Anticancer phytochemical nanodelivery systems using graphene derivatives: A review

2.1. Vinca alkaloids

The vinca alkaloids consist of around 130 terpenoid indole alkaloids, which are naturally derived from the leaves of C. roseus [36]. Vincristine, a prominent vinca alkaloid, was first approved by the United States Food and Drug Administration (U.S. FDA) in July 1963 under the brand name Oncovin^® (1 and 5 mg/vial as vincristine sulfate) by Eli Lilly and Company. Preclinical studies demonstrated its ability to cure P-1534 leukemia in DBA/2 mice and induce remission in childhood acute leukemias [37]. A preservative-free formulation, vincristine sulfate injection (vincristine sulfate PFS^®, 1 mg/ml, single-dose vial), later gained FDA approval in September 1999 for marketing by Teva Pharms USA (NDA 075493), following demonstration of bioequivalence to Eli Lilly’s Oncovin^® [38]. The liposomal formulation vincristine sulfate liposome (Marqibo^®), marketed by Talon Therapeutics, Inc., was approved in September 2012 (NDA 202497) for adult patients with Philadelphia chromosome-negative (Ph–) acute lymphoblastic leukemia in second or later relapse or with disease progression after at least two prior anti-leukemia regimens [39]. However, Marqibo^® was voluntarily withdrawn from the U.S. market in November 2021 at the request of Acrotech Biopharma, and the FDA formally withdrew approval of NDA 202497 in May 2022 (https://www.federalregister.gov/documents/2022/05/02/2022-09235).

In parallel, vinblastine sulfate (Velban^®, 10 mg/vial) received FDA approval under NDA 012665 in November 1965 for generalized Hodgkin’s disease, lymphocytic lymphoma, and advanced testicular carcinoma, marketed by Eli Lilly and Company [40]. Vinorelbine tartrate (Navelbine^®), a semisynthetic derivative marketed by Glaxo Wellcome Inc., was approved in August 2000 (NDA 020388) for intravenous treatment of nonsmall cell lung cancer. Subsequent clinical studies in November 2002 demonstrated that Navelbine^® in combination with cisplatin was effective in chemotherapy-naive patients with Stage IV or Stage IIIb nonsmall cell lung cancer, including those with malignant pleural effusion or multilobar disease [41].

Vinca alkaloids bind to tubulin, disrupting microtubule polymerization in the mitotic spindle and inducing prolonged cell cycle arrest, ultimately leading to cell death [42]. Vincristine binds more strongly to tubulin compared to vinblastine and the vinca derivatives vindesine and vinorelbine. This higher binding affinity enhances its ability to disrupt microtubule formation and inhibit cell division [43]. The dimeric structure of these alkaloids mediates their activity through two mechanisms: the vindoline moiety interacts with the β-subunit of tubulin heterodimers, inhibiting mitosis and inducing apoptosis, whereas the catharanthine moiety exhibits weaker effects on α/β-tubulin polymerization [44]. In a murine carcinoma model, vinblastine (7.5–10 mg/kg) caused a marked and sustained reduction in tumor blood flow, reaching ~10% of baseline within 2 hours and remaining below 20% at 24 hours, leading to early necrosis and an 11-day tumor growth delay. Vincristine (3 mg/kg) produced similar but less pronounced effects. In contrast, normal tissues (skin, kidney, liver, and muscle) exhibited blood flow reductions of <40%, with full recovery within 6 hours [45].

Toxicity issues from a clinical perspective are evident from two trials. In a phase III trial (NCT00059839) for advanced anaplastic large cell lymphoma, vinblastine consolidation caused notable adverse effects, including febrile neutropenia (29.5%), anemia (39.3%), gastrointestinal events such as vomiting (16.4%) and diarrhea (8.2%), and infections (32.8%), while vincristine was associated with slightly higher serious cardiac events (3.1%). In a phase III trial (NCT00003389) for Hodgkin’s lymphoma, ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) was linked to serious toxicities including infections with neutropenia (5.6%), anemia (4.8%), and febrile neutropenia (1.7%). The Stanford V regimen (DOX, vinblastine, vincristine, bleomycin, mechlorethamine, etoposide, prednisone) showed higher rates of anemia (20.4%), infections with neutropenia (6.4%), febrile neutropenia (2.6%), and motor neuropathy (5.7%). Cardiac events were rare, with the ABVD regimen causing conduction abnormalities (0.2%) and cardiac ischemia (0.7%), while the Stanford V regimen caused sinus tachycardia (0.7%) and supraventricular arrhythmias (0.5%).

