A Quantum Chemical, Biological, and Experimental Analysis of Newly Synthesized Schiff-based PEI CA/N-GQDs Nanomaterials: Evaluation of Anticancer Potential in Human Neuroblastoma Cell

A Quantum Chemical, Biological, and Experimental Analysis of Newly Synthesized Schiff-based PEI CA/N-GQDs Nanomaterials: Evaluation of Anticancer Potential in Human Neuroblastoma Cell | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Quantum Chemical, Biological, and Experimental Analysis of Newly Synthesized Schiff-based PEI CA/N-GQDs Nanomaterials: Evaluation of Anticancer Potential in Human Neuroblastoma Cell Murat Kilic, Buket Altinok Gunes, Omer Faruk Kirlangic, Aysenur Aslan, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5352941/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this study, we synthesized a new material, Schiff base PEI-CA/N-GQDs, from the reaction of 4-hydroxy-3-methoxy-cinnamaldehyde (CA) with polyethyleneimine-functionalized N-doped graphene quantum dots (PEI N-GQDs). The synthesized material was experimentally characterized by FT-IR, UV-Vis, SEM, EDX, AFM, XRD, and TGA, as well as theoretically by DFT method. In addition to their biological activities, their cytotoxic, apoptotic and cell cycle arresting effects were investigated in human epithelial neuroblastoma (SH-SY5Y) cells. According to the UV-Vis data, we observed that the material exhibits phenol-imine/keto-amine tautomerism, a phenomenon common in 2-hydroxy Schiff bases that help to explain the various properties of material. Furthermore, this material predominantly exists in the keto-amine form. The material demonstrated favorable electron transfer properties, making it suitable for electrochemical applications. We showed that it binds to DNA through an electrostatic interaction and causes oxidative and hydrolytic cleavage in DNA, which results in an increase in ROS in the cell, an activation of the CASPASE-3, leading the cells undergo apoptosis, and inhibiting cell division in the G1/S phase. We believe that the chemical properties of the Schiff-based PEI N-GQDs make them a superior carrier molecule for cancer treatment. Furthermore the anticancer properties of the Schiff-based PEI-CA/N-GQDs suggest their potential as a therapeutic agent for neuroblastoma. Biological sciences/Cancer/Cancer therapy Biological sciences/Chemical biology/Drug delivery Biological sciences/Cancer Physical sciences/Chemistry/Chemical biology Physical sciences/Chemistry/Chemical engineering Physical sciences/Chemistry/Materials chemistry/Biomaterials Biological sciences/Drug discovery Biological sciences/Drug discovery/Drug delivery Coniferaldehyde (CA) Schiff base PEI N-GQDs Apoptosis Neuroblastoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introductıon Cancer is a devastating illness and the leading cause of death, and given the rise in new occurrences of cancer and the number of cancer-related fatalities seen and recorded in almost every region of the world, it is clear that cancer will remain a major threat to human health 1 , 2 . As it is known, although the treatment of cancer varies depending on the person and the type of cancer, it generally includes radiotherapy, chemotherapy, immunotherapy, surgical methods and at least two combinations of these. Among these, chemotherapy is the main and the most commonly used treatment method, but it is not successful in many tumor types and sometimes causes serious side effects 3 . For this reason, drug research in cancer treatment has turned to natural compounds, primarily of plant origin, which may be less harmful or perhaps harmless to healthy cells, but may cause more damage to cancer cells 4 , 5 . Researches conducted in this field show that natural compounds can be used in humans since they have high dose tolerance with less toxic effects 6 and are effective in various molecular processes such as cellular proliferation, differentiation, apoptosis, metastasis, DNA damage and repair 7 . Cinnamaldehyde, one of these natural molecules, is a flavonoid found in the bark/twig of the cinnamon plant, which gives its taste and aroma to the cinnamon plant and has a proven effect in preventing or reducing the severity of many diseases, including diabetes, atherosclerosis, inflammation and cardiovascular diseases 8 . Furthermore, research highlights the antiproliferative properties of cinnamon that affect cancer cells, including those from leukemia 9 , 10 , melanoma 11 , breast cancer 12 , colon cancer 12 – 15 , ovarian cancer 16 , and hepatoma 17 , 18 , by causing apoptosis and halting the cell cycle via a variety of molecular mechanisms 19 . Essentially similar to cinnamaldehyde when replaced with a hydroxy group at position 4 and a methoxy group at position 3 20 , coniferaldehyde (CA), is a naturally occurring phenolic chemical and a member of the family known as cinnamaldehydes (4-Hydroxy-3-methoxycinnamaldehyde: (4H3MC)) 21 . A limited number of studies have focused on the cytotoxic and apoptotic effects of CA extracted from plant extracts for cancer treatment. Consediring this, CA derived from Patrinia heterophylla Bunge extract has been shown to exhibit strong cytotoxic effects at low concentrations on cell lines representing melanoma, hepatocellular carcinoma, gastric cancer, cervical cancer, colon cancer, and breast cancer. Additionally, in the same study CA has been reported to cause melanoma cells to undergo significant rates of apoptosis 22 . Recent advancements in nanotechnology have shown significant potential in mitigating the negative impacts associated with conventional chemotherapies, as well as enhancing the effectiveness of cancer treatment 23 – 25 . Due to their unique features, these nanosized drug delivery systems (DDS) including carbon-type materials as well as dendrimers and nanoparticles made up of polymers or lipids are among the first-line options for constructing straightforward platforms with tailored functionalities that enable targeted anticancer agent release by overcoming chemotherapeutic limitations in cancer. The DDSs are designed to reduce safety or act on specific active effects in cancerous cells/tissues compared to noncancer-treating free cytotoxic agents 26 . Graphene quantum dots (GQDs) are a constituent of QD materials derived from graphene sheets and showing both carbon dot-like properties and graphene features. They are recognized for their widespread use in many areas of technology research, including sensor systems, bio-imaging applications, diagnostics, drug carriers, gene delivery vectors, and energy storage devices 27 . Graphene-based nanocomposites have a cytoplasm–nucleus shuttling system, making them effective drug delivery carriers for pharmacological applications 28 . Moreover, research on GQDs targeting their antiproliferative and anticancer properties indicates that these materials can internalize into cells and interact with DNA, RNA, and proteins, leading to cytotoxicity 29 . Concomitantly, intracellular ROS generated by mitochondria accumulate in graphene-treated cells. This increase in ROS levels then triggers a cascade of metabolic events from the mitochondria to the cytoplasm, ultimately causing the cell to undergo apoptosis and die 30 , 31 . The heteroatom doping can modify the properties of GQDs, thereby broadening their range of applications and facilitating their utilization. One specific type of this modification is represented by Nitrogen-Doped Graphene Quantum Dots (N-GQDs). In contrast to nitrogen-free GQDs, N-GQDs have a strong blue fluorescence and operate electrocatalytically in the oxygen reduction reaction (ORR) under alkaline conditions in a manner akin to the Pt/C catalyst 32 . In addition to being a unique combination of biotechnology and state-of-the-art materials science with excellent photoluminescence and biocompatibility, N-GQDs are also useful tools because of their unique properties that improve imaging and drug delivery in the context of cancer treatment and diagnosis 33 – 36 . We assessed N-GQDs' capacity to attach to DNA, maintain cell viability, and possess antioxidant qualities in our research. As a result, our research demonstrated that N-GQDs can attach to DNA through electrostatic and intercalation routes, and that high quantum dot concentrations can damage cancer cell DNA and decrease the survival of ovarian and breast cancer cells 27 , 36 . Schiff bases, which are substances created by the condensation reaction between primary amines and aldehydes, are well-liked ligand precursors because of their adaptability and simplicity of production. Other donor functional groups like -Cl, -OH, -CH3, etc. are also present in these primary amines and aid in the regulation and enhancement of biological functions. These characteristics make schiff bases useful in pharmacological, therapeutic, biological, medicinal, and analytical fields 37 . According to studies, schiff bases have analgesic, depressive, anticancer, and antimycobacterial properties 38 . A malignant tumor of the peripheral nervous system that develops in children, neuroblastoma is caused by neural crest sympathoadrenal progenitor cells. Since patients with advanced neuroblastoma have a dismal prognosis despite recent advancements in combination therapy, more potent, less cytotoxic therapies are required. It is crucial to create an efficient treatment plan as a result 39 . Although information is given in the literature about the anticancer activities of cinemaldehyde and its analogues obtained from plant extracts, studies on the anticancer activity of CA, a derivative of cinemaldehyde, especially in neuroblastoma, are very limited. In this study, Schiff base modified N-GQDs nanoparticles were prepared by reacting CA with PEI N-GQDs in ethanol medium. The structures of Schiff base PEI-CA/N-GQDs were experimentally and theoretically characterized. We have also used cyclic voltammetry (CV) to investigate the electrochemical properties of material. Then their biological activity and antiproliferative, apoptotic and cell cycle arresting properties in human epithelial neuroblastoma (SH-SY5Y) cells were investigated. Results And Discussion Characterization of Schiff base PEI-CA/N-GQDs (UV-Vis, FT-IR, XRD, SEM, AFM EDX and TGA) The absorption spectrum of Schiff base PEI-CA/N-GQDs was measured in water (Fig. 1 ). As can be seen in Fig. 2 , in the UV-Vis spectrum, peaks assigned to the π-π* transitions of C = C at 268 nm and to the n-π* transitions of C = N (imine and pyridinic) and O-C = O (carboxylic acid) at 420 nm are observed. Surprisingly, another peak at 496 nm was observed in the spectrum. This peak is probably due to the phenol-imine/keto-amine tautomerization of the 4-hydroxy-cinnamaldehyde Schiff base 40 . It is known that absorptions above 400 nm in 2-hydroxy-Schiff bases are due to the phenol-imine/keto-amine tautomerization 40 . This phenomenon is also observed in 2-hydroxy-Schiff bases. Many properties of 2-hydroxy-Schiff bases are explained by this phenomenon 41 – 43 . Notably, this research highlights the first observation of keto-amine tautomerism occurring in a 4-hydroxy-Schiff base. An extensive comparison of the characteristic group vibrations between the starting material, PEI N-GQDs, and its Schiff base counterpart, Schiff base PEI-CA/N-GQDs, was conducted using the FT-IR spectra, and is presented in Figure S1 . In this comparative analysis, the synthesized materials were examined, and their vibrational frequencies were determined. It also uncovered ways in which the newly synthesized materials differed from their original compositions. The spectral data contained subtle variations and elevations that were exclusive to the synthesized material, potentially indicating structural modifications resulting from the formation of Schiff bases. Figure S1 illustrates the graphic representation of these results, which provided a description of the spectroscopic signatures associated with the chemical changes during synthesis. This greatly enhanced our understanding of the structural properties and molecular makeup of these materials. PEI N-GQDs with vibrational bands were detected at the following locations: 3831 − 3736 (OH), 3421 − 3270 (NH 2 + N + COOH) and 2942 − 2848 (C-H), 1686 (COO), 1651(C = N), 1551(C = C) and 1452 cm - 1 (C-N + C-O) 27 . The Schiff base PEI-CA/N-GQDs had bands at the same positions, 3852 − 3738 (OH), 3419 − 3228(NH 2 + NH + COOH + Ar-H), 2942 − 2848, 1732, 1651, 1546 and 1447 cm - 1 ( Figure S1 ). Comparing the vibrations in the FT-IR spectra of Schiff base PEI-CA/N-GQDs with the spectrum of pure PEI N-GQDs, it can be seen that the vibrations of the OH and COO functional groups are shifted to higher frequencies, while the NH 2 + NH + COOH, C = C, and C-N + C-O vibrations are shifted to lower frequencies. Surprisingly, in both materials, C-H and C = N vibrations were observed at the same frequency. X-ray diffraction (XRD) pattern presents a broad peak at 20 0 to 30 0 of 2 θ , characteristic of amorphous materials (Fig. 2 ). The broadness of the peak suggests poor long-range ordered nature, typical for carbon-based materials which could resemble unprocessed graphene oxide or N-GQDs 44 . The absence of sharp peaks and the gradual decrease in intensities beyond 30 0 indicate a low degree of crystallinity, which can be linked to the amorphous nature of this material. This behavior is characteristic of materials that have an amorphous structure, such as the Schiff base complexed with PEI-CA/N-GQDs. Further absence of sharp peaks supports the observation that synthesized Schiff base PEI-CA/N-GQDs are dominantly amorphous in nature with randomly oriented graphitic layers typical for N-GQDs. The elemental compositions and bonding configurations of Schiff base PEI-CA/N-GQDs were analyzed by energy dispersive X-ray spectroscopy (EDX). The amount of elements detected using EDX methods are given in Fig. 3 . According to the EDX spectrum, the detection of carbon (64.1 Wt%), oxygen (27.0 Wt%) and nitrogen (8.7 Wt%) on the surface of the samples confirms that the material has been successfully synthesized. The structure and morphology of Schiff base PEI-CA/N-GQDs were also confirmed by SEM images as shown in Fig. 4 (a) . As can be seen from Fig. 4 (a) , it is confirmed that Schiff base PEI-CA/N-GQDs retain the spherical shape morphology and do not form agglomerates even at 5 µm. Table 1 AFM results of Schiff base PEI-CA/N-GQDs Region Min(nm) Max(nm) Mid(nm) Mean(nm) Rpv(nm) Rq(nm) Ra(nm) Rz(nm) Rsk Rku All -43.574 50.604 3.515 0.001 94.178 9.712 7.041 87.766 -1.136 5.856 The morphology of Schiff base PEI-CA/N-GQDs was also characterized by atomic force microscopy (AFM). A 2D view of sample surface topography in Fig. 4 (b) reveals the height fluctuation range across a 5 µm × 5 µm scanned area. The left hand side shows a colour scale for elevation changes, going from the highest blobs around 50 nm high through to the lowest round − 40 nm. AFM results of Schiff base PEI-CA/N-GQDs is also given in Table 1 . The presence of a large number of height variations reflects surface roughness, and the Rpv (peak-to-valley) value for this is as high as 94.178 nm. The surface of the material is heterogeneous, showing brighter (higher) features mixed with a darker (lower) background. The bright areas represent the peaks or material clusters, while the dark regions indicate valleys or lower surfaces. The 3D image provides a profile of the surface in Fig. 4 (c) . Some of the peaks are quite sharp and tall compared to neighboring areas, which corresponds with an Rz (ten-point average roughness) measurement of 87.766 nm. It has a medium texture with few smooth areas and many prominent features, some of which are sharp peaks; all of this contributes to the high kurtosis (Rku = 5.856) characteristic of sharp sub-micron height distributions. These sharp peaks and deep valleys align with the negative skewness (Rsk = -1.136), meaning there are more valleys than peak areas. These multiple-height-layered-surface characteristics are obviously important for surface interactions in applications like coatings, sensors, or electrodes. It can be said that the material has specific surface forms which could be advantageous for drug delivery and cancer therapy use cases. The moderate roughness with sharp peaks, and deep valleys highly supports higher incorporation of the drug molecules for increased cellular internalization, thereby improved release in addition to cancer cell targeting 45 . However, our in vitro research results support that it has a material structure consistent with an apoptotic or other anti-cancer cell death mechanism. Figure 5 illustrates the thermal decomposition of Schiff base PEI-CA/N-GQDs. The figure presents combined data on TG, DTG, and DTA. As can be seen from Fig. 5 , the TG curve of Schiff base PEI-CA/N-GQDs, it degrades in four (IV) steps. The first (I) step of degradation took place at 57–148°C and resulted in a weight loss of 3.76%, which may be due to the removal of hydroxyl and crystalline water. The second (II) decomposition step took place at 148–287°C and resulted in a total weight loss of 11.20%, which may be due to the removal of carboxyl groups. The third (III) decomposition step occurred at 287–304°C, which may have resulted from the elimination of amine groups in the PEI chain, with a total weight loss of 2.74%. The last step (IV) took place at 304–900°C with a weight loss of 19.08%, probably due to the elimination of pyridine and pyrrolidine groups in the ring. DTG shoulders at 79°C and 180°C characterise steps I, II and III with lower mass loss attributed to PEI-CA decomposition, while the strong endothermal effect at 315–379°C characterizes step IV with high mass loss attributed to the fragmentation of GQDs containing prolidine and pyridine groups in the ring. When the DTA curve is analyzed, it can be seen that the exothermic effect at 25–200°C in stages I and II is different from DTG, whereas in stages III and IV it is endothermically identical to DTG. Small mass losses in stages I, II and III are characterised by an exothermic DTA peak, while high mass losses in stage IV are characterized by an endothermic DTG peak. As seen from the DTA curve, a broad, endothermic peak is observed at 200–600°C, which is believed to be due to thermal expansion of the Schiff base PEI-CA/N-GQDs. No other thermal changes were observed in Schiff base PEI-CA/N-GQDs after 700°C. As a result, in this case, it can be said that Schiff base PEI-CA/N-GQDs is thermally stable. Electrochemical properties of Schiff base PEI-CA/N-GQDs The cyclic voltammetry (CV) graph shows the well-known redox-active and consequently facile electrochemical behavior of the Schiff base PEI-CA/N-GQDs nanomaterial (Fig. 6 ). The x-axis represents the potential (V) applied during the measurement, and y-axis represents the current (mA) response of the system. A notable anodic peak appears around + 0.20 V, indicating the oxidation of electroactive species, likely related to the imine or phenolic groups present in the Schiff base structure. The corresponding cathodic peak around − 0.12 V signifies the reduction of the oxidized species, forming a redox couple typical of conjugated organic systems. The voltammogram shows a quasi-reversible nature, as evidenced by the asymmetry between the anodic and cathodic peaks and the relatively large potential separation, suggesting slower electron transfer kinetics. The maximum oxidative and reductive currents, approximately 0.12 mA and − 0.12 mA respectively, point to significant interaction between the material and