunolocalization of PCNA, SOD2, Catalase and b-Catenin in the Chicken Embryonic Livers

Fragments of the examined liver tissues (thickness: 6 μm, sliced using a semi-automatic RM2245 rotary microtome, Leica, Wetzlar, Germany) were deparaffinized in xylene, then hydrated by passing through alcohol solutions of decreasing concentrations (from 100% absolute to 75%). The slides were rinsed with water, heated in a citrate buffer (pH 6.0) at 75 °C for 5 min, then rinsed with a TBS buffer. After that, goat serum was applied to the slides at room temperature for 90 min. The serum was removed and the slides were then incubated overnight at 4 °C with antibodies against PCNA (proliferating cells nuclear antigen) (PCNA Polyclonal Antibody (PA1-38424, ThermoFisher Scientific, Waltham, MA, USA), oxidative stress regulators catalase (PA5-23246; Thermo Fisher Scientific, Waltham, MA, USA) and SOD2 (PA5-30604; Thermo Fisher Scientific, Waltham, MA, USA), and b-catenin (71-2700; Thermo Fisher Scientific, Waltham, MA, USA). The tissues were then washed three times in TBS and incubated for 1.5 h with the secondary fluorescent antibody. In the last step, the slides were sealed with (VEC-TASHIELD^®^HardsetTM Antifade Mounting, Vector Laboratories, Burlingame, CA, USA) Medium with DAPI. The preparations were observed and analyzed (n = 6 in each group) using an AxioScope fluorescence light microscope (ZEISS, Oberkochen, Germany) with an Axiocam 503 camera and ZEN 2.3 pro software (Carl Zeiss, Jena, Germany).

Immunofluorescence images were captured using AxioScope microscopy at 400× magnification from liver sections of all embryos in each group (n = 6 per group, representative fields of view). In this preliminary study, immunohistochemical analysis was performed qualitatively, with patterns of PCNA nuclear localization and β-catenin subcellular distribution (membrane vs. cytoplasmic/nuclear) visually compared across experimental groups. Formal quantitative morphometric analysis of tissue preparations was not performed in this preliminary investigation.

Analysis of protein expression of PCNA, protein expressed in the proliferating cells, key regulators of oxidative stress response (catalase and SOD2), and β-catenin, playing a crucial role in development by acting as a central mediator of WNTsignaling essential for cell proliferation and tissue organization, is a very important step toward knowledge of biological potential and toxicity of BzIm-DEA for the developing embryo.

4.4.7. Statistical Analysis of the Results

Statistical analysis of the results was performed using one-way ANOVA followed by Duncan’s test. Values were expressed as means ± SEM and were considered significantly different at p < 0.05. Calculations were performed using SigmaPlot (version 16, Systat Software Inc., San Jose, CA, USA).

5. Conclusions

This preliminary toxicological assessment of chitosan-based nanoparticles containing the imidazole derivative BzIm-DEA, conducted using the CAM model as an ethically advantaged alternative to mammalian in vivo testing, provides initial evidence regarding the acute biocompatibility of this novel nanoparticle delivery system under the specific experimental conditions evaluated.

Key findings:

• (1)Absence of gross acute embryotoxicity: no morphological abnormalities, embryonic lethality directly attributable to treatment, or overt disruption of basic developmental processes were observed across the five tested dose levels (D1–D5) in the CAM model.

• (2)Successful nanoparticle carrier synthesis: scanning electron microscopy confirmed successful synthesis of spherical, core–shell chitosan nanoparticles with uniform morphology and entrapped active pharmaceutical ingredient, indicating structural integrity and stability of the delivery system throughout the experimental period.

• (3)Predominantly stable molecular profiles: quantitative RT-qPCR analysis revealed no significant dose-dependent changes in mRNA expression of most apoptotic and oxidative stress regulatory genes (caspase-3, catalase, SOD2) in hepatic tissue. A statistically significant increase in Apaf-1 mRNA was observed specifically in CAM tissue at the D3 dose, warranting further mechanistic investigation.

