Pioneering the Photoactive Relevance of Quinazolinone–Fullereropyrrolidine Nanohybrids To Address Chemotherapeutic Resistance in Cancer

Abstract

This study investigates the impact of C₇₀ and C₆₀ fullerenes on quinazolinone, specifically in quinazolinone–fulleropyrrolidine nanohybrids. The nanohybrids Q₃C₇₀M and Q₃C₆₀M exhibit distinct spectral shifts and have significant photobiological antineoplastic properties. Q₃C₆₀M enhances apoptosis, while Q₃C₇₀M reduces Cyclin A levels and counteracts oncogenic effects by promoting cell differentiation. Q₃C₇₀M demonstrates heightened cytotoxicity by overcoming chemotherapy resistance by modulating BAX and BCL-2 levels. This innovative approach, distinguishing between C₇₀ and C₆₀, represents a novel contribution to the existing scientific literature.

Quinazolinones are a category of privileged pyrimidine analogues that serve as scaffolds in the manifestations of organic molecules to develop medicinal chemistry.^1,2 These compounds are abundant in natural products, and their synthetic analogues are pharmacologically active. Quinazolinones have been popularized in medicinal chemistry through extensive synthetic approaches to obtain structural and electronic variations to explore their possible biological activities.^1,3 While the biological chemistry of quinazolinone pharmacophores is valuable for medicinal chemistry, their photobiological relevance remains unexplored. Quinazolinones are effective chemotherapeutic agents against solid tumors, with structural modifications enhancing their anticancer activities by improving receptor binding. Different linkers attached to the ring structure result in diverse anticancer mechanisms, including the inhibition of DNA repair enzymes, tubulin polymerization, thymidylate enzyme, and epidermal growth factor receptor (EGFR). FDA-approved pyrimidine anticancer drugs such as gefitinib and erlotinib are developed based on these mechanisms. Additionally, compounds such as ceritinib, ruxolitinib, and others show promise as potent anticancer agents by targeting various enzymes and signaling pathways involved in cancer growth and differentiation.^4,5 By synthetically harnessing other bioactive synthons with these active pharmacophores, molecular hybrids capable of stronger biological potency can be achieved. Since photodynamic therapy (PDT) has revolutionized noninvasive target cancer therapy, our approach has been to cultivate new hybrid molecular systems by covalently functionalizing quinazolinones with competent bioactive chromophores such as fullerenes.

Fullerenes, in particular, have unique properties, such as high electron affinity, high thermal stability, and potent antioxidant activity, making them a popular study focus in recent years.⁶ Hydrophilic derivatives of C₆₀ and C₇₀, in particular, have been investigated for their potential as anticancer and anti-HIV agents. Hydrophilicity has been a significant challenge concerning the biomedical assessment of fullerenes (C₆₀ or C₇₀). Research groups worldwide have attempted to impart water solubility to fullerenes to exploit their properties to develop innovative and effective biological solutions. Hydrophilic fullerene derivatives have since been evaluated for their antibacterial effects,^7,8 antiviral effects,⁹⁻¹¹ and use as radical scavengers^12,13 and photosensitizers (PSs)¹⁴ in cancer PDT. The common strategies employed for imparting water solubility include the incorporation of −COOH-containing moieties,^15,16 −OHs,^17,18 quaternary ammonium salts,¹⁹⁻²¹ peptides,^9,22 sugars,^9,23 cyclodextrins,^14,24 and PEG,^25,26 among others.^27,28 The development of synthetic methodologies like Prato²⁹ and Bingel reaction³⁰ allowed for easier derivatization of fullerenes with higher yields and better isolation strategy. Functionalized fullerenes show promise in addressing systemic toxicity and drug resistance commonly encountered in traditional chemotherapy.⁶ C₆₀ and C₇₀ are two of the most commonly studied fullerene molecules, and they have been investigated for their potential as anticancer agents.^9,31 Functionalized fullerenes have properties that are beneficial for anticancer therapy, including inhibiting tumors and addressing issues such as systemic toxicity and drug resistance that are commonly encountered in traditional chemotherapy.⁶ By synthesizing the molecular hybrid of quinazolinone and fullerenes (Figure 1), we intend to introduce a photosensitive quinazolinone–fullerene (Q-F) nanohybrid, wherein the influence of the fullerenes on the quinazolinone pharmacophore as anticancer agents under PDT will be investigated.