Traditional intravenous (i.v.) delivery of vincristine, vinblastine, and vindesine exhibits significant pharmacokinetic differences in humans that influence their toxicity. After rapid administration (i.v.), all three showed a triphasic serum decay pattern. Vincristine had a much longer and more variable terminal half-life (85±69hours) compared to vindesine (24±10hours) and vinblastine (25±7hours), and the slowest clearance (0.106l/Kg/h vs. 0.252 and 0.740l/Kg/h, respectively) [46]. In mice, vinorelbine showed a three-compartment pharmacokinetic profile with a longer elimination half-life compared to vinblastine, with tissue concentrations 5–10 times higher than vinblastine, especially in lymphatic and testicular tissues. About 58% of the administered dose was excreted via urine (17%) and feces (41%), indicating prolonged tissue retention and distinct disposition [47]. Vinflunine (fluorinated vinca alkaloid) showed dose-limiting toxicities at 400mg/m² included mucositis, constipation, and short neutropenia [48]. Vincristine also alters the biodistribution of radiopharmaceuticals, as demonstrated in BALB/c mice, where it significantly reduced uptake of technetium-99m methylene diphosphonate in all major organs, indicating broad systemic biological effects beyond its cytotoxic activity [49]. In addition to toxicity and pharmacokinetic limitations, traditional intravenous delivery of vinca alkaloids faces challenges in formulation stability. A study showed that vincristine, vinblastine, and vindesine remain stable for up to 7 days at 4°C in both 5% glucose and 0.9% sodium chloride, while vinorelbine is more stable in 5% glucose (7 days) than 0.9% sodium chloride (3 days), indicating formulation and storage constraints for clinical use [50].

2.2. PTX

PTX is a diterpenoid natural product discovered by Wall and Wani in 1966 as the principal bioactive constituent of T. brevifolia (Pacific yew) bark, with its chemical structure fully elucidated in 1971 [51]. The first clinical formulation of PTX, injectable Taxol^® (6 mg/ml), was approved by the U.S. FDA (NDA 020262) and marketed by Bristol-Myers Squibb Co in December 1992 for refractory ovarian cancer. Later, it received indications for refractory or anthracycline-resistant breast cancer (April 1994), Kaposi’s sarcoma (August 1997), nonsmall cell lung cancer (April 1998), and advanced ovarian carcinoma in combination with cisplatin (June 1998) [52]. To reduce hypersensitivity reactions associated with the Cremophor EL (polyoxyethylated castor oil)-based formulation, a revised premedication regimen was introduced in June 2000, requiring oral dexamethasone (20 mg at 12 and 6 hours prior), along with intravenous diphenhydramine (50 mg) and either cimetidine (300 mg) or ranitidine (50 mg) administered 30–60 minutes before Taxol^® infusion [52]. A major advancement in PTX formulation emerged with the development of albumin-bound PTX (Abraxane^®), a Cremophor-free nanodroplet formulation designed to enhance solubility and reduce toxicity. Abraxane^® received FDA approval in July 2005 (NDA 021660), marketed by Abraxis BioScience, Inc., for refractory, metastatic, or relapsed breast cancer, representing a significant innovation in PTX delivery technology [53].

Extracts of Taxus cuspidata (containing 2.25% PTX) have demonstrated broad-spectrum anticancer activity, inhibiting 70%–90% of human cancer cell growth (HL-60, BGC-823, KB, Bel-7402, HeLa) by inducing apoptosis and G2/M cell cycle arrest, and synergistically enhancing the efficacy of 5-fluorouracil (5-FU) with a combination index (CI) of 0.93. Pharmacokinetic studies in Sprague–Dawley rats showed that PTX did not alter 5-FU absorption or plasma parameters [54]. PTX has also shown synergistic effects when combined with other compounds. For example, combination with β-elemene (derived from Curcuma zedoaria) enhanced apoptosis in human lung cancer cells (H23 and H358) via mitochondrial pathways independent of p53 and Fas. This involved cytochrome c release, activation of caspase-8 and -3, and downregulation of Bcl-2 [55]. In vivo studies demonstrated its potent anticancer effects: administration (i.v.) in Swiss mice had a maximum tolerated dose of 12mg/kg and a lethal dose of 19.5mg/kg. In NCI-H460 xenograft-bearing nude mice, PTX reduced tumour volume by a mean of 25.3% and extended the median time to reach 600mm³ tumour volume to 16days [56].

PTX at a low dose (4 mg/kg), when combined with a macrophage surface-modified tumour-cell vaccine, showed the greatest tumour regression and strongest CD8⁺ T-cell immune response, especially when the vaccine was given 2 days after PTX [57]. In vitro (MDA-MB-231 and primary tumor cells), sub-lethal doses (1–5 nM) combined with conditioned medium from human uterine cervical stem cells (CM-hUCESC) reduced tumor cell proliferation and invasiveness by 50%–70% and increased apoptosis by 40%–60%. In mouse xenografts, the combination significantly suppressed tumor growth [58].