the electrode surface. Additionally, the tailing at higher potentials (beyond 0.3 V) suggests some capacitive behavior, likely due to the high surface area of the N-GQDs contributing to double-layer charging. Overall, this CV indicates that the Schiff base PEI-CA/N-GQDs is electrochemically active with a quasi-reversible redox process, characteristic of such nanocomposite materials. Computational analysis Schiff base PEI-CA/N-GQDs (Optimization, FTIR, FMOs, MEP isosurfaces, UV-Vis) Initially, geometric optimization calculations were performed in the gas phase to determine the lowest energy structure representing the most stable configuration and thermodynamic properties of the Schiff base PEI-CA/N-GQDs for both phenol-imine and keto-amine forms (Table 2 ). The keto and phenol forms are tautomers, which are interconvertible isomers typically involving the movement of a proton and the shift of a double bond 46 . The characteristics of these tautomers—particularly their electron structures—can differ greatly. The keto form generally has a higher dipole moment, as is indicated in Table 2 , and it is because the keto structure usually contains a carbonyl group (C = O) where carbon and oxygen have what amounts to very different electronegativities. This creates a strong dipole, with oxygen being highly electronegative and carbon being relatively positive, giving the overall structure a high dipole moment 47 . Table 2 Electronic and thermal properties of Schiff base PEI CA/N-GQDs for both phenol-imine and keto-amine forms at T = 298.15K in gas phase and different solvents. Gas Phase (ε = 1) Ethanol (ε = 24,55) Water ( ε = 78,39) Phenol-Imine E total (Hartree) -4215.309 -4215.411 -4215.416 Dipole (Debye) 8.206 15.748 15.847 Polarizability (a.u.) 617.610 574.263 580.673 E thermal (eV) 1004.410 1004.378 1004.302 Heat Capacity (cal/mol.K) 352.568 349.774 349.816 Entropy (cal/mol.K) 528.398 498.373 498.456 Keto-Amine E total (Hartree) -4215.352 -4215.435 -4215.440 Dipole (Debye) 18.328 28.097 28.834 Polarizability (a.u.) 667.723 927.427 944.435 E thermal (eV) 1006.020 1005.308 1005.289 Heat Capacity (cal/mol.K) 348.891 348.097 348.057 Entropy (cal/mol.K) 502.236 502.253 501.586 In the phenol form, the hydroxyl group carries a stabilizing "O-H" interaction that as a group is not as much dipole-dipole oriented as the keto group. Because the structure as a whole "phenol" carries resonance indicating that the electron cloud is also partial more uniformly distributed, the dipole moment is lower. Conversely, in the keto form, the structure is more polar. The reason for the higher thermal energy and smaller heat capacity of the keto-amine tautomer compared to the phenol form is as follows: The keto form usually contains double bonds (C = O); these bonds are stronger and shorter, which means lower potential energy. Less flexible bonds, on the other hand, result in higher thermal energy and vibrational levels 48 . There are more vibrational modes in the phenol form due to its more flexible C = C bond and OH group. The phenol form can withstand higher temperatures because it has more vibrational modes, which allow the molecule to store more energy 49 . Because the keto-amine tautomer usually contains a carbonyl group (C = O), it has less entropy than the phenol-imine tautomer due to a lower degree of disorder. Essentially, at room temperature and in the gas phase, the optimized keto-amine and phenol-imine tautomers exhibit different and intriguing thermal behaviors, each with specific appeals and properties. This yields more ordered structures with more powerful hydrogen bonding or dipole interactions. In both water- and ethanol-based solutions, the energetic differences between the two forms are much smaller than in the gas phase. This is due to the combinations of hydrogen bonds that solvent molecules form with the solute, which lead to stabilizations of both forms that are on the same order of magnitude (in other words, both forms are solvated comparably well). Hydroxyl groups in both water and ethanol can also form hydrogen bonds with the nitrogen atom of the imine and with the phenolic oxygen atom, which equally stabilizes both the phenol-imine and keto-amine forms. Thus, the actual distinct energetic environment around each solute can dampen the differences in solvation energies, leading to minimal distinctions in the highs and lows of the energetic landscape for each form between the two solvent types. One advantage of the gas phase is that the free movement of molecules leads to ignoring external factors, thus obtaining a better understanding of the intrinsic parameters and reactions. It is also applied to investigate the basic scales of energies. Thus, this phase helps to make the observation of the fundamental energy level of molecular structures easier due to less intermolecular interaction and free movements. It is because the material was evaluated experimentally in aqueous media that the quantum chemical calculations were also run in the relevant water solvent, so that they will be compared reliably with the experimental data. The energy level in the gas phase is a critical index of structural stability. As shown in Figs. 7 (a) and (b) , the Schiff base PEI-CA/N-GQDs took extremely low energy in the gas phase (-4215.327 and − 4215.352 Hartree respectively) for both keto-amine and phenol-imine forms. The optimized structure proved the strong stability of this material, which means in some cases this could be the most stabler formed structure if it were possible. Their performance in real applications depend on the stability. It guaranties that material could operative robustly and reliably in various environment. The Schiff base PEI-CA/N-GQDs formed a much lower energy level than the material, the PEI N-GQDs (shown in our previous work 27 ). The Schiff base PEI-CA/N-GQDs material showed a great promise in drug development by enhancing the binding affinity. Theoretically calculating FTIR spectra are relevant: predictive power, structural elucidation, complement to experiments for interpretation purposes, functional group analysis; help in understanding vibrational modes and; method development/validation. In the next step, we generated an FTIR spectrum to explore molecular vibrations in more detail. The spectroscopic technique used to get an insight into the structural arrangement, functional group, and chemical nature of the molecule was very effective. For the two tautomers, we found that the vibrational shifts of PEI N-GQDs 27 original material vibration (FT-IR spectrum) are a little bit less compared to our theoretical analysis for Schiff base PEI-CA/N-GQD vibrations; When analyzing the FTIR spectrum ( Figure S2 ), the frequency of maximum absorption is represented by the largest peak. Moreover, the experimental spectra are compared with vibrational frequencies predicted by DFT to identify which tautomer is present according to their characteristic peaks. It was corroborated by an FTIR analysis, identifying certain peaks of the phenol-imine tautomer present at 3695 cm − 1 (O-H stretch) and 1651 cm − 1 (C = N stretch). The keto-amine tautomer type showed absorptions at 3557 cm − 1 (N-H stretch) and 1596 cm − 1 (C = O stretch). It indicated that the keto-amine tautomer was prevalent as in the aryl region, its values exhibited a pronounced singlet. It is reasonable to assume that a major peak at 2697 cm − 1 indicated the formation of hydrogen bonding between cinnamaldehyde with the COOH group and acting together as an acceptor OCH₃·(··O)H. They are both acceptors and donors of hydrogen bonds, especially in the case of the carboxyl group with a strong ability to form H-bonds because it is able to donate one through OH but also can accept up to two due to its properties as carbonyl oxygen. Both the phenol-imine and keto-amine tautomers have unique FTIR absorbances but are in equilibrium so can interconvert. The most representative absorbance bands of the phenol-imine form were observed to be OH, NH, and NH 2 stretching (3714–3323 cm − 1 ), CH, CH 2 , and CH 3 stretching (3300–2931 cm − 1 ); C = N stretching band at 1632 cm − 1 in addition to a strong sharp peak for the –C = C— aromatic ring vibration mode around 1579 cm − 1 ; finally more peaks appeared corresponding COOH group with (1668 cm − 1 ). On the other hand, the keto-amine form showed absorbance bands at 3718–3396 cm − 1 (OH, NH, and NH 2 ), 3321 − 2929 cm − 1 (CH, CH 2 , and CH 3 ), 1639 cm − 1 (C = N), 1581 cm − 1 (C = C), and 1658 cm − 1 (COOH). FMOs are made up of two very important parts: HOMO and LUMO. Normally, the HOMO (indicated by a blue line in Fig. 8 (a) ) is full of electrons with the highest energy and behaves like an electron donor. On the other hand, LUMO, illustrated with red lines, is empty and possesses the lowest energy level, working as an electron acceptor 50 . The energy gap (∆E) is a key factor in the reactivity of molecules. Conversely, the smaller Δ E that generally tends to correlate with higher chemical reactivity is beneficial when targeting specific biological molecules or pathways in cancer cells during drug development. Such a strategy allows us to conclude the likelihood for a molecule to undergo chemical reactions, under the assumption that elements of electron transfer mechanisms can be translated into an improved view of its overall stability. Especially in chemical reactions, those electrons are initially taken from the HOMO and moved to the LUMO of reacting materials 27 . In Fig. 8 (a) , for Schiff base PEI-CA/N-GQDs phenol-imine and keto-amine, we see red and green lobes in the HOMO level by the same phase sense inside the housing of PEI, indicating a more electrophilic nature. This indicates that PEI can easily donate electrons to other parts. In contrast, the red and green lobes in the N-GQDs structure LUMO show its electron-accepting capability, which means that N-GQDs may be an acceptor. Simply, nucleophilic attacks involve the donation of an electron pair from a nucleophile (rich in electrons) to any electrophile deficient. This means the HOMO is displayed in red, denoting positive values, and this corresponds to positive exchange regions, which show areas with fewer electrons. Electrophiles, on the other hand, are drawn to areas with more electrons, such as the negative portions of LUMO, which appear green. On the other hand, electrophiles are attracted to regions that have extra electrons, such as the negative parts of LUMO like green. As a result, an electrophile hitting a molecule will most likely hit the green lobes of its LUMO, and nucleophilic attacks have been associated with red HOMOs. The energy level difference between LUMO and HOMO in Fig. 8 (a) gives information about the properties of antioxidant activity. A smaller band gap means better antioxidant capabilities of Schiff base PEI-CA/N-GQDs phenol-imine form. This is because the compound became more reactive in electron transfer reactions, which are important for it to function as an antioxidant 51 . The increased reactivity due to the smaller band gap makes this form more effective in stabilizing reactive oxygen species (ROS). It has superior antioxidant activity in comparison to PEI N-GQDs, as reported in Ref. 27 . The MEP approach assesses a susceptibility of a molecule to electrophilic (electron-attracting) and nucleophilic (electron-donating) reactions by examining the distribution of electric charge. MEP surfaces visualize these regions with color gradients: red for areas with negative electrostatic potential (electrophilic) and blue for areas with positive potential (nucleophilic), ranging from − 0.02 to 0.02 atomic units, as shown in Fig. 8 b. In electrophilic areas (red), the molecule has a high electron density that renders these spaces attractive to electron-seeking species. In nucleophilic regions (blue), low electron density is found, which is why these places attract more electron-rich species 36 , 37 . This method is of great assistance in identifying the reactive sites within the molecule and is always applied for studying the antioxidant activity 52 , 53 . For antioxidants, MEP analysis reveals electron-rich regions (negative potential) that can donate electrons to neutralize free radicals, thus protecting cells from oxidative damage. The ability of an antioxidant to serve as a reducing agent is because it can donate one electron instead of electrons or other protons and provide high negative potential regions (-OH) (or -NH2 groups). On the other hand, positive regions (blue) might interact with opposite sides of electron-deficient partners to halt deleterious oxidative reactions 54 – 56 . Nucleophilic activity in the phenol-imine form can be seen from a high electron density localized around both OH and C = N groups. Keto-amine form has a high electron density around the carbonyl (C = O) group. This contributes to predicting antioxidant reactivity and stability, showing how tautomers behave in different chemical environments through the understanding of these electron distribution patterns. For the phenolic part, this was high electron density areas near hydroxyl oxygen atoms and imine nitrogen of both phenol-imines. For the keto-amine bond, the electron pair is found mostly between the carbon and oxygen atom of the carbonyl group, which in turn pulls high amounts of electron density onto an even higher portion of space surrounding its own orbital. Lower electron density is shown in blue colors on the hydrogen atoms, especially those associated with nitrogen. This means that in the case of the phenol-imine tautomer, the negative charge is disproportionately held on to at these particular donor sites (the O and N), making them even more prone to nucleophilic attack than under ordinary circumstances. The keto-amine tautomer possesses greater electron density in the vicinity of its carbonyl oxygen, rendering that region more nucleophilic 57 – 59 . These surfaces were employed to explain the reactivity and stability with regard to antioxidants, thereby revealing the behavior of addition-elimination tautomers in different chemical surroundings. The UV-Vis spectra of the material in aqueous media were thoroughly analyzed theoretically. This comprehensive analysis examined a number of spectroscopic characteristics associated with electronic transitions, including absorption wavelengths, excitation energies, and important transitions 60 . These aspects were meticulously examined and documented, including the highest oscillator strength, which was meticulously tabulated in Table 3 . Table 3. Optical properties of Schiff base PEI CIN N-GQDs for both phenol-imine and keto-amine forms in water media. According to the analysis in Table 3 , the Schiff base PEI-CA/N-GQDs in its phenol-imine form shows a prominent contribution at 467 nm and three additional significant contributions at 408, 321, and 273 nm. These correspond to excitation energies of 2.65, 3.04, 3.86, and 4.55 eV, respectively. Specifically, the transition at 467 nm has a 69% contribution from H-6 to L + 1. At 408 nm, the transitions include 31% from H-11 to L + 1, 11% from H-6 to L + 2, and 27% from H-1 to L + 4. For the 321 nm transition, there is an 11% contribution each from H-14 to L + 3 and H-13 to L + 3, and 73% from H-10 to L + 3. The contributions at 273 nm are as follows: 23% from H-28 to L + 1, 22% from H-22 to L + 1, and 22% from H-17 to L + 4. Similar to this, the keto-amine form of the PEI-CA/N-GQDs Schiff base exhibits notable contributions at 483 nm, 423 nm, 408 nm, 338 nm, and one at 262 nm, which correspond to excitation energies of 2.57, 2.93, 3.67, and 4.73 eV, respectively. Specifically, the 483 nm transition involves 53% from H-11 to LUMO and 26% from H-5 to L + 2. At 423 nm, transitions include 14% from H-16 to L + 1, 23% from H-13 to L + 1, 15% from H-7 to L + 1, and 41% from H-3 to L + 1. For the 338 nm transition, contributions are 44% from H-20 to LUMO and 23% from H-17 to L + 2. The 262 nm transition involves a 47% contribution from H-26 to LUMO. As can be seen from Table 3 , the absorption wavelengths with characteristic peaks indicating electronic transitions in the UV-Vis analysis of the keto amine form are more in agreement with the experimental results compared to the phenol imine form. This supports that the material is in keto-amine form. Antioxidant activity The antioxidant activity of Schiff base PEI-CA/N-GQDs is shown in Figure S3 as the average of three experiments. As observed in Figure S3 , Schiff base PEI-CA/N-GQDs exhibited higher activity compared to stable BHT. The antioxidant activity of Schiff base PEI-CA/N-GQDs was found to be 42%, 51%, 65%, 78%, and 79%. The activity increased up to a material concentration of 100 ppm and then remained stable at 200 ppm with no significant difference. Notably, it demonstrated greater activity than the standard BHT, which is likely attributable to the Schiff base PEI-CA bond (C = N). The high antioxidant activity of Schiff base PEI-CA/N-GQDs can be attributed to the presence of OH and COOH groups in N-GQDs, as well as the coniferaldehyde and the imine bond. DNA-Binding and DNA Cleavage Absorption spectra of nanomaterial, Schiff base PEI-CA/N-GQDs in the absence and presence of CT-DNA are shown in Fig. 9 . As can be seen from Fig. 9 , with increasing CT-DNA concentrations, 4–79% hyperchromicity at 248 nm and 4–9% hyperchromicity at 348 nm is observed in the material. So, Schiff base PEI-CA/N-GQDs interacts with DNA in an electrostatic mode. Probably in this interaction the hydroxyl proton in the investigated material is taken up by the negatively charged phosphate groups in the DNA helix and part of the charge of the DNA is neutralized. The charge density of the DNA is thus disturbed, and as the proton leaves the material, the materials become negatively charged and exert an electrostatic repulsive force on the DNA helix, accelerating the degradation of the DNA 61 . Both hydrolytic and oxidative DNA breakage were investigated in this work. Figure 10 displays the outcomes of the two cleavage tests. When exposed with increasing concentrations of nanocomposite (from 50 ppm to 800 ppm), Lanes 1–6 in Fig. 10 demonstrate hydrolytic breakage of DNA. DNA chain breakage are caused by the material, as evidenced by the emergence of new bands and the primary band's (Form I) decline in intensity. A concentration-dependent interaction is shown by the fact that these effects intensify as concentration rises. DNA + H 2 O 2 oxidative stress conditions are used in Lanes 7–12 to assess the nanocomposite's impact on DNA. It is consistent with enhanced DNA degradation that greater breakage in the presence of oxidative agents (especially in lanes 7 to 12) may be due to both direct chemical interactions with DNA and the nanocomposite producing ROS. Prior research has demonstrated the ability of graphene-based nanomaterials to interact with DNA via intercalation, electrostatic forces, and π-π interactions 62 . This particular Schiff base mechanism offers a more nuanced perspective, indicating that this material may also promote targeted DNA cleavage in an oxidative environment. This is in line with the characteristics of Schiff base PEI-CA/N-GQDs that have been shown in other settings, including drug delivery systems and biosensors. Determining the safety of these nanocomposites in biological applications requires an understanding of the circumstances behind DNA damage. The DNA damage that is discovered to be concentration-dependent emphasizes the necessity of cautious dosing and delivery