Figure 1.

Target hydrophilic quinazolinone-C₆₀/C₇₀ dyads.

The target hydrophilic Q-F nanohybrids and their quinazolinone precursors were synthesized through a multistep synthetic pathway as outlined in Figure S1. The initial synthetic step involved the synthesis of 2-(pyridin-3-yl)-2,3-dihydroquinazolin-4(1H)-one (DHQP₃) by reacting anthranilamide and 3-pyridinecarboxaldehyde in refluxing ethanol in the presence of CoCl₂ as the catalyst. The product DHQP₃ was subjected to oxidation by KMnO₄ in acetone to obtain 2-(pyridin-3-yl)quinazolin-(4H)-one (Q₃). The compounds DHQP₃ and Q₃ have been reported earlier, prepared via different synthetic methods.^32,33Q₃ was then derivatized with 4-(3-bromopropoxy)benzaldehyde through a nucleophilic substitution reaction carried out in dimethylformamide (DMF) in the presence of K₂CO₃ and KI. The aldehyde 4-(3-(4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-ylpropoxy)benzaldehyde (QP₃Al) so formed was isolated through silica gel gravity percolation column chromatography. The Q-F nanohybrids were then synthesized through a modified Prato reaction involving QP₃Al, fullerene (C₆₀/C₇₀), and sarcosine. The three-component one-pot synthesis was carried out in refluxing toluene under an inert atmosphere of gaseous Ar. The progress of the reaction was monitored by thin-layer chromatography (TLC). While the formation of the C₆₀-quinazolinone nanohybrid Q₃C₆₀ required 8 h of reaction time, the formation of the C₇₀-quinazolinone nanohybrid Q₃C₇₀ required 13 h. The compounds were isolated by column chromatography using 60–200 mesh silica gel as the stationary phase. Sequential elution was performed using toluene and chloroform as eluents. The final step to obtain the hydrophilic Q-F nanohybrids was the room-temperature alkylation of Q₃C₆₀ and Q₃C₇₀ with methyl iodide to obtain the corresponding pyridinium salts Q₃C₆₀M and Q₃C₇₀M.

The isolated compounds were characterized by nuclear magnetic resonance (¹H NMR and ¹³C NMR), matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF), ultraviolet–visible light (UV-vis) spectroscopy, and emission spectroscopy, whichever is applicable. The final step to obtain the hydrophilic Q-F nanohybrids was the room-temperature alkylation of Q₃C₆₀ and Q₃C₇₀ with methyl iodide to obtain the corresponding pyridinium salts Q₃C₆₀M and Q₃C₇₀M.

The UV-vis spectra of QP₃Al, Q₃C₆₀, and Q₃C₇₀ were recorded by using a 20 μM solution of the compounds in CHCl₃. The spectral plot (Figure S2A) exhibited clear variations, indicating structural differences among the compounds. The Q-F nanohybrids, Q₃C₆₀ and Q₃C₇₀, exhibited strong absorption peaks at 254 and 246 nm, respectively, which can be attributed to the appended fullerene (C₆₀/C₇₀) moiety. In the pyridinium salts (Q₃C₆₀M and Q₃C₇₀M) of the Q-F nanohybrids and the quinazolinone derivative Q₃M, the corresponding absorption peaks (Figure S3A) were observed at 282, 326; 275, 323; and 278 nm, respectively, indicating bathochromic shifts. The emission spectra of QP₃Al, Q₃C₆₀, and Q₃C₇₀ dissolved in CHCl₃ display several weak emission bands with maxima at 385, 769, and 743 nm (see Table S1 and Figure S2B), respectively. The spectral data was recorded by excitation of a 20 μM solution of the compounds QP₃Al, Q₃C₆₀, and Q₃C₇₀ at 302, 323, and 396 nm, respectively.