• (4)Practical utility of the CAM model: the chicken embryonic CAM model demonstrated significant practical value as a preliminary in ovo screening system, offering major advantages over mammalian toxicological models, including:

• -substantially lower cost per animal

• -full compliance with the 3Rs ethical principles (Replacement, Reduction, Refinement);

• -high experimental efficiency and throughput

• -reduced waste generation compared to in vitro alternatives

Important qualifications and limitations requiring acknowledgment:

Dose characterization gap: the selected concentrations were not rigorously justified based on anticipated therapeutic or toxic exposure levels in clinical contexts. Future studies incorporating quantitative pharmacokinetic profiling and comparison with established reference doses for structurally similar compounds are essential for risk assessment.

Limited molecular panel: assessment of only a restricted set of apoptotic and oxidative stress markers in this preliminary study may not fully capture the spectrum of potential adverse biological effects. The observed Apaf-1 upregulation at D3—notably without corresponding caspase-3 activation—represents an atypical pattern requiring further mechanistic investigation through expanded gene expression profiling, protein-level analysis, and functional apoptosis assays.

Qualitative morphological and angiogenic assessment: evaluation of CAM morphology and vascular development relied on visual inspection and qualitative imaging. Quantitative vascular analysis (vessel density, branching metrics, fractal dimension) would substantially strengthen conclusions regarding potential effects on developmental angiogenesis.

Single developmental timepoint: assessment at embryonic day 11 provides information only for acute, early-stage exposure effects. Longer-term developmental windows, repeated dosing scenarios, and assessment at later gestational stages may reveal delayed or stage-specific toxicological responses not captured in this single-timepoint study.

Restricted hepatic characterization: gene expression analysis in liver tissue was restricted to a minimal panel of molecular markers and did not assess phase I/II/III xenobiotic metabolism enzymes, inflammatory mediators, or extended stress response pathways. Such a comprehensive transcriptomic analysis would be necessary to detect subtle metabolic perturbations or early hepatotoxicological signals.

Pathway forward: within the scope of this preliminary investigation, the chitosan-BzIm-DEA nanoparticle system demonstrated biocompatibility in the CAM embryonic model without evidence of gross acute embryotoxicity under the specific dose range and conditions evaluated. The CAM platform proved serviceable as a cost-effective screening tool for preliminary assessment of novel nanoparticulate drug carriers. However, these findings should not be interpreted as definitive evidence of safety, and substantial additional work is required before translation toward therapeutic development or clinical applications:

Expanded molecular and toxicological characterization:

• -RNA-sequencing or comprehensive gene expression profiling
• -multi-timepoint sampling to capture temporal dynamics of gene expression responses-Protein-level validation via Western blotting and immunofluorescence
• -functional apoptosis and oxidative stress assays-Investigation of alternative stress pathways (autophagy, ferroptosis, necrosis)
• -quantitative immunohistochemical analysis.

Pharmacokinetic and dose–response characterization:

• -pharmacokinetic studies to establish absorption, distribution, metabolism, and elimination profiles
• -determination of dose–response relationships using extended dose ranges
• -NOAEL and LOAEL establishment through structured dose-escalation studies
• -comparison with established reference doses for structurally analogous compounds.

Extended developmental and temporal assessment:

• -multi-stage embryonic assessment (earlier and later developmental stages)
• -chronic or repeated exposure models
• -postnatal outcome evaluation in mammalian models.

Transition to regulatory-relevant models:

• -in vitro toxicity assessment using hepatocytes, intestinal epithelial cells, and other relevant cell types
• -mammalian in vivo toxicological studies in rodent models with regulatory relevance
• -GLP-compliant toxicology studies if clinical translation is intended.

In conclusion, the present preliminary findings establish the feasibility and basic biocompatibility of the chitosan-based BzIm-DEA nanoparticle system in the CAM model, providing a foundation for more rigorous and comprehensive investigations. The successful synthesis and stability of the nanoparticle carrier system, combined with the absence of gross acute embryotoxicity, support progression to the next tier of toxicological evaluation. However, the numerous limitations acknowledged above must be addressed through targeted future studies before this system can be advanced toward therapeutic applications or clinical translation.