methods in order to reduce the risk of genotoxicity. The specificity of DNA interaction sites should be investigated further, and any potential off-target effects should be evaluated. Furthermore, examining these nanocomposites' biocompatibility and in vivo degradation behavior might offer vital information for therapeutic uses. Effect of Schiff base PEI-CA/N-GQDs on cellular viability The MTT test, also known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, is a laboratory test that is used in various fields, including drug research, toxicity evaluations, and cell culture studies, to determine cell viability and metabolic activity 63 . We also used the MTT assay in our investigation to examine the antiproliferative effect of the Schiff base PEI-CA/N-GQDs on human neuroblastoma cells, SH-SY5Y. The test results indicated that for 24 and 48 hours, Schiff base PEI-CA/N-GQDs demonstrated cytotoxicity within the measured dosage range. The most cytotoxic effect on SH-SY5Y cells was demonstrated by Schiff-based PEI-CA/N-GQDs applied at a concentration of 1.25 µg/µL for 48 hours, as shown in Fig. 11 . For both 24 and 48 hours, the IC 50 values of Schiff base PEI-CA/N-GQDs were determined to be 0.1407 µg/µL and 0.0257 µg/µL, respectively. The anticancer properties of GQDs are well-established 28 , 29 , but these compounds also exhibit cytotoxic effects on normal cells. Consequently, scientists sought to create compounds that specifically target cancer cells while exhibiting reduced cytotoxicity on normal cells. This was achieved by creating N-GQDs 64 . In this regard, Senel et al.'s 2019 work demonstrated that N-GQDs had antioxidant and antibacterial qualities in addition to their capacity to attach to DNA and maintain cell viability. According to the study, N-GQDs can bind to DNA by electrostatic and intercalation mechanisms; formulations containing GQDs can penetrate cells and lower the number and amount of lung cancer cells that express ephrin type-A receptor 2 (EpHA2); and high concentrations of quantum dots can cause DNA damage in cancer cells, thereby lowering the viability of those cells 36 . The cytotoxic, apoptotic, and cell cycle arrest effects of PEI N-GQDs on ovarian cancer cell lines were examined for the first time in our earlier work 27 . Using the same serial dilution approach, we were able to demonstrate that in the cell culture and viability test, PEI N-GQDs killed 50% of the cells at a concentration of 16.52 ± 1.69 µg/µL. The dosage rate that killed half of these cells dropped to 13.76 ± 4.48 µg/µL and the cytotoxic impact enhanced when palladium nanoparticles (PdNPs) were added to PEI N-GQDs. In this work, we demonstrated that the synthesized Schiff based PEI-CA/N-GQDs can bind to DNA through an electrostatic effect by displaying an exonuclease activity, which is consistent with the findings of Senel et al. 36 . At a treatment concentration of 0.0257 µg/µL, Schiff-base PEI-CA/N-GQDs demonstrated the capability to eliminate 50% of the neuroblastoma cells, as indicated by the findings from the cytotoxicity part of our study. This results indicate that the cytotoxic impact of Schiff base PEI-CA/N-GQDs on cancer cells is approximately 643 times larger than that of PEI N-GQDs, despite the fact that we attempted a different cancer cell line in our prior work. The Schiff base and CA in the created nanoparticle may be the cause of this discrepancy. Apoptotic Effects of Schiff base PEI-CA/N-GQD on SH-SY5Y A substance or chemical that may precisely stop the proliferation of cancer cells by changing the aberrant signaling pathway involved in the cell cycle and/or apoptotic mechanism is usually regarded as a crucial chemotherapeutic tool in the field of anticancer research. Therefore, causing cancer cells to undergo apoptosis is one of the primary objectives of chemotherapy 65 . In this work, we used three different methods to assess the apoptotic effects of newly manufactured Schif base PEI-CA/N-GQDs on the SH-SY5Y cell line. Using flow cytometry, we first demonstrated whether Schiff base PEI-CA/N-GQDs increased intracellular ROS. Furthermore, we used flow cytometry to demonstrate the apoptotic effects of the material, and immunohistochemistry labeling allowed us to identify the variations in protein expression between apoptotic and antiapoptotic. The effect of Schiff base PEI-CA/N-GQDs treatment on ROS generation in SH-SY5Y cells is depicted in Fig. 12 . As a result, ROS generation increased dramatically to 52.3% (p < 0.001) in cells treated with Schiff base PEI-CA/N-GQDs compared to 41.4% in untreated cells (control group). The findings of the flowcytometric analysis showed that, in comparison to the cells in the untreated control group, the viability rate of SH-SY5Y cells treated with Schiff base PEI-CA/N-GQDs dropped significantly to an average of 65.5% ( p < 0.05). Total apoptosis in treated cells increased significantly by 108.5-fold to 21.7%, while the number of necrotic cells increased significantly by 12.8%. When cells treated with Schiff base PEI-CA/N-GQDs, the average proportion of cells undergoing early and late apoptosis was 12.2% and 9.5%, respectively ( p < 0.05) (Fig. 13 ). Apoptotic protein CASPASE-3 and antiapoptotic protein BCl-2 immunohistochemistry expression differences in SH-SY5Y cells treated and untreated with produced Schiff base PEI-CA/N-GQDs are presented in Fig. 14 . Consequently, it is seen that CASPASE-3 expression is negative in untreated SH-SY5Y cells (Figs. 14 A and 14 B), but CASPASE-3 protein is expressed with a diffuse staining and a staining intensity of + 3 in treated cells (Figs. 14 C and 14 D). However, it was observed that Bcl-2 protein was expressed at 10% prevalence and + 1 staining intensity in untreated cells (Figs. 14 E and 14 F), while BCL-2 protein expression was inhibited and showed negative expression in treated cells (Figs. 14 G and 14 H). According to Ou et al. (2017), exposure to graphene increased the amount of ROS that mitochondria produced inside the cell. As a result, following a sequence of metabolic processes, proapoptotic molecules were released from mitochondria into the cytoplasm, causing cells to undergo apoptosis 66 . Furthermore, it was demonstrated by Ramachandran et al. (2022) that the treatment of human breast cancer cells with N-GQDs/titanium dioxide nanocomposites (N-GQDs/TiO2) increased ROS and resulted in mitochondria-associated apoptotic cell death 31 . Parallel to these two studies that demonstrate that GQDs and N-GQDs raise ROS in individual cells, we have demonstrated in this work that Schiff base PEI-CA/N-GQDs raise ROS in human neuroblastoma SH-SY5Y cells, causing the cells to undergo apoptosis. According to Qin et al. (2015), activating the apoptotic proteins in the CASPASE family causes the apoptotic process to be initiated in macrophages treated with GQDs via a mitochondria-associated mechanism 67 . In this work, we demonstrated that the upregulation of ROS in cells triggers the activation of the CASPASE-3 protein, which in turn drives the cell towards death by inhibiting BCL-2. Furthermore, the overall apoptosis rate in cells treated with PEI NGQDs rose by 45.8% compared to untreated cells in our prior work, where the apoptotic impact of PEI NGQDs in ovarian cancer was first demonstrated 27 . In this study, compared to the control, the number of cells treated with Schiff base PEI-CA/N-GQDs increased by 21.7%. While both studies demonstrated statistically significant increases, we believe this is mostly because the different cancer cells employed in the research had distinct metabolic features. In addition, the excellent antioxidant activity of Schiff base PEI-CA/N-GQDs was demonstrated in this investigation. We revealed that the CA itself and the imine bond that NGQDs have with each other are the sources of the nanoparticle's strong antioxidant capability. Due to the increased antioxidant activities of Schiff base PEI-CA/N-GQDs compared to PEI N-GQDs, we believe that the apoptosis rates in our prior investigation were higher than the results in this study. Cell cycle arrest effect of Schiff base PEI-CA/N-GQDs in SH-SY5Y cell Cyclin-dependent kinases (CDKs) and Cyclin-dependent kinase inhibitors (CKIs) are enzymes that control the cell cycle in living cells. By limiting the number of healthy cells that can pass through the G0/G1, S, and G2/M phases of the cell cycle, these enzymes control the proliferation of cells. During this process, injured cells are either destined to die by apoptosis or undergo irreversible damage repair. Uncontrolled cell proliferation results from a breakdown in this system; uncontrolled growth is a well-known characteristic that sets cancer apart from other diseases 68 . In order to get more insight into the cytotoxicity caused by Schiff base PEI-CA/N-GQDs, the distribution of cell cycle stages in SH-SY5Y was assessed following a 48-hour incubation period with 0.0257 µg/µL Schiff base PEI-CA/N-GQDs. Figure 15 shows that following a 48-hour treatment with Schiff base PEI-CA/N-GQDs, the cell population was much more concentrated in the G1 phase (41.2%) ( p < 0,001) as compared to the untreated cells (13.6%), and the G2 population also decreased concurrently ( p < 0,01). Following a 48-hour treatment with Schiff base PEI-CA/N-GQDs, 56.1% of SH-SY5Y cells were found to be in the G2 phase, compared to 82.1% of control cells. Additionally, there was no statistically significant variation in S phase across the groups. The results demonstrate that Schiff base PEI-CA/N-GQDs stopped the cell cycle in the G1 phase in SH-SY5Y, which inhibited cell division and caused apoptosis. Tian et al. 69 claim that GQDs are genotoxic substances. The cell responds to the genotoxicity induced by GQDs by undergoing DNA damage. In reaction to DNA damage, the cell undergoes this response, which involves stopping the cell cycle and giving the damaged DNA adequate time to heal. According to Ku et al. 70 , GQDs caused apoptosis in breast cancer and stopped the cell cycle at the G2/M checkpoint. In our earlier work 27 , we looked at the impact of PEI NGQDs on ovarian cancer cells' ability to arrest the cell cycle. The results indicated that, in comparison to untreated (control group) cells, cells treated with PEI N-GQDs were arrested in the sub-G0/G1 phase of the cell cycle. In this work, we used DNA-Binding and DNA-Cleavage assays to elucidate the connection between Schiff base PEI-CA/N-GQDs and DNA. As a result, we demonstrated that Schiff base PEI-CA/N-GQDs can bind to DNA via an electrostatic mode interaction. This binding can also result in both hydrolytic and oxidative cleavage, with oxidative cleavage being more potent and occurring at a lower dose than hydrolytic cleavage. When chromosomes are damaged or destroyed, G1/S and G2/M checkpoints are triggered in response to DNA damage to halt cell division. Currently, damaged DNA may be repaired by cells thanks to DNA damage checkpoints. A broken DNA molecule has the potential to interrupt a cell's growth or perhaps initiate its demise. Damage to DNA triggers the cell death process. While the G2/M checkpoint stops cells from proliferating with damaged DNA, the G1/S checkpoint stops cells from copying damaged DNA 71 . This work has demonstrated that Schiff base PEI-CA/N-GQDs, which oxidatively and hydrolytically cleave DNA and attach to DNA in an electrostatic fashion, halt cell cycle at the G1/S checkpoint and induce apoptotic cell death through the CASPASE pathway. Conclusion The Schiff base PEI-CA/N-GQDs nanomaterial novel developed in this study has shown a strong potential for anticancer activity, mainly against human SH-SY5Y neuroblastoma cells. This shows the efficacy of the material to induce oxidative stress, DNA cleavage, and apoptosis, as well as cell cycle arrest at an early phase named G1/S, making this a promising tool for future therapies. Upon extreme experimental and theoretical characterization, the structure was found to be primarily in a keto-amine form dominated by very good electron transfer properties. The nanomaterial was responsible for both cytotoxic effects, mainly linked to its electrostatic binding with DNA and ROS generation, but it also serves as an apoptotic compound through CASPASE-3 activation. Enhanced antioxidant properties of Schiff base PEI-CA/N-GQDs in comparison to the standard antioxidants also confirm its medicinal potential. These conclusions indicated that the combination of PEI-CA/N-GQDs may be promising as a cancer therapy in the treatment of neuroblastoma and suggest a possible pathway for how nanomaterial can work at the cellular level. MATERIALS AND METHODS Materials FT-IR spectra were measured with a Perkin Elmer BX II spectrometer with KBr discs and UV-Visible spectra were measured in water with a Varioskan Flash Multimode spectrometer. Morphology of material were characterized using Park System Atomic Force Microscope (AFM) XE100. Chemical composition of the samples were determined by Hitachi SU5000 FE-SEM instrument. Thermogravimetric analyses were performed on a Hitachi STA 7300 simultaneous TG/DTA Instrument. The sample (10 mg) were placed in aluminium crucibles and heated between 25°C and 900°C to determine the thermal stability. The experiments were carried out in nitrogen at different heating rates of 5 0 C min − 1 . X-ray diffraction (XRD) patterns were obtained through a Bruker D8 Advance X-ray diffractometer using Cu Kα radiation (λ = 1.54051 Å) in the 2θ range of 20–90° with a step size of 0.02 at 2 min – 1 steps, operating at an accelerating voltage of 40 kV and a current of 40 mA. Electrochemical measurements were taken with the C4 Cell Stand experimental model. Glassy carbon electrode was used as the working electrode, and MW 1032 electrodes were used as the counter electrode. All of the chemicals were obtained commercially from Sigma-Aldrich and used without further purification, including citric acid (CA), silver nitrate (AgNO3), polyethyleneimine (PEI) (average Mw ~ 25,000 by LS, average Mn ~ 10,000 by GPC, branched), 4-hydroxy-3-methoxycinnamaldehyde (coniferaldehyde), ethidium bromide (EB), calf thymus DNA (CT-DNA), pBR322 DNA, 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), and ethylalcohol (EtOH). Synthesis of Shiff base PEI-CA/N-GQDs PEI N-GQDs 72 – 75 (7.02 g) were added to a 250 mL round bottom flask and 150 mL of EtOH was added. Then 50 mL alcohol solution of coniferaldehyde (CA) was added to this mixture and the mixture was boiled under reflux for 2 h to form Schiff base PEI-CA/N-GQDs. After the mixture was filtered and allowed to evaporate, the dark red solid was kept for later use in a vacuum desiccator (Scheme 1 ). Computational details This section presents an extensive investigation into the characteristics of Schiff base PEI-CA/N-GQDs, building upon our previous research 27 , 72 . The purpose of this work is to assess the electronic properties of the material in order to determine its sensitivity and selectivity. We accomplish this with the help of many calculations, among which are the geometry optimizations. These optimizations provide a very good approximation of the actual arrangement and dimensions of atoms in a given material and the nature of their atomic-scale bonding, including bond lengths, angles, and the types of bonds they form. After the structures are optimized, we can then carry out a complete exploration of their electronic structures and use the results to perform a deep-dive analysis of what their electronic properties really are 76 . At first glance, Scheme 2 offers a clearer, more detailed view of the theoretical structure of the Schiff base PEI-CA/N-GQDs material. Both the phenol-imine and keto-amine forms are shown in Scheme 2 (a) and 2(b) , respectively. Both forms are presented in a way that makes the tautomeric relationship between them visually accessible, with the hydrogen involved in the tautomerization highlighted in green. In order to gain a thorough understanding of the molecular dynamics involved in this tautomerization, DFT method is frequently used to optimize geometries, calculate energies, analyze vibrational frequencies, thermodynamic properties and take solvent effects into account. To determine the most stable structural configurations of this theoretical model, DFT/B3LYP optimization was performed using the Gaussian 09W software package 77 with the Lanl2dz basis set 78 , 79 , which is known to be useful for the functionalized graphene quantum dot calculations 27 , 72 . The structures were visualized with GaussView 5.0 software 80 , which aided the calculations of the structural and electronic properties that were critical. We also recorded and analyzed Fourier Transform Infrared (FTIR) spectra, which provided information on molecular vibrations and helped infer the structure and chemical functionality of the nanomaterials. Additionally, important characteristics like FMOs, MEP, and the HOMO-LUMO energy gap were determined using the LanL2DZ basis set. Also, calculations of the UV-Vis spectra were done using Time-Dependent Density Functional Theory (TD-DFT) 81 , with a single consistent basis set used throughout the calculations 82 , 83 . Antioxidant activity The antioxidant properties of the material were evaluated using the DPPH method [54] as described in reference [53], with butylated hydroxytoluene (BHT) serving as the standard antioxidant according to reference [55]. Methanol solutions with DPPH (20 mg/mL) and PEI-CA/N-GQDs Schiff base at different concentrations (20–200 ppm) were utilized to achieve this. The Schiff base PEI-CA/N-GQDs was mixed with a DPPH solution and left to remain in the dark for 30 minutes. The absorption level was determined at 517 nm. The scavenging impact was assessed as the proportion of radical reduction. DNA-Binding and DNA-Cleavage UV-Vis titration in room-temperature Tris-HCl/NaCl buffer was used to investigate the binding affinity of the Schiff base PEI-CA/N-GQDs with CT-DNA, in accordance with the protocol outlined in reference 84 . An aqueous solution of the Schiff base PEI-CA/N-GQDs (100 ppm) was utilized in this experiment. The Schiff base PEI-CA/N-GQDs -DNA solutions were allowed to stand at room temperature for 5 min before each measurement. The effectiveness of the material for DNA cleavage was evaluated using gel electrophoresis 84 . The substrate in this analysis was supercoiled (SC) pBR322 DNA. We investigated the DNA cleavage activity of the Schiff base PEI-CA/N-GQDs under oxidative conditions (in the presence of H2O2) and hydrolytic conditions (in the absence of H 2 O 2 ). To initiate the oxidative cleavage activity, we combined pBR322 and the Schiff base PEI-CA/N-GQDs with 2 µL of H 2 O 2 , acting as an oxidizing agent. For three hours, we incubated pBR322 (0.1 µg/mL) in 10 µM Tris-HCl buffer (pH:7.2) at 37°C using the Schiff base PEI-CA/N-GQDs. Following incubation, the samples were treated with loading buffer, and we electrophoresed them for one hour at 60 V on a 1% agarose gel in TBE (Tris-Boric acid-EDTA, pH: 8.0). The bands were then captured on camera and made visible with UV light. Cell Culture Cell Culture: Dulbecco's modified Eagle's medium F12 (DMEM/F12, Gibco, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS, Gibco, CA, USA) containing 100 U/mL penicillin-streptomycin was used to culture the human-derived epithelial neuroblastoma (SH-SY5Y) cells (ATCC number: CRL-2266, Manassas, VA, USA). The cells were grown at 37°C with 5% CO2 in a humidified incubator. A combination of adherent and floating cells makes up the growth of SH-SY5Y cells. Consequently, the medium holding the floating cells was taken out, and centrifugation was used to save the cells. 