The emission spectra of Q₃M, Q₃C₆₀M, and Q₃C₇₀M, when dissolved in DMF, display several emission bands at 507, 645, 677, and 715 nm; 444, 645, 676, and 718 nm; and 437, 468, and 708 nm, respectively (see Table S1 and Figure S3B). For data collection, 20 μM solutions of the compounds in DMF were employed, and solutions of compounds Q₃M, Q₃C₆₀M, and Q₃C₇₀M were excited at 326, 323, and 379 nm, respectively.

As indicated by the NMR analysis (Figures S6–S13), the formation and purity of the compounds are well-established by identifying the characteristic signals for the quinazolinones, the aldehyde-linked quinazolinone, and its fullerene derivatives identified in the experimental section. The ¹H NMR spectra of the quinazolinone aldehyde QP₃Al (Figure S7) indicate the successful attachment of the formyl linker, the aldehydic proton giving rise to a singlet centered at 9.88 (s, 1H) ppm. The methylene protons of the three C atom linker give rise to the expected triplet, quartet, and triplet peak combinations at 4.94, 2.59, and 4.33 ppm, respectively. The aromatic protons of QP₃Al resonate between 8.81 and 7.02 ppm. The presence of the fulleropyrrolidine ring for the nanohybrid species, Q₃C₆₀, and its alkylated salt, Q₃C₆₀M, is proved by the presence of the two doublets at the highly deshielded aliphatic regions due to the presence of the enantiotopic CH₂– protons of the five-membered fulleropyrrolidine ring condensed with the fullerenes (Figure S4). Furthermore, the resonance of −N–CH₃ and the single proton present at the C atom of the linker species that attaches the fulleropyrrolidine to the phenyl ring of the flexible linker establishes the formation of the desired product. In species Q₃C₆₀M, the methylated N atom is characterized at δ = 2.70 ppm as a singlet. The protons of the fused benzene ring of the quinazolinone moiety in Q₃C₆₀ resonate at 8.17 (d, J = 8.0 Hz), 7.53 (t, J = 7.6 Hz), 7.85 (t, J = 7.7 Hz), and 7.98 (d, J = 8.3 Hz) ppm, while those of the 3-pyridyl ring exhibit peaks at 9.75 (s), 8.7 (d, J = 3.6 Hz), 7.42 (dd, J = 7.9, 4.39 Hz), and 8.81 (d, J = 8.0 Hz) ppm (Figure S4).

The peak assignments are ably supported by the COSY spectra (Figure S5), the cross peaks suggesting through space coupling. The three C atom linker’s methylene protons display a characteristic pattern in the NMR spectrum, with peaks appearing as a triplet, quartet, and triplet at chemical shifts of 4.92, 2.45, and 4.25 ppm, respectively. Additionally, the ¹³C signals indicate the presence of C₆₀ fullerene (Figure S10). The quinazolinone moiety in Q₃C₆₀M exhibits proton resonances (Figure S11) in the fused benzene ring at 8.29 (d, J = 7.6 Hz), 7.55 (m), and 8.06 (d, J = 8.0 Hz). Additionally, the 3-pyridinium ring displays peaks at 9.85 (s), 9.12 (d, J = 5.7 Hz), 7.77 (m), and 9.45 (d, J = 8.0 Hz) ppm. A noticeable downshift in the proton resonances of the 3-pyridinium group in Q₃C₆₀M, compared to the 3-pyridyl group in Q₃C₆₀, demonstrates the effect of the additional positive charge on electron density around the resonating protons. The NMR spectrum also reveals a characteristic pattern for the methylene protons of the three C atom linker, with peaks appearing as triplet, multiplet, and triplet at chemical shifts of 4.85, 2.39, and 4.29 ppm, respectively.

The C₇₀ molecule possesses D₅h symmetry with 20 hexagonal and 12 pentagonal rings, forming a closed-cage structure with icosahedral symmetry. Each polygon’s vertices are carbon atoms, bonded along each edge. It has two types of bond lengths: the 6:6 ring bonds (considered “double bonds”) are shorter than the 6:5 bonds. Structurally similar to C₆₀, it has a belt of 5 hexagons inserted at the equator.^34,35 However, unlike C₆₀, C₇₀ lacks “super aromaticity” due to the absence of double bonds in pentagonal rings, leading to poor electron delocalization. Consequently, they behave like electron-deficient alkenes, readily reacting with electron-rich species.

The molecule’s stability can be attributed to factors such as its geodesic shape and electronic bonding. The C₇₀ molecule exhibits eight different bond lengths, ranging between 0.137 and 0.146 nm.³⁶ C₇₀ derivatives respond to various regioselective isomers upon covalent functionalization due to the different types of C=C bonds, unlike C₆₀ derivatives. These regioselective isomers have similar R_f values and contribute to signal overlapping in NMR. This overlap makes distinguishing and assigning specific signals to individual carbon atoms or functional groups within the molecule difficult. Furthermore, C₇₀ derivatives can display broadened NMR signals due to molecular motion, molecular dynamics, or interactions with the solvent. These broadened signals can hinder the resolution and interpretation of the NMR spectrum, making it challenging to obtain clear and distinct peaks.³⁷ Therefore, the NMR of the compound Q₃C₇₀M could not be obtained in a pure form for the characterization of signals.

All the nanohybrids and their precursors quinazolinone derivatives were successfully characterized by MALDI-TOF mass spectroscopy (Figures S14–S21), taken in dithranol as a matrix. MALDI-TOF mass spectrometry utilizes laser desorption and ionization of matrix–analyte cocrystals to generate ions, which are then accelerated and measured based on their flight time in a time-of-flight (TOF) detector. This technique allows for the rapid and accurate analysis of biomolecules, making it valuable in various fields such as proteomics, clinical diagnostics, and drug discovery.

We next investigated the effect of these nanohybrids on cell systems to propose their plausible physiological relevance. For this, we adopted three distinct experimental approaches. First, Q-F nanohybrid-induced cytotoxicity on U87-MG glioblastoma cells was assayed by Western blot analyses by verifying changes in the expression of different proteins affecting apoptosis, such as CASAPASE 3 and 7, BAX, BCL2, and a cell cycle marker CYCLIN A upon PDT exposure. Second alterations in cytomorphology and cell survival were evaluated by imaging through phase contrast microscopy. Furthermore, fluorescence imaging studies were undertaken to evaluate nuclear condensation, ROS generation, and increase in the number of apoptotic cells, in response to PDT in cells with Q-F nanohybrids; flow cytometry was also utilized to score for ROS generation.

Q₃M, Q₃C₆₀M, and Q₃C₇₀M were accessed for their potential to elicit changes in the cell cycle and apoptosis-associated proteins to establish their anticancer effects (Figure 2). The nanohybrids Q₃C₆₀M and Q₃C₇₀M demonstrated predominant anticancer activities, compared to their precursor quinazolinone Q₃M salt. Photoactivation of Q₃M did not elicit significant changes in the levels of CYCLIN A2, BAX, BCL-2, and cleaved CASPASES 7 and 3. For this, U87-MG cells were treated with Q₃M and either subjected to PDT or left untreated; cell lysates generated were analyzed by Western blots for the indicated proteins and compared against β-TUBULIN. To analyze the advantage of the Q-F nanohybrids over the Q₃M precursor, U87-MG cell lysates treated with the indicated drugs were either subjected to PDT or left untreated (Figure 2). Western blot analyses showed that treatment with Q₃C₆₀M or Q₃C₇₀M, even without PDT, led to significant upregulation of the pro-apoptosis-associated protein BAX and also cleavage of CASPASE-7, signifying its activation. PDT further increased the levels of these proteins, indicating the elicitation of apoptosis after the photoactivation of the Q-F nanohybrids. Both BAX and cleaved CASPASE-7 are well-established markers of apoptosis.³⁸ On the other hand, BCL-2 protein levels were seen to be lower in lysates derived from cells with Q₃C₆₀M exposed to PDT. BCL-2 is a pro-survival regulator; hence, a decrease in its levels upon photoactivation indicates the onset of apoptosis.³⁹ However, PDT of Q₃C₇₀M did not lead to a significant change in BCL-2 levels. The BAX:BCL-2 ratio also showed an increasing trend, further supporting a pro-apoptotic condition. There may be a compensatory mechanism to justify this anomaly—the amount of CASPASE-7 activation is probably sufficient to overcome a lack of effect from BCL-2. Furthermore, a significant decrease in the levels of CYCLIN A2 confirmed our hypothesis regarding the anticancerous effects of the Q-F nanohybrids upon PDT. CYCLIN A2 levels are known to be upregulated in gliomas and positively correlate with their metastasis.⁴⁰ Hence, the downregulation of this factor supports the antitumorigenic properties of the Q₃C₆₀M and Q₃C₇₀M nanohybrids. Our results suggest a notable advantage of Q₃C₆₀M and Q₃C₇₀M over the precursor salt Q₃M.