1 to 2 mL of 0.25% trypsin solution was added to the adherent cells after they had been rinsed with 1x PBS solution. The flask was then left at 37°C until the cells detached. After adding and aspirating fresh media, floating cells that had been gathered above were mixed in and placed to fresh flasks 27 . Cell Viability Assay A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric test (Beyotime Biotech, Haimen, China) was used to measure the proliferation of SH-SY5Y cells. To put it briefly, these cells were suspended in 150 µL and planted at a density of 1×10 4 cells/well in 96-well plates. They were then kept overnight at 37°C in a CO2 incubator to facilitate adhesion. Different quantities of Schiff-base PEI-CA/N-GQDs (0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, and 1.25 µg/µL) were applied to SH-SY5Y cells. Following treatment for 24 and 48 hours, the medium was taken out of each well and MTT solution (0.5 mg/mL) from Sigma-Aldrich, Saint Louis, Missouri, USA was added. The solution was left for 4 hours at 37°C. Following the incubation period, 200 µL of isopropyl alcohol was added to each well and pipetted up and down many times to dissolve the purple formazan crystals. Each well's absorbance was then measured at 570 nm using a plate reader (BioTek, USA) 27 , 85 . Flow Cytometric Analysis of Intracellular ROS Generation In accordance with the manufacturer's instructions, the ROS-sensitive fluorescent dye dichloro-dihydrofluorescein diacetate (DCF-DA) probe was employed to measure ROS production. Subsequently, Schiff base PEI-CA/N-GQDs (0.0257 µg/µL) were applied to SH-SY5Y cells for 48 hours. Following this, the cells were suspended in PBS and incubated with DCF-DA (10 µM) for 30 minutes at 37°C. Following two PBS washes, the cells were stained with DCF fluorescence, recorded using a BD Accuri C6 Flow Cytometer at 485 and 535 nm for excitation and emission wavelengths. CellQuest software was then used to analyze the data 86 . Flow Cytometric Analysis of Apoptosis An Annexin V/7-aminoactinomycin (7-AAD) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to measure the apoptosis rate. In 6-well culture plates, 0.0257 µg/µL Schiff-base PEI-CA/N-GQDs were applied for 48 hours to SH-SY5Y cells. Following cell harvesting, an equivalent amount of PE-Annexin V was immunostained for 15 minutes, after which the cells were suspended in Annexin V binding buffer and cleaned with cold PBS. Tubes were incubated for 30 minutes in the dark with the addition of 7-AAD (5 µg/ml, Sigma-Aldrich) and binding buffer, prior to flow cytometric analysis. Tubes were incubated for 30 minutes in the dark with the addition of 7-AAD (5 µg/ml, Sigma-Aldrich) and binding buffer, prior to flow cytometric analysis. When cells were stained with both PE-Annexin V and 7-AAD, they were considered to be late apoptotic, while cells labeled with PE-Annexin V alone were considered to be early apoptotic. With the use of a flow cytometer (BD Accuri C6 Flow Cytometer, USA), the percentage of apoptotic cells was determined 27 , 85 . Cell Blocking and Immunohistochemical Staining Schiff-base PEI-CA/N-GQDs at a concentration of 0.0257 µg/µL were applied to human neuroblastoma cells (SH-SY5Y) and incubated for a duration of 48 hours. Following incubation, a cell pellet was obtained by centrifuging the collected cells and trypsinizing them. The cell pellet was embedded in paraffin, sectioned at 4 µm thickness, and fixed for 24 hours in 10% neutral buffered formalin 87 . Leica Bond-Max automated staining system was used for immunohistochemical staining under typical operating circumstances. The DAB-compatible Bond Polymer Refine Detection Kit (DS9800, Buffalo Grove, USA) was employed. Antibodies that were ready for usage were applied for Bcl-2 (clone bcl-2/100/D5 cod. PA0117, Leica Biosystems, UK) and CASPASE-3 (clone EP410 cod. AC-0364RUO, Cell Marque, USA). After deparaffinizing, rehydrating, and applying citrate buffer (pH 6.0) for antigen retrieval, slides were processed. The sections were incubated with the primary antibodies for a whole night at 4°C after non-specific binding was blocked with normal goat serum. DAB substrate was used for color development after the biotinylated secondary antibody and ABC reagent were incubated 87 . Figures S4 and S5 display internal positive control stainings of the Bcl-2 and CASPASE-3 proteins in lymphoid tissue. Apoptotic and anti-apoptotic marker expression was evaluated by microscopic inspection of the sections. Flow Cytometric Analysis of Cell Cycle The cells were treated for 48 hours with Schiff-base PEI-CA/N-GQDs (0.0257 µg/µL). Following this, they were harvested using 0.05% trypsin, twice rinsed with assay buffer, and fixed in 70% ethanol/PBS at 4°C for the whole night. After the ethanol was removed by centrifugation, the fixed cells were resuspended in PBS and stained for 30 minutes at 37°C in the dark using propidium iodide (PI) that included 20 µg/mL of RNase. The samples were examined on a flow cytometer (BD Bioscience, USA) using the CellQuest software to quantify the forward and side scatter in order to determine the cell cycle distribution 27 . Declarations Acknowledgement This work has been supported by Ankara University Scientific Research Projects Coordination Unit under grant number: FBA-2024-3174 . The numerical calculations reported in this paper were fully performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources). Declaration of competing interest The authors declare no conflict of interest. Author contributions statement Murat Kilic: conceptualization, data curation, formal analysis, investigation, visualization, writing – original draft, writing – review & editing; Buket Altinok Gunes: conceptualization, data curation, formal analysis, investigation, visualization, writing – original draft, writing – review & editing; Omer Faruk Kirlangic: conceptualization, data curation, formal analysis, investigation, visualization; Aysenur Aslan: investigation, resources, visualization; Didem Ketenoğlu: investigation, visualization, project administration, writing – original draft, writing – review & editing; Fatma Zeynep Kirlangic: formal analysis, investigation, resources, visualization; Pinar Mualla Elci : formal analysis, investigation, resources, visualization; Faruk Mert : formal analysis, investigation, resources, visualization; Neslihan Demir : formal analysis, investigation, resources, visualization, writing – original draft; Bahadir Boyacioglu: formal analysis, investigation, visualization, project administration, supervision, writing – original draft, writing – review & editing; Huseyin Unver: formal analysis, investigation, visualization, project administration, writing – review & editing; Ashok Chatterjee: supervision, writing – review & editing; Mustafa Yildiz: conceptualization, data curation, formal analysis, investigation, project administration, visualization,supervision, writing – original draft, writing – review & editing Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. 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base\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/c528070179478db8991d1848.png"},{"id":71650585,"identity":"a268ea85-1411-4fb4-b071-13162f63c313","added_by":"auto","created_at":"2024-12-17 12:10:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73599,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of Schiff base PEI-CA/N-GQDs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/f4348d37f841295398d2215c.png"},{"id":71650582,"identity":"fd90e18a-872c-41c4-a168-39dc89f2f9ab","added_by":"auto","created_at":"2024-12-17 12:10:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":93346,"visible":true,"origin":"","legend":"\u003cp\u003eEDX spectra of Schiff base PEI-CA/N-GQDs\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/28e691d49cbf75395e9541b9.png"},{"id":71651006,"identity":"203a6acf-9064-407c-a894-6b5f392348f8","added_by":"auto","created_at":"2024-12-17 12:18:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":411056,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SEM, (b) 2D and (c) 3D\u003c/p\u003e\n\u003cp\u003eAFM images of Schiff base PEI-CA/N-GQDs\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/206f286785a5912fc84d3b74.png"},{"id":71652290,"identity":"87e58320-11d0-440e-81f0-9e8c1f43fed8","added_by":"auto","created_at":"2024-12-17 12:26:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101640,"visible":true,"origin":"","legend":"\u003cp\u003eDTA-TG-DTG curves of Schiff base PEI-CA/N-GQDs.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/15b0ed64c3cd43ba10ea4b96.png"},{"id":71651007,"identity":"2749b5a5-af3c-4841-9180-58ac50f368a3","added_by":"auto","created_at":"2024-12-17 12:18:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57356,"visible":true,"origin":"","legend":"\u003cp\u003eCV voltammograms of Schiff base PEI-CA/N-GQDs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/f339fa6a7aebd62dee126166.png"},{"id":71650586,"identity":"6e05f3f8-d66e-456f-9647-d55f59e8c1e5","added_by":"auto","created_at":"2024-12-17 12:10:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":212813,"visible":true,"origin":"","legend":"\u003cp\u003eThe optimized molecular structures of Schiff base PEI CA/N-GQDs for both phenol-imine (a) and keto-amine (b) forms in the gas phase.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/f2928b85de8cf4ceaa2fea26.png"},{"id":71650578,"identity":"5bf425f5-aad8-4e65-9dd1-31db90fceaf9","added_by":"auto","created_at":"2024-12-17 12:10:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":250491,"visible":true,"origin":"","legend":"\u003cp\u003e(a) FMOs and (b) MEP isosurfaces of Schiff base PEI CA/N-GQDs for both phenol-imine and keto-amine forms.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/4fddd350bc0d40ef5dc06eb0.png"},{"id":71650584,"identity":"939cc92d-a7c1-4aa9-81bb-ca11737f3a26","added_by":"auto","created_at":"2024-12-17 12:10:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":87742,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption spectra of Schiff base PEI-CA/N-GQDs in the absence and presence of increasing-decreasing amounts of CT-DNA at room temperature in Tris–HCl/NaCl buffer (pH 7.2)\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/0ff51e9405947ba6a4d2bc5e.png"},{"id":71650576,"identity":"e8641ba5-524c-4a1b-b89f-1d294b45a939","added_by":"auto","created_at":"2024-12-17 12:10:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":117047,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis patterns for the hydrolytic and oxidative cleavage of pBR322 DNA by Schiff base PEI-CA/N-GQDs.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/dc755c6b9553d5acd77bbf12.png"},{"id":71650583,"identity":"2a813865-9678-49d6-ae6a-b2528d6e3904","added_by":"auto","created_at":"2024-12-17 12:10:03","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":67837,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effect of Schiff-based PEI-CA/N-GQDs for SH-SY5Y cells. The mean ± SD of the three separate experiments (n = 3) is presented in the results. In contrast to the control group, #p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/d2d1ef4bb151aa13e859e32e.png"},{"id":71650580,"identity":"8420b741-0e8d-4028-9228-a8f47ccfa832","added_by":"auto","created_at":"2024-12-17 12:10:03","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":175040,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of 48 h of Schiff base PEI-CA/N-GQDs treatment on SH-SY5Y cell ROS production by flow cytometric analysis. Mean fluorescence intensity was illustrated in the lower left panel. The data are shown as the mean ± SD. ***p \u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/0b60085632ccbeec76ea2f85.png"},{"id":71650588,"identity":"6e16b60d-2a98-4d87-9193-0289b703cbce","added_by":"auto","created_at":"2024-12-17 12:10:04","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":350053,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic cell death induced by Schiff base PEI-CA/N-GQDs via Annexin V/7-AAD double staining. SH-SY5Y cells were treated with 0.0257 μg/μL of Schiff base PEI-CA/N-GQDs for 48 h, stained with Annexin V and 7-AAD, then measured by flow cytometry. The percentage of viable, early, late and necrotic cells was shown in upper right panel. Four populations of cells were observed: viable cells, Annexin V negative and 7-AAD negative; early apoptotic cells, Annexin V positive and 7-AAD negative; late apoptotic cells, Annexin V positive and 7-AAD positive; necrotic cells, Annexin V negative and PI positive.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/6c5bd1e831854bb2d6ab6f60.png"},{"id":71650587,"identity":"2ea772cf-a010-48a0-958c-ae26d759701f","added_by":"auto","created_at":"2024-12-17 12:10:04","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":534689,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in CASPASE-3 and Bcl-2 expression in SH-SY5Y cells treated and untreated (control) with Schiff base PEI-CA/N-GQDs. A: Negative CASPASE-3 expression in control samples (100X); B: Negative CASPASE-3 expression in control samples (400X); C: Strong CASPASE-3 expression in treated cells (100X); D: Strong CASPASE-3 expression in treated cells (400X); E: Mild Bcl-2 expression in control samples (100X); F: Mild Bcl-2 expression in control samples (400X); G: Negative Bcl-2 expression in treated cells (100X); H: Negative Bcl-2 expression in treated cells (400X); I: Negative control staining for CASPASE-3 (without antibody) (400X); J: Negative control staining for Bcl-2 (without antibody) (400X).\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/72f90612a07721a448fe3946.png"},{"id":71650581,"identity":"8d6bd1b8-bef6-4d0e-8911-8583f6f3b887","added_by":"auto","created_at":"2024-12-17 12:10:03","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":248716,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Schiff base PEI-CA/N-GQDs Schiff base nanocomposites on the cell cycle distribution in SH-SY5Y cells. SH-SY5Y cells treated with Schiff base PEI-CA/N-GQDs for 48 h were stained with PI and subjected to flow cytometry analysis to assess the cell cycle distribution. Analysis of the percentage of cell number % of G1, S and G2 phases were shown in right panel. Data are mean ± SD. **p \u0026lt; 0.01, and ***p \u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/a6870a25d96a623cc58e6317.png"},{"id":82838912,"identity":"bcc831fd-e4d3-4792-8430-3b74487d5617","added_by":"auto","created_at":"2025-05-15 20:16:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4176333,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/90ca0782-500b-45af-a7a1-c27232fe8e41.pdf"},{"id":71650573,"identity":"9b46b91c-9672-4129-948c-877788e59b9a","added_by":"auto","created_at":"2024-12-17 12:10:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5682186,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationR1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/e964558c8169a7037e93602f.docx"},{"id":71651005,"identity":"21d1ba7c-c477-47c3-87be-c1b8e8179b04","added_by":"auto","created_at":"2024-12-17 12:18:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":172041,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme.docx","url":"https://assets-eu.researchsquare.com/files/rs-5352941/v1/751194fab259eec7348116e3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Quantum Chemical, Biological, and Experimental Analysis of Newly Synthesized Schiff-based PEI CA/N-GQDs Nanomaterials: Evaluation of Anticancer Potential in Human Neuroblastoma Cell","fulltext":[{"header":"Introductıon","content":"\u003cp\u003eCancer is a devastating illness and the leading cause of death, and given the rise in new occurrences of cancer and the number of cancer-related fatalities seen and recorded in almost every region of the world, it is clear that cancer will remain a major threat to human health \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. As it is known, although the treatment of cancer varies depending on the person and the type of cancer, it generally includes radiotherapy, chemotherapy, immunotherapy, surgical methods and at least two combinations of these. Among these, chemotherapy is the main and the most commonly used treatment method, but it is not successful in many tumor types and sometimes causes serious side effects \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For this reason, drug research in cancer treatment has turned to natural compounds, primarily of plant origin, which may be less harmful or perhaps harmless to healthy cells, but may cause more damage to cancer cells \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Researches conducted in this field show that natural compounds can be used in humans since they have high dose tolerance with less toxic effects \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and are effective in various molecular processes such as cellular proliferation, differentiation, apoptosis, metastasis, DNA damage and repair \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCinnamaldehyde, one of these natural molecules, is a flavonoid found in the bark/twig of the cinnamon plant, which gives its taste and aroma to the cinnamon plant and has a proven effect in preventing or reducing the severity of many diseases, including diabetes, atherosclerosis, inflammation and cardiovascular diseases \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, research highlights the antiproliferative properties of cinnamon that affect cancer cells, including those from leukemia \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, melanoma \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, breast cancer \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, colon cancer \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, ovarian cancer \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and hepatoma \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, by causing apoptosis and halting the cell cycle via a variety of molecular mechanisms \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEssentially similar to cinnamaldehyde when replaced with a hydroxy group at position 4 and a methoxy group at position 3 \u003csup\u003e20\u003c/sup\u003e, coniferaldehyde (CA), is a naturally occurring phenolic chemical and a member of the family known as cinnamaldehydes (4-Hydroxy-3-methoxycinnamaldehyde: (4H3MC)) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. A limited number of studies have focused on the cytotoxic and apoptotic effects of CA extracted from plant extracts for cancer treatment. Consediring this, CA derived from \u003cem\u003ePatrinia heterophylla\u003c/em\u003e Bunge extract has been shown to exhibit strong cytotoxic effects at low concentrations on cell lines representing melanoma, hepatocellular carcinoma, gastric cancer, cervical cancer, colon cancer, and breast cancer. Additionally, in the same study CA has been reported to cause melanoma cells to undergo significant rates of apoptosis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent advancements in nanotechnology have shown significant potential in mitigating the negative impacts associated with conventional chemotherapies, as well as enhancing the effectiveness of cancer treatment \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Due to their unique features, these nanosized drug delivery systems (DDS) including carbon-type materials as well as dendrimers and nanoparticles made up of