Figure 2.

Altered expression of proapoptotic and antiapoptotic proteins with PDT of Q-F nanohybrids. (A, B) Precursor salts do not elicit significant proapoptotic or antiapoptotic effects. Western blots of the indicated proteins were quantified and plotted. Data represent the mean ± standard error of measurement (SEM) of three independent experiments. (C) Lysates from U87-MG cells treated with Q-F nanohybrids upon PDT were analyzed for the indicated antibodies; CYCLIN A2 indicated as CYCLIN A. (D) Histogram plotting in BAX:BCL2 ratio from data generated in panel C. (E) Graph with data from panel A. [Legend: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001, using an unpaired two-tailed Student’s t-test with data from at least three independent experiments. Error bars indicate ± SEM. DMSO denotes DMSO-treated control, unexposed cells, DARK represents treated, unexposed cells, and LIGHT denotes cells treated with PDT. Note a significant increase in proapoptotic proteins with PDT.

Shrinkage of cell volume and membrane deformities are characteristic changes observed during apoptosis. More than 70% of U87-MG glioblastoma cells treated with Q-F dyads, but not exposed to light, display a uniform shape and margins when imaged under phase contrast microscopy (Figure 3). However, PDT treatment led to drastic cytomorphological changes, with distinct shrinkage in cellular area and volume, membrane blebbing, and loss of neuronal morphology in ∼95% of the cells. However, these Q-F nanohybrids did not elicit significant phenotypic changes in an immortalized cell line, COS7 (<5%). Cell death in U87-MG cells was characterized as apoptosis, evidenced by condensed DNA and fragmented nuclei.⁴¹ Nuclear fragmentation and chromatin condensation, established markers of apoptosis, were evaluated in U87-MG cells treated with Q-F nanohybrids by using a DAPI staining assay (Figure 4). The morphological changes in the nucleus and its condensation in U87-MG cells treated with Q-F drugs during PDT were significantly greater than those in cells treated in darkness, confirming the higher cytotoxic effect of these nanohybrids upon PDT. The apoptosis of cells in the presence of Q-F nanohybrids upon PDT was further confirmed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Figure 5). This assay specifically detects DNA fragmentation, which is an established phenotype of late apoptotic cells.⁴² We observed that treatment with these nanohybrids upon PDT resulted in a significant increase in the percentage of apoptotic cells, compared to the DMSO control and samples left unexposed to light (darkness). As a positive control, one set of cells was treated with 0.5 mM CoCl₂, which is a reagent routinely used to simulate conditions of hypoxia in cells and likely to induce apoptosis. It has been reported that PDT induces oxidative stress and activates various antiapoptotic and proapoptotic pathways.⁴³ Our results suggest that the antiapoptotic activities of the nanohybrids are enhanced upon light treatment. Therefore, we aimed to understand whether these compounds could induce reactive oxygen species (ROS) in the glioma cell line. ROS production in cells was analyzed using two methods: flow cytometry and fluorescence microscopy, employing the cell-permeant reagent 2′,7′-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA). U87-MG cells were treated with Q₃C₆₀M and Q₃C₇₀M drugs at a final concentration of 10 μM. After 1 h of PDT, cellular ROS production was examined using a CM-H2DCFDA probe, as described in the manufacturer’s protocol. FACS results showed an increase in ROS production in Q₃C₆₀M and Q₃C₇₀M drug-treated cells, compared to DMSO-treated ones (Figure 6). The increase in ROS population from 4% ± 0.4% (Q₃C₆₀M_Dark) and 4% ± 0.18% (Q₃C₇₀M_Dark) to 29.5% ± 3.8% (Q₃C₆₀M_Light) and 12.4% ± 1.5% (Q₃C₇₀M_Light) was observed. This analysis indicates that Q₃C₆₀M and Q₃C₇₀ nanohybrids induce ROS generation in the U87 glioma cell line, and their activity is enhanced by photodynamic therapy or PDT.

Figure 3.

Q₃C₆₀M and Q₃C₇₀M induce cytomorphological changes in tumor-derived cells upon PDT. (A) U87-MG cells treated with Q-F nanohybrids upon PDT induced changes in cellular morphology. Scale bar = 150 μm. The expanded image represents Q₃C₆₀M and Q₃C₇₀M drug-treated U87_MG cells and shows the significant morphological differences between the PDT-treated condition, where the cells were spherical in shape and showed loss of elongated neuronal morphology, compared to the untreated condition. Approximately 400 cells were analyzed for each sample from at least 3 independent experiments. (B) Histogram plotted from data generated in panel (A). (C, D) COS7 cells treated with Q-F nanohybrids with or without PDT treatment. No significant morphological changes were observed, irrespective of PDT. [Legend: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001 using an unpaired two-tailed Student’s t-test. Error bars = ±SEM. Approximately 300 cells were analyzed for each sample from at least 3 independent experiments.]

Figure 4.

Q-F nanohybrids induce nuclear fragmentation and chromatin condensation. U87-MG cells treated with Q-F nanohybrids either exposed to light or left unexposed were stained with 10 μM DAPI and imaged. Scale bar = 10 μm. Expanded images are represented as insets. Upper panel inset shows changes in cellular morphology; lower panel inset shows apoptotic nuclei (condensed and fragmented) marked by red arrows. The inset is marked with a red arrow. Expanded image represents Q₃C₇₀M drug-treated U87_MG cells. Apoptotic nuclei (condensed and fragmented) marked by a red arrow. (B) Histogram plotted with data from panel (C). Note the significant increase in apoptotic cells with PDT. [Legend: (***) p ≤ 0.001, using an unpaired two-tailed Student’s t-test. Error bars = ±SEM. Approximately 350 cells were analyzed across all samples from at least 3 independent experiments.]

Figure 5.

TUNEL assay of Q-F nanohybrids in U87-MG cells. (A) U87-MG cells were treated with Q₃C₆₀M and Q₃C₇₀M subjected to PDT (LIGHT) or left unexposed (DARK). As a negative control, cells were treated with DMSO and as a positive control for apoptosis, cells were treated with 0.5 mM CoCl₂. Cell death by apoptosis was assayed by TUNEL. Q-F nanohybrid-treated cells had a higher number of TUNEL-positive nuclei, which significantly increased upon PDT treatment both in Q₃C₆₀M and Q₃C₇₀M samples. The cells were counterstained with DAPI to mark the nuclei. Scale bar = 50 μM. (B) Graph with data from panel (A). [Legend: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001, using an unpaired two-tailed Student’s t-test. Error bars = ±SEM. Approximately 20 fields were analyzed for each sample from at least 3 independent experiments.]

Figure 6.

The Q-F nanohybrids induce ROS production in cells after PDT. The levels of ROS production in U87_MG cells were detected using CM-H2DCFDA. (A) Flow cytometric analyses of nanohybrid-treated U87_MG cells show increased ROS production upon PDT. Data represents the mean value ± SEM of 3 experiments. The population P2 represents cells with lower ROS levels, and population P3 represents cells with enhanced fluorescence indicating higher ROS levels. (B) Graph with data from panel (A) acquired from at least 3 independent experiments. Note the significantly higher P3 population in cells with Q₃C₆₀M in PDT condition (LIGHT), compared to control DMSO-treated or unexposed cells (DARK). Q₃C₇₀M treatment with PDT conditions resulted in increased ROS levels, although less than those with Q3C60M. (C) Note that U87-MG after PDT with Q-F nanohybrids showed enhanced fluorescence of CM-H2DCFDA, compared with the unexposed controls. 0.5 mM CoCl₂ treatment of cells was used as a positive control for ROS production in cells. [Scale bar = 100 μm. Approximately 15 fields were analyzed for each sample from at least 3 independent experiments.]