polymers or lipids are among the first-line options for constructing straightforward platforms with tailored functionalities that enable targeted anticancer agent release by overcoming chemotherapeutic limitations in cancer. The DDSs are designed to reduce safety or act on specific active effects in cancerous cells/tissues compared to noncancer-treating free cytotoxic agents \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGraphene quantum dots (GQDs) are a constituent of QD materials derived from graphene sheets and showing both carbon dot-like properties and graphene features. They are recognized for their widespread use in many areas of technology research, including sensor systems, bio-imaging applications, diagnostics, drug carriers, gene delivery vectors, and energy storage devices \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Graphene-based nanocomposites have a cytoplasm\u0026ndash;nucleus shuttling system, making them effective drug delivery carriers for pharmacological applications \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, research on GQDs targeting their antiproliferative and anticancer properties indicates that these materials can internalize into cells and interact with DNA, RNA, and proteins, leading to cytotoxicity \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Concomitantly, intracellular ROS generated by mitochondria accumulate in graphene-treated cells. This increase in ROS levels then triggers a cascade of metabolic events from the mitochondria to the cytoplasm, ultimately causing the cell to undergo apoptosis and die \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe heteroatom doping can modify the properties of GQDs, thereby broadening their range of applications and facilitating their utilization. One specific type of this modification is represented by Nitrogen-Doped Graphene Quantum Dots (N-GQDs). In contrast to nitrogen-free GQDs, N-GQDs have a strong blue fluorescence and operate electrocatalytically in the oxygen reduction reaction (ORR) under alkaline conditions in a manner akin to the Pt/C catalyst \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In addition to being a unique combination of biotechnology and state-of-the-art materials science with excellent photoluminescence and biocompatibility, N-GQDs are also useful tools because of their unique properties that improve imaging and drug delivery in the context of cancer treatment and diagnosis \u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We assessed N-GQDs' capacity to attach to DNA, maintain cell viability, and possess antioxidant qualities in our research. As a result, our research demonstrated that N-GQDs can attach to DNA through electrostatic and intercalation routes, and that high quantum dot concentrations can damage cancer cell DNA and decrease the survival of ovarian and breast cancer cells \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSchiff bases, which are substances created by the condensation reaction between primary amines and aldehydes, are well-liked ligand precursors because of their adaptability and simplicity of production. Other donor functional groups like -Cl, -OH, -CH3, etc. are also present in these primary amines and aid in the regulation and enhancement of biological functions. These characteristics make schiff bases useful in pharmacological, therapeutic, biological, medicinal, and analytical fields \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. According to studies, schiff bases have analgesic, depressive, anticancer, and antimycobacterial properties \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA malignant tumor of the peripheral nervous system that develops in children, neuroblastoma is caused by neural crest sympathoadrenal progenitor cells. Since patients with advanced neuroblastoma have a dismal prognosis despite recent advancements in combination therapy, more potent, less cytotoxic therapies are required. It is crucial to create an efficient treatment plan as a result \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Although information is given in the literature about the anticancer activities of cinemaldehyde and its analogues obtained from plant extracts, studies on the anticancer activity of CA, a derivative of cinemaldehyde, especially in neuroblastoma, are very limited. In this study, Schiff base modified N-GQDs nanoparticles were prepared by reacting CA with PEI N-GQDs in ethanol medium. The structures of Schiff base PEI-CA/N-GQDs were experimentally and theoretically characterized. We have also used cyclic voltammetry (CV) to investigate the electrochemical properties of material. Then their biological activity and antiproliferative, apoptotic and cell cycle arresting properties in human epithelial neuroblastoma (SH-SY5Y) cells were investigated.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of Schiff base PEI-CA/N-GQDs (UV-Vis, FT-IR, XRD, SEM, AFM EDX and TGA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe absorption spectrum of Schiff base PEI-CA/N-GQDs was measured in water (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). As can be seen in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, in the UV-Vis spectrum, peaks assigned to the \u0026pi;-\u0026pi;* transitions of C\u0026thinsp;=\u0026thinsp;C at 268 nm and to the n-\u0026pi;* transitions of C\u0026thinsp;=\u0026thinsp;N (imine and pyridinic) and O-C\u0026thinsp;=\u0026thinsp;O (carboxylic acid) at 420 nm are observed. Surprisingly, another peak at 496 nm was observed in the spectrum. This peak is probably due to the phenol-imine/keto-amine tautomerization of the 4-hydroxy-cinnamaldehyde Schiff base \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. It is known that absorptions above 400 nm in 2-hydroxy-Schiff bases are due to the phenol-imine/keto-amine tautomerization \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This phenomenon is also observed in 2-hydroxy-Schiff bases. Many properties of 2-hydroxy-Schiff bases are explained by this phenomenon \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Notably, this research highlights the first observation of keto-amine tautomerism occurring in a 4-hydroxy-Schiff base.\u003c/p\u003e\n\u003cp\u003eAn extensive comparison of the characteristic group vibrations between the starting material, PEI N-GQDs, and its Schiff base counterpart, Schiff base PEI-CA/N-GQDs, was conducted using the FT-IR spectra, and is presented in \u003cstrong\u003eFigure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e. In this comparative analysis, the synthesized materials were examined, and their vibrational frequencies were determined. It also uncovered ways in which the newly synthesized materials differed from their original compositions. The spectral data contained subtle variations and elevations that were exclusive to the synthesized material, potentially indicating structural modifications resulting from the formation of Schiff bases. \u003cstrong\u003eFigure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e illustrates the graphic representation of these results, which provided a description of the spectroscopic signatures associated with the chemical changes during synthesis. This greatly enhanced our understanding of the structural properties and molecular makeup of these materials.\u003c/p\u003e\n\u003cp\u003ePEI N-GQDs with vibrational bands were detected at the following locations: 3831\u0026thinsp;\u0026minus;\u0026thinsp;3736 (OH), 3421\u0026thinsp;\u0026minus;\u0026thinsp;3270 (NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;N\u0026thinsp;+\u0026thinsp;COOH) and 2942\u0026thinsp;\u0026minus;\u0026thinsp;2848 (C-H), 1686 (COO), 1651(C\u0026thinsp;=\u0026thinsp;N), 1551(C\u0026thinsp;=\u0026thinsp;C) and 1452 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e (C-N\u0026thinsp;+\u0026thinsp;C-O) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The Schiff base PEI-CA/N-GQDs had bands at the same positions, 3852\u0026thinsp;\u0026minus;\u0026thinsp;3738 (OH), 3419\u0026thinsp;\u0026minus;\u0026thinsp;3228(NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NH\u0026thinsp;+\u0026thinsp;COOH\u0026thinsp;+\u0026thinsp;Ar-H), 2942\u0026thinsp;\u0026minus;\u0026thinsp;2848, 1732, 1651, 1546 and 1447 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e (\u003cstrong\u003eFigure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). Comparing the vibrations in the FT-IR spectra of Schiff base PEI-CA/N-GQDs with the spectrum of pure PEI N-GQDs, it can be seen that the vibrations of the OH and COO functional groups are shifted to higher frequencies, while the NH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NH\u0026thinsp;+\u0026thinsp;COOH, C\u0026thinsp;=\u0026thinsp;C, and C-N\u0026thinsp;+\u0026thinsp;C-O vibrations are shifted to lower frequencies. Surprisingly, in both materials, C-H and C\u0026thinsp;=\u0026thinsp;N vibrations were observed at the same frequency.\u003c/p\u003e\n\u003cp\u003eX-ray diffraction (XRD) pattern presents a broad peak at 20\u003csup\u003e0\u003c/sup\u003e to 30\u003csup\u003e0\u003c/sup\u003e of 2\u003cem\u003e\u0026theta;\u003c/em\u003e, characteristic of amorphous materials (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The broadness of the peak suggests poor long-range ordered nature, typical for carbon-based materials which could resemble unprocessed graphene oxide or N-GQDs \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The absence of sharp peaks and the gradual decrease in intensities beyond 30\u003csup\u003e0\u003c/sup\u003e indicate a low degree of crystallinity, which can be linked to the amorphous nature of this material. This behavior is characteristic of materials that have an amorphous structure, such as the Schiff base complexed with PEI-CA/N-GQDs. Further absence of sharp peaks supports the observation that synthesized Schiff base PEI-CA/N-GQDs are dominantly amorphous in nature with randomly oriented graphitic layers typical for N-GQDs.\u003c/p\u003e\n\u003cp\u003eThe elemental compositions and bonding configurations of Schiff base PEI-CA/N-GQDs were analyzed by energy dispersive X-ray spectroscopy (EDX). The amount of elements detected using EDX methods are given in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. According to the EDX spectrum, the detection of carbon (64.1 Wt%), oxygen (27.0 Wt%) and nitrogen (8.7 Wt%) on the surface of the samples confirms that the material has been successfully synthesized.\u003c/p\u003e\n\u003cp\u003eThe structure and morphology of Schiff base PEI-CA/N-GQDs were also confirmed by SEM images as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003e(a)\u003c/strong\u003e. As can be seen from Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003e(a)\u003c/strong\u003e, it is confirmed that Schiff base PEI-CA/N-GQDs retain the spherical shape morphology and do not form agglomerates even at 5 \u0026micro;m.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAFM results of Schiff base PEI-CA/N-GQDs\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"11\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRegion\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMin(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMax(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMid(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRpv(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRq(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRa(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRz(nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRsk\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRku\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAll\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-43.574\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.856\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe morphology of Schiff base PEI-CA/N-GQDs was also characterized by atomic force microscopy (AFM). A 2D view of sample surface topography in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003e(b)\u003c/strong\u003e reveals the height fluctuation range across a 5 \u0026micro;m \u0026times; 5 \u0026micro;m scanned area. The left hand side shows a colour scale for elevation changes, going from the highest blobs around 50 nm high through to the lowest round \u0026minus;\u0026thinsp;40 nm. AFM results of Schiff base PEI-CA/N-GQDs is also given in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The presence of a large number of height variations reflects surface roughness, and the Rpv (peak-to-valley) value for this is as high as 94.178 nm. The surface of the material is heterogeneous, showing brighter (higher) features mixed with a darker (lower) background. The bright areas represent the peaks or material clusters, while the dark regions indicate valleys or lower surfaces.\u003c/p\u003e\n\u003cp\u003eThe 3D image provides a profile of the surface in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003e(c)\u003c/strong\u003e. Some of the peaks are quite sharp and tall compared to neighboring areas, which corresponds with an Rz (ten-point average roughness) measurement of 87.766 nm. It has a medium texture with few smooth areas and many prominent features, some of which are sharp peaks; all of this contributes to the high kurtosis (Rku\u0026thinsp;=\u0026thinsp;5.856) characteristic of sharp sub-micron height distributions. These sharp peaks and deep valleys align with the negative skewness (Rsk = -1.136), meaning there are more valleys than peak areas. These multiple-height-layered-surface characteristics are obviously important for surface interactions in applications like coatings, sensors, or electrodes. It can be said that the material has specific surface forms which could be advantageous for drug delivery and cancer therapy use cases. The moderate roughness with sharp peaks, and deep valleys highly supports higher incorporation of the drug molecules for increased cellular internalization, thereby improved release in addition to cancer cell targeting \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, our in vitro research results support that it has a material structure consistent with an apoptotic or other anti-cancer cell death mechanism.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the thermal decomposition of Schiff base PEI-CA/N-GQDs. The figure presents combined data on TG, DTG, and DTA. As can be seen from Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the TG curve of Schiff base PEI-CA/N-GQDs, it degrades in four (IV) steps. The first (I) step of degradation took place at 57\u0026ndash;148\u0026deg;C and resulted in a weight loss of 3.76%, which may be due to the removal of hydroxyl and crystalline water. The second (II) decomposition step took place at 148\u0026ndash;287\u0026deg;C and resulted in a total weight loss of 11.20%, which may be due to the removal of carboxyl groups. The third (III) decomposition step occurred at 287\u0026ndash;304\u0026deg;C, which may have resulted from the elimination of amine groups in the PEI chain, with a total weight loss of 2.74%. The last step (IV) took place at 304\u0026ndash;900\u0026deg;C with a weight loss of 19.08%, probably due to the elimination of pyridine and pyrrolidine groups in the ring. DTG shoulders at 79\u0026deg;C and 180\u0026deg;C characterise steps I, II and III with lower mass loss attributed to PEI-CA decomposition, while the strong endothermal effect at 315\u0026ndash;379\u0026deg;C characterizes step IV with high mass loss attributed to the fragmentation of GQDs containing prolidine and pyridine groups in the ring. When the DTA curve is analyzed, it can be seen that the exothermic effect at 25\u0026ndash;200\u0026deg;C in stages I and II is different from DTG, whereas in stages III and IV it is endothermically identical to DTG. Small mass losses in stages I, II and III are characterised by an exothermic DTA peak, while high mass losses in stage IV are characterized by an endothermic DTG peak. As seen from the DTA curve, a broad, endothermic peak is observed at 200\u0026ndash;600\u0026deg;C, which is believed to be due to thermal expansion of the Schiff base PEI-CA/N-GQDs. No other thermal changes were observed in Schiff base PEI-CA/N-GQDs after 700\u0026deg;C. As a result, in this case, it can be said that Schiff base PEI-CA/N-GQDs is thermally stable.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eElectrochemical properties of Schiff base PEI-CA/N-GQDs\u003c/h2\u003e\n \u003cp\u003eThe cyclic voltammetry (CV) graph shows the well-known redox-active and consequently facile electrochemical behavior of the Schiff base PEI-CA/N-GQDs nanomaterial (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The x-axis represents the potential (V) applied during the measurement, and y-axis represents the current (mA) response of the system. A notable anodic peak appears around +\u0026thinsp;0.20 V, indicating the oxidation of electroactive species, likely related to the imine or phenolic groups present in the Schiff base structure. The corresponding cathodic peak around \u0026minus;\u0026thinsp;0.12 V signifies the reduction of the oxidized species, forming a redox couple typical of conjugated organic systems. The voltammogram shows a quasi-reversible nature, as evidenced by the asymmetry between the anodic and cathodic peaks and the relatively large potential separation, suggesting slower electron transfer kinetics. The maximum oxidative and reductive currents, approximately 0.12 mA and \u0026minus;\u0026thinsp;0.12 mA respectively, point to significant interaction between the material and the electrode surface. Additionally, the tailing at higher potentials (beyond 0.3 V) suggests some capacitive behavior, likely due to the high surface area of the N-GQDs contributing to double-layer charging. Overall, this CV indicates that the Schiff base PEI-CA/N-GQDs is electrochemically active with a quasi-reversible redox process, characteristic of such nanocomposite materials.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eComputational analysis Schiff base PEI-CA/N-GQDs (Optimization, FTIR, FMOs, MEP isosurfaces, UV-Vis)\u003c/h3\u003e\n\u003cp\u003eInitially, geometric optimization calculations were performed in the gas phase to determine the lowest energy structure representing the most stable configuration and thermodynamic properties of the Schiff base PEI-CA/N-GQDs for both phenol-imine and keto-amine forms (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The keto and phenol forms are tautomers, which are interconvertible isomers typically involving the movement of a proton and the shift of a double bond \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The characteristics of these tautomers\u0026mdash;particularly their electron structures\u0026mdash;can differ greatly. The keto form generally has a higher dipole moment, as is indicated in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, and it is because the keto structure usually contains a carbonyl group (C\u0026thinsp;=\u0026thinsp;O) where carbon and oxygen have what amounts to very different electronegativities. This creates a strong dipole, with oxygen being highly electronegative and carbon being relatively positive, giving the overall structure a high dipole moment\u0026nbsp;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eElectronic and thermal properties of Schiff base PEI CA/N-GQDs for both phenol-imine and keto-amine forms at T\u0026thinsp;=\u0026thinsp;298.15K in gas phase and different solvents.