In this study, we aimed to demonstrate the distinctive and promising photobiological characteristics of Q-F nanohybrids in the context of antineoplastic PDT. Our analysis thoroughly examines how the incorporation of C₆₀ and C₇₀ components affects the anticancer efficacy of quinazolinones. Integrating quinazolinones into photobiologically active nanohybrids featuring C₆₀ and C₇₀ fullerenes enhances their effectiveness as potent antineoplastic agents. These amphiphilic Q-F nanohybrids show potential in targeting glioblastoma cells, disrupting oncogenic processes, and modulating crucial proteins involved in cell cycle regulation and apoptosis. The Q₃C₇₀M nanohybrid exhibits anticancer efficacy by inducing cell apoptosis. Q₃C₆₀M prompts cell apoptosis, triggering cell death. It is noteworthy that the distinct functions of C₆₀ and C₇₀ derivatives, specifically Q₃C₆₀M and Q₃C₇₀M, in provoking apoptosis and cell differentiation, underscore the versatility of these nanohybrids. ROS analysis revealed that, compared to Q₃C₇₀M, the Q₃C₆₀M nanohybrid significantly induced more ROS-generated cells, compared to the untreated control. While Q₃C₇₀M proves to be a potent candidate for anticancer treatment, it appears to induce cellular apoptosis through a pathway independent of ROS generation. Furthermore, the amphiphilic properties of Q₃C₆₀M and Q₃C₇₀M enhance their solubility and bioavailability, improving the systemic distribution and the potential for intravenous administration.

Our research work presents compelling evidence for the potential of Q-F nanohybrids as innovative anticancer agents, offering enhanced photodynamic properties, targeted cytotoxicity, and refined drug delivery mechanisms. It highlights the importance of exploring C₇₀ as a potential anticancer pharmacophore, setting a solid foundation for future research. The study introduces amphiphilic Q-F nanohybrids with strong affinity for targeted antineoplastic effects while remaining nontoxic to noncancerous cells. By integration of quinazolinones into nanohybrids featuring C₇₀ and C₆₀ fullerenes, it significantly advances their role as potent antineoplastic agents. These nanohybrids target cancer cells, hinder oncogenic processes, and regulate crucial proteins involved in cell cycle regulation and apoptosis. Q₃C₇₀M and Q₃C₆₀M induce cell apoptosis, with Q₃C₇₀M demonstrating robust anticancer effectiveness, overcoming resistance associated with conventional chemotherapy by inducing cancer cell differentiation. The distinct roles of C₆₀ and C₇₀ derivatives underscore the versatility of these nanohybrids. This study urges further exploration of C₇₀ as a promising anticancer pharmacophore, paving the way for future advancements in this field.

Data Availability Statement

All data generated or analyzed during this study are included in this published article (and its Supporting Information files).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00187.

• Materials and methods, UV-vis, emission, ¹H NMR, and mass spectral data, unedited protein blots (PDF)

Author Contributions

^∇ These authors contributed equally to the respective sections. D. Sengupta conceptualized and standardized a methodology for designing, synthesizing, and applying compounds as antineoplastic agents, securing funding and interpreting results. D. Sharma and R.K.D. performed synthesis, isolation, and characterization of quinazolinones and Q-F nanohybrids, with equal contributions to investigation, validation, and analysis. D. Sharma also compiled and interpreted data. P.R. assisted in acquiring data for photophysical studies. P.D. and M.H. conducted antineoplastic studies and contributed equally to methodology, investigation, analysis, and writing. O.C. oversaw and funded assays analyzed results, and contributed to writing and supervision of the biological evaluation. All authors contributed to and approved the final manuscript.

This work was supported by the DBT project (Reg. No. BT/PR25024/NER/95/961/2017) granted to D. Sengupta by the Department of Biotechnology, Ministry of Science and Technology, Government of India. O.C. is funded by intramural funding from the Department of Atomic Energy, Government of India.

The authors declare the following competing financial interest(s): D. Sengupta, D. Sharma, R.K.D., P.D., M.H., and O.C. are inventors on Indian Patent Application No. 202331048869, submitted by Assam University titled, "Amphiphilic Quinazolinone-C70 and Quinazolinone-C60 Nanohybrids as New Generation Antineoplastic Agents under Photoactivated Conditions". P.R. does not have any conflicts of interest to declare.