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGas Phase\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(\u0026epsilon;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(\u0026epsilon;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24,55)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003e\u0026epsilon;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;78,39)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenol-Imine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003etotal\u003c/sub\u003e (Hartree)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4215.309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4215.411\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4215.416\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDipole (Debye)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.847\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolarizability (a.u.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e617.610\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e574.263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e580.673\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003ethermal\u003c/sub\u003e (eV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1004.410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1004.378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1004.302\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeat Capacity (cal/mol.K)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e352.568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e349.774\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e349.816\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEntropy (cal/mol.K)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e528.398\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e498.373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e498.456\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKeto-Amine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003etotal\u003c/sub\u003e (Hartree)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4215.352\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4215.435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4215.440\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDipole (Debye)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.834\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolarizability (a.u.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e667.723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e927.427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e944.435\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003ethermal\u003c/sub\u003e (eV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1006.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1005.308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1005.289\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeat Capacity (cal/mol.K)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e348.891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e348.097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e348.057\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEntropy (cal/mol.K)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e502.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e502.253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e501.586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn the phenol form, the hydroxyl group carries a stabilizing \u0026quot;O-H\u0026quot; interaction that as a group is not as much dipole-dipole oriented as the keto group. Because the structure as a whole \u0026quot;phenol\u0026quot; carries resonance indicating that the electron cloud is also partial more uniformly distributed, the dipole moment is lower. Conversely, in the keto form, the structure is more polar. The reason for the higher thermal energy and smaller heat capacity of the keto-amine tautomer compared to the phenol form is as follows: The keto form usually contains double bonds (C\u0026thinsp;=\u0026thinsp;O); these bonds are stronger and shorter, which means lower potential energy. Less flexible bonds, on the other hand, result in higher thermal energy and vibrational levels \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. There are more vibrational modes in the phenol form due to its more flexible C\u0026thinsp;=\u0026thinsp;C bond and OH group. The phenol form can withstand higher temperatures because it has more vibrational modes, which allow the molecule to store more energy \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Because the keto-amine tautomer usually contains a carbonyl group (C\u0026thinsp;=\u0026thinsp;O), it has less entropy than the phenol-imine tautomer due to a lower degree of disorder. Essentially, at room temperature and in the gas phase, the optimized keto-amine and phenol-imine tautomers exhibit different and intriguing thermal behaviors, each with specific appeals and properties. This yields more ordered structures with more powerful hydrogen bonding or dipole interactions. In both water- and ethanol-based solutions, the energetic differences between the two forms are much smaller than in the gas phase. This is due to the combinations of hydrogen bonds that solvent molecules form with the solute, which lead to stabilizations of both forms that are on the same order of magnitude (in other words, both forms are solvated comparably well). Hydroxyl groups in both water and ethanol can also form hydrogen bonds with the nitrogen atom of the imine and with the phenolic oxygen atom, which equally stabilizes both the phenol-imine and keto-amine forms. Thus, the actual distinct energetic environment around each solute can dampen the differences in solvation energies, leading to minimal distinctions in the highs and lows of the energetic landscape for each form between the two solvent types.\u003c/p\u003e\n\u003cp\u003eOne advantage of the gas phase is that the free movement of molecules leads to ignoring external factors, thus obtaining a better understanding of the intrinsic parameters and reactions. It is also applied to investigate the basic scales of energies. Thus, this phase helps to make the observation of the fundamental energy level of molecular structures easier due to less intermolecular interaction and free movements. It is because the material was evaluated experimentally in aqueous media that the quantum chemical calculations were also run in the relevant water solvent, so that they will be compared reliably with the experimental data. The energy level in the gas phase is a critical index of structural stability. As shown in Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cstrong\u003e(a) and (b)\u003c/strong\u003e, the Schiff base PEI-CA/N-GQDs took extremely low energy in the gas phase (-4215.327 and \u0026minus;\u0026thinsp;4215.352 Hartree respectively) for both keto-amine and phenol-imine forms. The optimized structure proved the strong stability of this material, which means in some cases this could be the most stabler formed structure if it were possible. Their performance in real applications depend on the stability. It guaranties that material could operative robustly and reliably in various environment. The Schiff base PEI-CA/N-GQDs formed a much lower energy level than the material, the PEI N-GQDs (shown in our previous work \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e). The Schiff base PEI-CA/N-GQDs material showed a great promise in drug development by enhancing the binding affinity.\u003c/p\u003e\n\u003cp\u003eTheoretically calculating FTIR spectra are relevant: predictive power, structural elucidation, complement to experiments for interpretation purposes, functional group analysis; help in understanding vibrational modes and; method development/validation. In the next step, we generated an FTIR spectrum to explore molecular vibrations in more detail. The spectroscopic technique used to get an insight into the structural arrangement, functional group, and chemical nature of the molecule was very effective. For the two tautomers, we found that the vibrational shifts of PEI N-GQDs \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e original material vibration (FT-IR spectrum) are a little bit less compared to our theoretical analysis for Schiff base PEI-CA/N-GQD vibrations; When analyzing the FTIR spectrum (\u003cstrong\u003eFigure S2\u003c/strong\u003e), the frequency of maximum absorption is represented by the largest peak. Moreover, the experimental spectra are compared with vibrational frequencies predicted by DFT to identify which tautomer is present according to their characteristic peaks. It was corroborated by an FTIR analysis, identifying certain peaks of the phenol-imine tautomer present at 3695 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (O-H stretch) and 1651 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;N stretch). The keto-amine tautomer type showed absorptions at 3557 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretch) and 1596 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretch). It indicated that the keto-amine tautomer was prevalent as in the aryl region, its values exhibited a pronounced singlet. It is reasonable to assume that a major peak at 2697 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated the formation of hydrogen bonding between cinnamaldehyde with the COOH group and acting together as an acceptor OCH₃\u0026middot;(\u0026middot;\u0026middot;O)H. They are both acceptors and donors of hydrogen bonds, especially in the case of the carboxyl group with a strong ability to form H-bonds because it is able to donate one through OH but also can accept up to two due to its properties as carbonyl oxygen. Both the phenol-imine and keto-amine tautomers have unique FTIR absorbances but are in equilibrium so can interconvert. The most representative absorbance bands of the phenol-imine form were observed to be OH, NH, and NH\u003csub\u003e2\u003c/sub\u003e stretching (3714\u0026ndash;3323 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CH, CH\u003csub\u003e2\u003c/sub\u003e, and CH\u003csub\u003e3\u003c/sub\u003e stretching (3300\u0026ndash;2931 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); C\u0026thinsp;=\u0026thinsp;N stretching band at 1632 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in addition to a strong sharp peak for the \u0026ndash;C\u0026thinsp;=\u0026thinsp;C\u0026mdash; aromatic ring vibration mode around 1579 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; finally more peaks appeared corresponding COOH group with (1668 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). On the other hand, the keto-amine form showed absorbance bands at 3718\u0026ndash;3396 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (OH, NH, and NH\u003csub\u003e2\u003c/sub\u003e), 3321\u0026thinsp;\u0026minus;\u0026thinsp;2929 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (CH, CH\u003csub\u003e2\u003c/sub\u003e, and CH\u003csub\u003e3\u003c/sub\u003e), 1639 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;N), 1581 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;C), and 1658 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (COOH).\u003c/p\u003e\n\u003cp\u003eFMOs are made up of two very important parts: HOMO and LUMO. Normally, the HOMO (indicated by a blue line in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cstrong\u003e(a)\u003c/strong\u003e) is full of electrons with the highest energy and behaves like an electron donor. On the other hand, LUMO, illustrated with red lines, is empty and possesses the lowest energy level, working as an electron acceptor \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The energy gap (∆E) is a key factor in the reactivity of molecules. Conversely, the smaller \u0026Delta;\u003cem\u003eE\u003c/em\u003e that generally tends to correlate with higher chemical reactivity is beneficial when targeting specific biological molecules or pathways in cancer cells during drug development. Such a strategy allows us to conclude the likelihood for a molecule to undergo chemical reactions, under the assumption that elements of electron transfer mechanisms can be translated into an improved view of its overall stability. Especially in chemical reactions, those electrons are initially taken from the HOMO and moved to the LUMO of reacting materials \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cstrong\u003e(a)\u003c/strong\u003e, for Schiff base PEI-CA/N-GQDs phenol-imine and keto-amine, we see red and green lobes in the HOMO level by the same phase sense inside the housing of PEI, indicating a more electrophilic nature. This indicates that PEI can easily donate electrons to other parts. In contrast, the red and green lobes in the N-GQDs structure LUMO show its electron-accepting capability, which means that N-GQDs may be an acceptor. Simply, nucleophilic attacks involve the donation of an electron pair from a nucleophile (rich in electrons) to any electrophile deficient. This means the HOMO is displayed in red, denoting positive values, and this corresponds to positive exchange regions, which show areas with fewer electrons. Electrophiles, on the other hand, are drawn to areas with more electrons, such as the negative portions of LUMO, which appear green. On the other hand, electrophiles are attracted to regions that have extra electrons, such as the negative parts of LUMO like green. As a result, an electrophile hitting a molecule will most likely hit the green lobes of its LUMO, and nucleophilic attacks have been associated with red HOMOs. The energy level difference between LUMO and HOMO in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cstrong\u003e(a)\u003c/strong\u003e gives information about the properties of antioxidant activity. A smaller band gap means better antioxidant capabilities of Schiff base PEI-CA/N-GQDs phenol-imine form. This is because the compound became more reactive in electron transfer reactions, which are important for it to function as an antioxidant \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The increased reactivity due to the smaller band gap makes this form more effective in stabilizing reactive oxygen species (ROS). It has superior antioxidant activity in comparison to PEI N-GQDs, as reported in Ref. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe MEP approach assesses a susceptibility of a molecule to electrophilic (electron-attracting) and nucleophilic (electron-donating) reactions by examining the distribution of electric charge. MEP surfaces visualize these regions with color gradients: red for areas with negative electrostatic potential (electrophilic) and blue for areas with positive potential (nucleophilic), ranging from \u0026minus;\u0026thinsp;0.02 to 0.02 atomic units, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb. In electrophilic areas (red), the molecule has a high electron density that renders these spaces attractive to electron-seeking species. In nucleophilic regions (blue), low electron density is found, which is why these places attract more electron-rich species \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This method is of great assistance in identifying the reactive sites within the molecule and is always applied for studying the antioxidant activity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. For antioxidants, MEP analysis reveals electron-rich regions (negative potential) that can donate electrons to neutralize free radicals, thus protecting cells from oxidative damage. The ability of an antioxidant to serve as a reducing agent is because it can donate one electron instead of electrons or other protons and provide high negative potential regions (-OH) (or -NH2 groups). On the other hand, positive regions (blue) might interact with opposite sides of electron-deficient partners to halt deleterious oxidative reactions \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Nucleophilic activity in the phenol-imine form can be seen from a high electron density localized around both OH and C\u0026thinsp;=\u0026thinsp;N groups. Keto-amine form has a high electron density around the carbonyl (C\u0026thinsp;=\u0026thinsp;O) group. This contributes to predicting antioxidant reactivity and stability, showing how tautomers behave in different chemical environments through the understanding of these electron distribution patterns. For the phenolic part, this was high electron density areas near hydroxyl oxygen atoms and imine nitrogen of both phenol-imines. For the keto-amine bond, the electron pair is found mostly between the carbon and oxygen atom of the carbonyl group, which in turn pulls high amounts of electron density onto an even higher portion of space surrounding its own orbital. Lower electron density is shown in blue colors on the hydrogen atoms, especially those associated with nitrogen. This means that in the case of the phenol-imine tautomer, the negative charge is disproportionately held on to at these particular donor sites (the O and N), making them even more prone to nucleophilic attack than under ordinary circumstances. The keto-amine tautomer possesses greater electron density in the vicinity of its carbonyl oxygen, rendering that region more nucleophilic \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. These surfaces were employed to explain the reactivity and stability with regard to antioxidants, thereby revealing the behavior of addition-elimination tautomers in different chemical surroundings.\u003c/p\u003e\n\u003cp\u003eThe UV-Vis spectra of the material in aqueous media were thoroughly analyzed theoretically. This comprehensive analysis examined a number of spectroscopic characteristics associated with electronic transitions, including absorption wavelengths, excitation energies, and important transitions \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. These aspects were meticulously examined and documented, including the highest oscillator strength, which was meticulously tabulated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eOptical properties of Schiff base PEI CIN N-GQDs for both phenol-imine and keto-amine forms in water media.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003eAccording to the analysis in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the Schiff base PEI-CA/N-GQDs in its phenol-imine form shows a prominent contribution at 467 nm and three additional significant contributions at 408, 321, and 273 nm. These correspond to excitation energies of 2.65, 3.04, 3.86, and 4.55 eV, respectively. Specifically, the transition at 467 nm has a 69% contribution from H-6 to L\u0026thinsp;+\u0026thinsp;1. At 408 nm, the transitions include 31% from H-11 to L\u0026thinsp;+\u0026thinsp;1, 11% from H-6 to L\u0026thinsp;+\u0026thinsp;2, and 27% from H-1 to L\u0026thinsp;+\u0026thinsp;4. For the 321 nm transition, there is an 11% contribution each from H-14 to L\u0026thinsp;+\u0026thinsp;3 and H-13 to L\u0026thinsp;+\u0026thinsp;3, and 73% from H-10 to L\u0026thinsp;+\u0026thinsp;3. The contributions at 273 nm are as follows: 23% from H-28 to L\u0026thinsp;+\u0026thinsp;1, 22% from H-22 to L\u0026thinsp;+\u0026thinsp;1, and 22% from H-17 to L\u0026thinsp;+\u0026thinsp;4. Similar to this, the keto-amine form of the PEI-CA/N-GQDs Schiff base exhibits notable contributions at 483 nm, 423 nm, 408 nm, 338 nm, and one at 262 nm, which correspond to excitation energies of 2.57, 2.93, 3.67, and 4.73 eV, respectively. Specifically, the 483 nm transition involves 53% from H-11 to LUMO and 26% from H-5 to L\u0026thinsp;+\u0026thinsp;2. At 423 nm, transitions include 14% from H-16 to L\u0026thinsp;+\u0026thinsp;1, 23% from H-13 to L\u0026thinsp;+\u0026thinsp;1, 15% from H-7 to L\u0026thinsp;+\u0026thinsp;1, and 41% from H-3 to L\u0026thinsp;+\u0026thinsp;1. For the 338 nm transition, contributions are 44% from H-20 to LUMO and 23% from H-17 to L\u0026thinsp;+\u0026thinsp;2. The 262 nm transition involves a 47% contribution from H-26 to LUMO. As can be seen from Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the absorption wavelengths with characteristic peaks indicating electronic transitions in the UV-Vis analysis of the keto amine form are more in agreement with the experimental results compared to the phenol imine form. This supports that the material is in keto-amine form.\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eAntioxidant activity\u003c/h3\u003e\n\u003cp\u003eThe antioxidant activity of Schiff base PEI-CA/N-GQDs is shown in \u003cstrong\u003eFigure S3\u003c/strong\u003e as the average of three experiments. As observed in \u003cstrong\u003eFigure S3\u003c/strong\u003e, Schiff base PEI-CA/N-GQDs exhibited higher activity compared to stable BHT. The antioxidant activity of Schiff base PEI-CA/N-GQDs was found to be 42%, 51%, 65%, 78%, and 79%. The activity increased up to a material concentration of 100 ppm and then remained stable at 200 ppm with no significant difference. Notably, it demonstrated greater activity than the standard BHT, which is likely attributable to the Schiff base PEI-CA bond (C\u0026thinsp;=\u0026thinsp;N). The high antioxidant activity of Schiff base PEI-CA/N-GQDs can be attributed to the presence of OH and COOH groups in N-GQDs, as well as the coniferaldehyde and the imine bond.\u003c/p\u003e\n\u003ch3\u003eDNA-Binding and DNA Cleavage\u003c/h3\u003e\n\u003cp\u003eAbsorption spectra of nanomaterial, Schiff base PEI-CA/N-GQDs in the absence and presence of CT-DNA are shown in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e. As can be seen from Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, with increasing CT-DNA concentrations, 4\u0026ndash;79% hyperchromicity at 248 nm and 4\u0026ndash;9% hyperchromicity at 348 nm is observed in the material. So, Schiff base PEI-CA/N-GQDs interacts with DNA in an electrostatic mode.\u003c/p\u003e\n\u003cp\u003eProbably in this interaction the hydroxyl proton in the investigated material is taken up by the negatively charged phosphate groups in the DNA helix and part of the charge of the DNA is neutralized. The charge density of the DNA is thus disturbed, and as the proton leaves the material, the materials become negatively charged and exert an electrostatic repulsive force on the DNA helix, accelerating the degradation of the DNA \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBoth hydrolytic and oxidative DNA breakage were investigated in this work. Figure \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e displays the outcomes of the two cleavage tests. When exposed with increasing concentrations of nanocomposite (from 50 ppm to 800 ppm), Lanes 1\u0026ndash;6 in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e demonstrate hydrolytic breakage of DNA. DNA chain breakage are caused by the material, as evidenced by the emergence of new bands and the primary band\u0026apos;s (Form I) decline in intensity. A concentration-dependent interaction is shown by the fact that these effects intensify as concentration rises. DNA\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e oxidative stress conditions are used in Lanes 7\u0026ndash;12 to assess the nanocomposite\u0026apos;s impact on DNA. It is consistent with enhanced DNA degradation that greater breakage in the presence of oxidative agents (especially in lanes 7 to 12) may be due to both direct chemical interactions with DNA and the nanocomposite producing ROS.\u003c/p\u003e\n\u003cp\u003ePrior research has demonstrated the ability of graphene-based nanomaterials to interact with DNA via intercalation, electrostatic forces, and \u0026pi;-\u0026pi; interactions \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. This particular Schiff base mechanism offers a more nuanced perspective, indicating that this material may also promote targeted DNA cleavage in an oxidative environment. This is in line with the characteristics of Schiff base PEI-CA/N-GQDs that have been shown in other settings, including drug delivery systems and biosensors. Determining the safety of these nanocomposites in biological applications requires an understanding of the circumstances behind DNA damage. The DNA damage that is discovered to be concentration-dependent emphasizes the necessity of cautious dosing and delivery methods in order to reduce the risk of genotoxicity. The specificity of DNA interaction sites should be investigated further, and any potential off-target effects should be evaluated. Furthermore, examining these nanocomposites\u0026apos; biocompatibility and in vivo degradation behavior might offer vital information for therapeutic uses.\u003c/p\u003e\n\u003ch3\u003eEffect of Schiff base PEI-CA/N-GQDs on cellular viability\u003c/h3\u003e\n\u003cp\u003eThe MTT test, also known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, is a laboratory test that is used in various fields, including drug research, toxicity evaluations, and cell culture studies, to determine cell viability and metabolic activity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. We also used the MTT assay in our investigation to examine the antiproliferative effect of the Schiff base PEI-CA/N-GQDs on human neuroblastoma cells, SH-SY5Y. The test results indicated that for 24 and 48 hours, Schiff base PEI-CA/N-GQDs demonstrated cytotoxicity within the measured dosage range. The most cytotoxic effect on SH-SY5Y cells was demonstrated by Schiff-based PEI-CA/N-GQDs applied at a concentration of 1.25 \u0026micro;g/\u0026micro;L for 48 hours, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e. For both 24 and 48 hours, the IC\u003csub\u003e50\u003c/sub\u003e values of Schiff base PEI-CA/N-GQDs were determined to be 0.1407 \u0026micro;g/\u0026micro;L and 0.0257 \u0026micro;g/\u0026micro;L, respectively.\u003c/p\u003e\n\u003cp\u003eThe anticancer properties of GQDs are well-established \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, but these compounds also exhibit cytotoxic effects on normal cells. Consequently, scientists sought to create compounds that specifically target cancer cells while exhibiting reduced cytotoxicity on normal cells. This was achieved by creating N-GQDs \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. In this regard, Senel et al.\u0026apos;s 2019 work demonstrated that N-GQDs had antioxidant and antibacterial qualities in addition to their capacity to attach to DNA and maintain cell viability. According to the study, N-GQDs can bind to DNA by electrostatic and intercalation mechanisms; formulations containing GQDs can penetrate cells and lower the number and amount of lung cancer cells that express ephrin type-A receptor 2 (EpHA2); and high concentrations of quantum dots can cause DNA damage in cancer cells, thereby lowering the viability of those cells \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe cytotoxic, apoptotic, and cell cycle arrest effects of PEI N-GQDs on ovarian cancer cell lines were examined for the first time in our earlier work \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Using the same serial dilution approach, we were able to demonstrate that in the cell culture and viability test, PEI N-GQDs killed 50% of the cells at a concentration of 16.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69 \u0026micro;g/\u0026micro;L. The dosage rate that killed half of these cells dropped to 13.76\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48 \u0026micro;g/\u0026micro;L and the cytotoxic impact enhanced when palladium nanoparticles (PdNPs) were added to PEI N-GQDs.\u003c/p\u003e\n\u003cp\u003eIn this work, we demonstrated that the synthesized Schiff based PEI-CA/N-GQDs can bind to DNA through an electrostatic effect by displaying an exonuclease activity, which is consistent with the findings of Senel et al. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. At a treatment concentration of 0.0257 \u0026micro;g/\u0026micro;L, Schiff-base PEI-CA/N-GQDs demonstrated the capability to eliminate 50% of the neuroblastoma cells, as indicated by the findings from the cytotoxicity part of our study. This results indicate that the cytotoxic impact of Schiff base PEI-CA/N-GQDs on cancer cells is approximately 643 times larger than that of PEI N-GQDs, despite the fact that we attempted a different cancer cell line in our prior work. The Schiff base and CA in the created nanoparticle may be the cause of this discrepancy.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eApoptotic Effects of Schiff base PEI-CA/N-GQD on SH-SY5Y\u003c/h2\u003e\n \u003cp\u003eA substance or chemical that may precisely stop the proliferation of cancer cells by changing the aberrant signaling pathway involved in the cell cycle and/or apoptotic mechanism is usually regarded as a crucial chemotherapeutic tool in the field of anticancer research. Therefore, causing cancer cells to undergo apoptosis is one of the primary objectives of chemotherapy \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. In this work, we used three different methods to assess the apoptotic effects of newly manufactured Schif base PEI-CA/N-GQDs on the SH-SY5Y cell line. Using flow cytometry, we first demonstrated whether Schiff base PEI-CA/N-GQDs increased intracellular ROS. Furthermore, we used flow cytometry to demonstrate the apoptotic effects of the material, and immunohistochemistry labeling allowed us to identify the variations in protein expression between apoptotic and antiapoptotic.\u003c/p\u003e\n \u003cp\u003eThe effect of Schiff base PEI-CA/N-GQDs treatment on ROS generation in SH-SY5Y cells is depicted in Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e. As a result, ROS generation increased dramatically to 52.3% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in cells treated with Schiff base PEI-CA/N-GQDs compared to 41.4% in untreated cells (control group).\u003c/p\u003e\n \u003cp\u003eThe findings of the flowcytometric analysis showed that, in comparison to the cells in the untreated control group, the viability rate of SH-SY5Y cells treated with Schiff base PEI-CA/N-GQDs dropped significantly to an average of 65.5% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Total apoptosis in treated cells increased significantly by 108.5-fold to 21.7%, while the number of necrotic cells increased significantly by 12.8%. When cells treated with Schiff base PEI-CA/N-GQDs, the average proportion of cells undergoing early and late apoptosis was 12.2% and 9.5%, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eApoptotic protein CASPASE-3 and antiapoptotic protein BCl-2 immunohistochemistry expression differences in SH-SY5Y cells treated and untreated with produced Schiff base PEI-CA/N-GQDs are presented in Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e. Consequently, it is seen that CASPASE-3 expression is negative in untreated SH-SY5Y cells (Figs. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eB), but CASPASE-3 protein is expressed with a diffuse staining and a staining intensity of +\u0026thinsp;3 in treated cells (Figs. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eD). However, it was observed that Bcl-2 protein was expressed at 10% prevalence and +\u0026thinsp;1 staining intensity in untreated cells (Figs. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eF), while BCL-2 protein expression was inhibited and showed negative expression in treated cells (Figs. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003eH).\u003c/p\u003e\n \u003cp\u003eAccording to Ou et al. (2017), exposure to graphene increased the amount of ROS that mitochondria produced inside the cell. As a result, following a sequence of metabolic processes, proapoptotic molecules were released from mitochondria into the cytoplasm, causing cells to undergo apoptosis \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Furthermore, it was demonstrated by Ramachandran et al. (2022) that the treatment of human breast cancer cells with N-GQDs/titanium dioxide nanocomposites (N-GQDs/TiO2) increased ROS and resulted in mitochondria-associated apoptotic cell death \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Parallel to these two studies that demonstrate that GQDs and N-GQDs raise ROS in individual cells, we have demonstrated in this work that Schiff base PEI-CA/N-GQDs raise ROS in human neuroblastoma SH-SY5Y cells, causing the cells to undergo apoptosis. According to Qin et al. (2015), activating the apoptotic proteins in the CASPASE family causes the apoptotic process to be initiated in macrophages treated with GQDs via a mitochondria-associated mechanism \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. In this work, we demonstrated that the upregulation of ROS in cells triggers the activation of the CASPASE-3 protein, which in turn drives the cell towards death by inhibiting BCL-2.\u003c/p\u003e\n \u003cp\u003eFurthermore, the overall apoptosis rate in cells treated with PEI NGQDs rose by 45.8% compared to untreated cells in our prior work, where the apoptotic impact of PEI NGQDs in ovarian cancer was first demonstrated \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In this study, compared to the control, the number of cells treated with Schiff base PEI-CA/N-GQDs increased by 21.7%. While both studies demonstrated statistically significant increases, we believe this is mostly because the different cancer cells employed in the research had distinct metabolic features. In addition, the excellent antioxidant activity of Schiff base PEI-CA/N-GQDs was demonstrated in this investigation. We revealed that the CA itself and the imine bond that NGQDs have with each other are the sources of the nanoparticle\u0026apos;s strong antioxidant capability. Due to the increased antioxidant activities of Schiff base PEI-CA/N-GQDs compared to PEI N-GQDs, we believe that the apoptosis rates in our prior investigation were higher than the results in this study.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCell cycle arrest effect of Schiff base PEI-CA/N-GQDs in SH-SY5Y cell\u003c/h3\u003e\n\u003cp\u003eCyclin-dependent kinases (CDKs) and Cyclin-dependent kinase inhibitors (CKIs) are enzymes that control the cell cycle in living cells. By limiting the number of healthy cells that can pass through the G0/G1, S, and G2/M phases of the cell cycle, these enzymes control the proliferation of cells. During this process, injured cells are either destined to die by apoptosis or undergo irreversible damage repair. Uncontrolled cell proliferation results from a breakdown in this system; uncontrolled growth is a well-known characteristic that sets cancer apart from other diseases \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn order to get more insight into the cytotoxicity caused by Schiff base PEI-CA/N-GQDs, the distribution of cell cycle stages in SH-SY5Y was assessed following a 48-hour incubation period with 0.0257 \u0026micro;g/\u0026micro;L Schiff base PEI-CA/N-GQDs. Figure \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e shows that following a 48-hour treatment with Schiff base PEI-CA/N-GQDs, the cell population was much more concentrated in the G1 phase (41.2%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,001) as compared to the untreated cells (13.6%), and the G2 population also decreased concurrently (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,01). Following a 48-hour treatment with Schiff base PEI-CA/N-GQDs, 56.1% of SH-SY5Y cells were found to be in the G2 phase, compared to 82.1% of control cells. Additionally, there was no statistically significant variation in S phase across the groups. The results demonstrate that Schiff base PEI-CA/N-GQDs stopped the cell cycle in the G1 phase in SH-SY5Y, which inhibited cell division and caused apoptosis.\u003c/p\u003e\n\u003cp\u003eTian et al. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e claim that GQDs are genotoxic substances. The cell responds to the genotoxicity induced by GQDs by undergoing DNA damage. In reaction to DNA damage, the cell undergoes this response, which involves stopping the cell cycle and giving the damaged DNA adequate time to heal. According to Ku et al.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, GQDs caused apoptosis in breast cancer and stopped the cell cycle at the G2/M checkpoint. In our earlier work \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, we looked at the impact of PEI NGQDs on ovarian cancer cells\u0026apos; ability to arrest the cell cycle. The results indicated that, in comparison to untreated (control group) cells, cells treated with PEI N-GQDs were arrested in the sub-G0/G1 phase of the cell cycle.\u003c/p\u003e\n\u003cp\u003eIn this work, we used DNA-Binding and DNA-Cleavage assays to elucidate the connection between Schiff base PEI-CA/N-GQDs and DNA. As a result, we demonstrated that Schiff base PEI-CA/N-GQDs can bind to DNA via an electrostatic mode interaction. This binding can also result in both hydrolytic and oxidative cleavage, with oxidative cleavage being more potent and occurring at a lower dose than hydrolytic cleavage. When chromosomes are damaged or destroyed, G1/S and G2/M checkpoints are triggered in response to DNA damage to halt cell division. Currently, damaged DNA may be repaired by cells thanks to DNA damage checkpoints. A broken DNA molecule has the potential to interrupt a cell\u0026apos;s growth or perhaps initiate its demise. Damage to DNA triggers the cell death process. While the G2/M checkpoint stops cells from proliferating with damaged DNA, the G1/S checkpoint stops cells from copying damaged DNA \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. This work has demonstrated that Schiff base PEI-CA/N-GQDs, which oxidatively and hydrolytically cleave DNA and attach to DNA in an electrostatic fashion, halt cell cycle at the G1/S checkpoint and induce apoptotic cell death through the CASPASE pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe Schiff base PEI-CA/N-GQDs nanomaterial novel developed in this study has shown a strong potential for anticancer activity, mainly against human SH-SY5Y neuroblastoma cells. This shows the efficacy of the material to induce oxidative stress, DNA cleavage, and apoptosis, as well as cell cycle arrest at an early phase named G1/S, making this a promising tool for future therapies. Upon extreme experimental and theoretical characterization, the structure was found to be primarily in a keto-amine form dominated by very good electron transfer properties. The nanomaterial was responsible for both cytotoxic effects, mainly linked to its electrostatic binding with DNA and ROS generation, but it also serves as an apoptotic compound through CASPASE-3 activation. Enhanced antioxidant properties of Schiff base PEI-CA/N-GQDs in comparison to the standard antioxidants also confirm its medicinal potential. These conclusions indicated that the combination of PEI-CA/N-GQDs may be promising as a cancer therapy in the treatment of neuroblastoma and suggest a possible pathway for how nanomaterial can work at the cellular level.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eFT-IR spectra were measured with a Perkin Elmer BX II spectrometer with KBr discs and UV-Visible spectra were measured in water with a Varioskan Flash Multimode spectrometer. Morphology of material were characterized using Park System Atomic Force Microscope (AFM) XE100. Chemical composition of the samples were determined by Hitachi SU5000 FE-SEM instrument. Thermogravimetric analyses were performed on a Hitachi STA 7300 simultaneous TG/DTA Instrument. The sample (10 mg) were placed in aluminium crucibles and heated between 25\u0026deg;C and 900\u0026deg;C to determine the thermal stability. The experiments were carried out in nitrogen at different heating rates of 5 \u003csup\u003e0\u003c/sup\u003eC min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. X-ray diffraction (XRD) patterns were obtained through a Bruker D8 Advance X-ray diffractometer using Cu Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.54051 \u0026Aring;) in the 2θ range of 20\u0026ndash;90\u0026deg; with a step size of 0.02 at 2 min\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e steps, operating at an accelerating voltage of 40 kV and a current of 40 mA. Electrochemical measurements were taken with the C4 Cell Stand experimental model. Glassy carbon electrode was used as the working electrode, and MW 1032 electrodes were used as the counter electrode.\u003c/p\u003e \u003cp\u003eAll of the chemicals were obtained commercially from Sigma-Aldrich and used without further purification, including citric acid (CA), silver nitrate (AgNO3), polyethyleneimine (PEI) (average Mw\u0026thinsp;~\u0026thinsp;25,000 by LS, average Mn\u0026thinsp;~\u0026thinsp;10,000 by GPC, branched), 4-hydroxy-3-methoxycinnamaldehyde (coniferaldehyde), ethidium bromide (EB), calf thymus DNA (CT-DNA), pBR322 DNA, 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), and ethylalcohol (EtOH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of Shiff base PEI-CA/N-GQDs\u003c/h2\u003e \u003cp\u003ePEI N-GQDs \u003csup\u003e\u003cspan additionalcitationids=\"CR73 CR74\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e (7.02 g) were added to a 250 mL round bottom flask and 150 mL of EtOH was added. Then 50 mL alcohol solution of coniferaldehyde (CA) was added to this mixture and the mixture was boiled under reflux for 2 h to form Schiff base PEI-CA/N-GQDs. After the mixture was filtered and allowed to evaporate, the dark red solid was kept for later use in a vacuum desiccator (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eComputational details\u003c/h2\u003e \u003cp\u003eThis section presents an extensive investigation into the characteristics of Schiff base PEI-CA/N-GQDs, building upon our previous research \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. The purpose of this work is to assess the electronic properties of the material in order to determine its sensitivity and selectivity. We accomplish this with the help of many calculations, among which are the geometry optimizations. These optimizations provide a very good approximation of the actual arrangement and dimensions of atoms in a given material and the nature of their atomic-scale bonding, including bond lengths, angles, and the types of bonds they form. After the structures are optimized, we can then carry out a complete exploration of their electronic structures and use the results to perform a deep-dive analysis of what their electronic properties really are \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt first glance, Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e offers a clearer, more detailed view of the theoretical structure of the Schiff base PEI-CA/N-GQDs material. Both the phenol-imine and keto-amine forms are shown in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e and \u003cb\u003e2(b)\u003c/b\u003e, respectively. Both forms are presented in a way that makes the tautomeric relationship between them visually accessible, with the hydrogen involved in the tautomerization highlighted in green. In order to gain a thorough understanding of the molecular dynamics involved in this tautomerization, DFT method is frequently used to optimize geometries, calculate energies, analyze vibrational frequencies, thermodynamic properties and take solvent effects into account. To determine the most stable structural configurations of this theoretical model, DFT/B3LYP optimization was performed using the Gaussian 09W software package \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e with the Lanl2dz basis set \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, which is known to be useful for the functionalized graphene quantum dot calculations \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. The structures were visualized with GaussView 5.0 software \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, which aided the calculations of the structural and electronic properties that were critical. We also recorded and analyzed Fourier Transform Infrared (FTIR) spectra, which provided information on molecular vibrations and helped infer the structure and chemical functionality of the nanomaterials. Additionally, important characteristics like FMOs, MEP, and the HOMO-LUMO energy gap were determined using the LanL2DZ basis set. Also, calculations of the UV-Vis spectra were done using Time-Dependent Density Functional Theory (TD-DFT) \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e, with a single consistent basis set used throughout the calculations \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e,\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant properties of the material were evaluated using the DPPH method [54] as described in reference [53], with butylated hydroxytoluene (BHT) serving as the standard antioxidant according to reference [55]. Methanol solutions with DPPH (20 mg/mL) and PEI-CA/N-GQDs Schiff base at different concentrations (20\u0026ndash;200 ppm) were utilized to achieve this. The Schiff base PEI-CA/N-GQDs was mixed with a DPPH solution and left to remain in the dark for 30 minutes. The absorption level was determined at 517 nm. The scavenging impact was assessed as the proportion of radical reduction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDNA-Binding and DNA-Cleavage\u003c/h2\u003e \u003cp\u003eUV-Vis titration in room-temperature Tris-HCl/NaCl buffer was used to investigate the binding affinity of the Schiff base PEI-CA/N-GQDs with CT-DNA, in accordance with the protocol outlined in reference \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. An aqueous solution of the Schiff base PEI-CA/N-GQDs (100 ppm) was utilized in this experiment. The Schiff base PEI-CA/N-GQDs -DNA solutions were allowed to stand at room temperature for 5 min before each measurement.\u003c/p\u003e \u003cp\u003eThe effectiveness of the material for DNA cleavage was evaluated using gel electrophoresis \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. The substrate in this analysis was supercoiled (SC) pBR322 DNA. We investigated the DNA cleavage activity of the Schiff base PEI-CA/N-GQDs under oxidative conditions (in the presence of H2O2) and hydrolytic conditions (in the absence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). To initiate the oxidative cleavage activity, we combined pBR322 and the Schiff base PEI-CA/N-GQDs with 2 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, acting as an oxidizing agent. For three hours, we incubated pBR322 (0.1 \u0026micro;g/mL) in 10 \u0026micro;M Tris-HCl buffer (pH:7.2) at 37\u0026deg;C using the Schiff base PEI-CA/N-GQDs. Following incubation, the samples were treated with loading buffer, and we electrophoresed them for one hour at 60 V on a 1% agarose gel in TBE (Tris-Boric acid-EDTA, pH: 8.0). The bands were then captured on camera and made visible with UV light.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eCell Culture: Dulbecco's modified Eagle's medium F12 (DMEM/F12, Gibco, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS, Gibco, CA, USA) containing 100 U/mL penicillin-streptomycin was used to culture the human-derived epithelial neuroblastoma (SH-SY5Y) cells (ATCC number: CRL-2266, Manassas, VA, USA). The cells were grown at 37\u0026deg;C with 5% CO2 in a humidified incubator. A combination of adherent and floating cells makes up the growth of SH-SY5Y cells. Consequently, the medium holding the floating cells was taken out, and centrifugation was used to save the cells. 1 to 2 mL of 0.25% trypsin solution was added to the adherent cells after they had been rinsed with 1x PBS solution. The flask was then left at 37\u0026deg;C until the cells detached. After adding and aspirating fresh media, floating cells that had been gathered above were mixed in and placed to fresh flasks \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability Assay\u003c/h2\u003e \u003cp\u003eA 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric test (Beyotime Biotech, Haimen, China) was used to measure the proliferation of SH-SY5Y cells. To put it briefly, these cells were suspended in 150 \u0026micro;L and planted at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-well plates. They were then kept overnight at 37\u0026deg;C in a CO2 incubator to facilitate adhesion. Different quantities of Schiff-base PEI-CA/N-GQDs (0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, and 1.25 \u0026micro;g/\u0026micro;L) were applied to SH-SY5Y cells. Following treatment for 24 and 48 hours, the medium was taken out of each well and MTT solution (0.5 mg/mL) from Sigma-Aldrich, Saint Louis, Missouri, USA was added. The solution was left for 4 hours at 37\u0026deg;C. Following the incubation period, 200 \u0026micro;L of isopropyl alcohol was added to each well and pipetted up and down many times to dissolve the purple formazan crystals. Each well's absorbance was then measured at 570 nm using a plate reader (BioTek, USA) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometric Analysis of Intracellular ROS Generation\u003c/h2\u003e \u003cp\u003eIn accordance with the manufacturer's instructions, the ROS-sensitive fluorescent dye dichloro-dihydrofluorescein diacetate (DCF-DA) probe was employed to measure ROS production. Subsequently, Schiff base PEI-CA/N-GQDs (0.0257 \u0026micro;g/\u0026micro;L) were applied to SH-SY5Y cells for 48 hours. Following this, the cells were suspended in PBS and incubated with DCF-DA (10 \u0026micro;M) for 30 minutes at 37\u0026deg;C. Following two PBS washes, the cells were stained with DCF fluorescence, recorded using a BD Accuri C6 Flow Cytometer at 485 and 535 nm for excitation and emission wavelengths. CellQuest software was then used to analyze the data \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometric Analysis of Apoptosis\u003c/h2\u003e \u003cp\u003eAn Annexin V/7-aminoactinomycin (7-AAD) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to measure the apoptosis rate. In 6-well culture plates, 0.0257 \u0026micro;g/\u0026micro;L Schiff-base PEI-CA/N-GQDs were applied for 48 hours to SH-SY5Y cells. Following cell harvesting, an equivalent amount of PE-Annexin V was immunostained for 15 minutes, after which the cells were suspended in Annexin V binding buffer and cleaned with cold PBS. Tubes were incubated for 30 minutes in the dark with the addition of 7-AAD (5 \u0026micro;g/ml, Sigma-Aldrich) and binding buffer, prior to flow cytometric analysis. Tubes were incubated for 30 minutes in the dark with the addition of 7-AAD (5 \u0026micro;g/ml, Sigma-Aldrich) and binding buffer, prior to flow cytometric analysis. When cells were stained with both PE-Annexin V and 7-AAD, they were considered to be late apoptotic, while cells labeled with PE-Annexin V alone were considered to be early apoptotic. With the use of a flow cytometer (BD Accuri C6 Flow Cytometer, USA), the percentage of apoptotic cells was determined \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCell Blocking and Immunohistochemical Staining\u003c/h2\u003e \u003cp\u003eSchiff-base PEI-CA/N-GQDs at a concentration of 0.0257 \u0026micro;g/\u0026micro;L were applied to human neuroblastoma cells (SH-SY5Y) and incubated for a duration of 48 hours. Following incubation, a cell pellet was obtained by centrifuging the collected cells and trypsinizing them. The cell pellet was embedded in paraffin, sectioned at 4 \u0026micro;m thickness, and fixed for 24 hours in 10% neutral buffered formalin \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. Leica Bond-Max automated staining system was used for immunohistochemical staining under typical operating circumstances. The DAB-compatible Bond Polymer Refine Detection Kit (DS9800, Buffalo Grove, USA) was employed. Antibodies that were ready for usage were applied for Bcl-2 (clone bcl-2/100/D5 cod. PA0117, Leica Biosystems, UK) and CASPASE-3 (clone EP410 cod. AC-0364RUO, Cell Marque, USA). After deparaffinizing, rehydrating, and applying citrate buffer (pH 6.0) for antigen retrieval, slides were processed. The sections were incubated with the primary antibodies for a whole night at 4\u0026deg;C after non-specific binding was blocked with normal goat serum. DAB substrate was used for color development after the biotinylated secondary antibody and ABC reagent were incubated \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. \u003cb\u003eFigures S4\u003c/b\u003e and \u003cb\u003eS5\u003c/b\u003e display internal positive control stainings of the Bcl-2 and CASPASE-3 proteins in lymphoid tissue. Apoptotic and anti-apoptotic marker expression was evaluated by microscopic inspection of the sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometric Analysis of Cell Cycle\u003c/h2\u003e \u003cp\u003eThe cells were treated for 48 hours with Schiff-base PEI-CA/N-GQDs (0.0257 \u0026micro;g/\u0026micro;L). Following this, they were harvested using 0.05% trypsin, twice rinsed with assay buffer, and fixed in 70% ethanol/PBS at 4\u0026deg;C for the whole night. After the ethanol was removed by centrifugation, the fixed cells were resuspended in PBS and stained for 30 minutes at 37\u0026deg;C in the dark using propidium iodide (PI) that included 20 \u0026micro;g/mL of RNase. The samples were examined on a flow cytometer (BD Bioscience, USA) using the CellQuest software to quantify the forward and side scatter in order to determine the cell cycle distribution \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been supported by Ankara University Scientific Research Projects Coordination Unit under grant number: \u003cstrong\u003eFBA-2024-3174\u003c/strong\u003e. The numerical calculations reported in this paper were fully performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurat Kilic:\u0026nbsp;\u003c/strong\u003econceptualization, data curation, formal analysis, investigation, visualization, writing – original draft, writing – review \u0026amp; editing; \u003cstrong\u003eBuket Altinok Gunes:\u0026nbsp;\u003c/strong\u003econceptualization, data curation, formal analysis, investigation, visualization, writing – original draft, writing – review \u0026amp; editing; \u003cstrong\u003eOmer Faruk Kirlangic:\u0026nbsp;\u003c/strong\u003econceptualization, data curation, formal analysis, investigation, visualization; \u003cstrong\u003eAysenur Aslan:\u0026nbsp;\u003c/strong\u003einvestigation, resources, visualization; \u003cstrong\u003eDidem Ketenoğlu:\u0026nbsp;\u003c/strong\u003einvestigation, visualization, project administration, writing – original draft, writing – review \u0026amp; editing; \u003cstrong\u003eFatma Zeynep Kirlangic:\u003c/strong\u003eformal analysis, investigation, resources, visualization; \u003cstrong\u003ePinar Mualla Elci\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eformal analysis, investigation, resources, visualization; \u003cstrong\u003eFaruk Mert\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e formal analysis, investigation, resources, visualization; \u003cstrong\u003eNeslihan Demir\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e formal analysis, investigation, resources, visualization, writing – original draft; \u003cstrong\u003eBahadir Boyacioglu:\u0026nbsp;\u003c/strong\u003eformal analysis, investigation, visualization, project administration, supervision, writing – original draft, writing – review \u0026amp; editing; \u003cstrong\u003eHuseyin Unver:\u0026nbsp;\u003c/strong\u003eformal analysis, investigation, visualization, project administration, writing – review \u0026amp; editing;\u003cstrong\u003e\u0026nbsp;Ashok Chatterjee:\u0026nbsp;\u003c/strong\u003esupervision, writing – review \u0026amp; editing; \u003cstrong\u003eMustafa Yildiz:\u0026nbsp;\u003c/strong\u003econceptualization, data curation, formal analysis, investigation, project administration, visualization,supervision, writing – original draft, writing – review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDhillon, P. 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The synthesized material was experimentally characterized by FT-IR, UV-Vis, SEM, EDX, AFM, XRD, and TGA, as well as theoretically by DFT method. In addition to their biological activities, their cytotoxic, apoptotic and cell cycle arresting effects were investigated in human epithelial neuroblastoma (SH-SY5Y) cells. According to the UV-Vis data, we observed that the material exhibits phenol-imine/keto-amine tautomerism, a phenomenon common in 2-hydroxy Schiff bases that help to explain the various properties of material. Furthermore, this material predominantly exists in the keto-amine form. The material demonstrated favorable electron transfer properties, making it suitable for electrochemical applications. We showed that it binds to DNA through an electrostatic interaction and causes oxidative and hydrolytic cleavage in DNA, which results in an increase in ROS in the cell, an activation of the CASPASE-3, leading the cells undergo apoptosis, and inhibiting cell division in the G1/S phase. We believe that the chemical properties of the Schiff-based PEI N-GQDs make them a superior carrier molecule for cancer treatment. Furthermore the anticancer properties of the Schiff-based PEI-CA/N-GQDs suggest their potential as a therapeutic agent for neuroblastoma.\u003c/p\u003e","manuscriptTitle":"A Quantum Chemical, Biological, and Experimental Analysis of Newly Synthesized Schiff-based PEI CA/N-GQDs Nanomaterials: Evaluation of Anticancer Potential in Human Neuroblastoma Cell","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-17 12:09:57","doi":"10.21203/rs.3.rs-5352941/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"42fdff57-f288-4166-b6f0-0dcc652db98a","owner":[],"postedDate":"December 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41390794,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":41390795,"name":"Biological sciences/Chemical biology/Drug delivery"},{"id":41390796,"name":"Biological sciences/Cancer"},{"id":41390797,"name":"Physical sciences/Chemistry/Chemical biology"},{"id":41390798,"name":"Physical sciences/Chemistry/Chemical engineering"},{"id":41390799,"name":"Physical sciences/Chemistry/Materials chemistry/Biomaterials"},{"id":41390800,"name":"Biological sciences/Drug discovery"},{"id":41390801,"name":"Biological sciences/Drug discovery/Drug delivery"}],"tags":[],"updatedAt":"2025-05-15T20:08:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-17 12:09:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5352941","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5352941","identity":"rs-5352941","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}