Despite its promising anticancer activity, the primary limitation is its extremely low aqueous solubility (0.30 ± 0.02 μg/ml), which complicates formulation and clinical administration. Hydrotropic agents such as N,N-Diethylnicotinamide, N-Picolylnicotinamide, N-allylnicotinamide, and sodium salicylate can significantly enhance its solubility [49]. In addition, PTX also exhibits notable toxicities. Intraperitoneal administration (i.p., 8 mg/kg) induced mechanical and cold hypersensitivity and reduced nerve conduction amplitude in both male and female C57BL/6J mice [59]. High doses (i.v., 10–20 mg/kg) caused severe toxicity and rapid death in Sprague–Dawley rats, whereas a low dose of 5 mg/kg (i.v.) was well tolerated [60]. In nude mice, 30 mg/kg for 3 weeks resulted in a 2.4-fold increase in micronucleated erythrocytes, indicating genotoxic effects [61]. Oral administration is limited by intestinal P-glycoprotein, which reduces absorption and increases direct excretion into the gut. In wild-type mice, oral PTX showed low bioavailability (11%) and high fecal excretion (87%). In contrast, mice lacking intestinal P-glycoprotein exhibited increased bioavailability (35%) and reduced fecal excretion (<3%) [62]. Priming doses of CUR for 4 days increased PTX absolute bioavailability from 2.78% to 4.49% and inhibited CYP2C8 and CYP3A4 activity in rat liver microsomes by 61% and 72%, respectively [63]. A chitosan-stearic acid micelle system, modified with L-carnitine and loaded with QUE (148nm), enhanced oral PTX delivery, with good drug loading (7.05%), increased bioavailability to 168%, and improved absorption while inhibiting CYP3A4 metabolism [64]. Glycyrrhizic acid micelles showed high encapsulation efficiency (≈90%) and drug loading (7.9%) and achieved approximately sixfold higher oral absorption of PTX in rats, mainly due to increased uptake in the jejunum and colon [65].

2.3. CPT and its derivatives

CPT was first isolated in 1966 from the bark of C. acuminata with potent anticancer activity against mouse lymphoid leukemia L-1210 [66]. While showing strong anti-tumor activity, the original CPT had poor water solubility (1.34 μg/ml) [67]. To improve clinical utility, water-soluble CPT derivatives such as irinotecan and topotecan were developed. Irinotecan hydrochloride (Camptosar^®) was approved by the U.S. FDA in June 1996 (NDA 020571) based on three open-label, single-agent trials involving 304 patients with metastatic colorectal carcinoma, primarily with disease progression after 5-FU therapy [68]. In April 2000, Camptosar^® gained an expanded indication as part of first-line combination therapy with 5-FU and leucovorin for metastatic colorectal cancer [68]. Another major derivative, topotecan hydrochloride (Hycamtin^®, 4 mg base/vial), was approved in May 1996 (NDA 020671) and marketed by SmithKline Beecham Pharmaceuticals for metastatic ovarian cancer, supported by four clinical trials involving 452 patients [69]. Subsequent regulatory approvals expanded the clinical use of topotecan. Hycamtin^® was approved in November 1998 for chemosensitive small cell lung cancer following failure of first-line therapy and in June 2006 in combination with cisplatin for Stage IV-B, recurrent, or persistent cervical cancer unsuitable for curative surgery or radiotherapy [69]. An oral formulation of Hycamtin^® (0.25 and 1 mg capsules) was approved in October 2007 (NDA 020981) for relapsed small cell lung cancer in patients achieving a prior complete or partial response at least 45 days after first-line chemotherapy [70].

CPT exerts its anticancer effects by interacting with the TOP1 cleavage complex, leading to an increase in DNA strand breaks during replication. These breaks induce apoptosis, particularly during the S phase of the cell cycle [71]. CPT (0.5–2 μM) significantly reduced cell viability and induced apoptosis in temozolomide-resistant glioblastoma cells (U87MG-R and GL261-R). CPT triggered caspase-3 activation, DNA fragmentation, and a marked increase in intracellular reactive oxygen species (ROS). Mechanistically, CPT upregulated p53, phospho-p53, and p21, while downregulating CDK6, cyclin D1, and Bcl-xL, confirming ROS-mediated activation of the p53-p21-CDK6/E2F1 apoptotic pathway [72]. CPT combined with DOX (molar ratio of 4.5:1) exhibited strong synergy, increasing early apoptosis by 24% in BT-474 cells compared with either drug alone, while showing antagonistic effects in healthy endothelial cells, indicating enhanced cancer selectivity. In vivo delivery significantly reduced tumor volume in 4T1 models without observable toxicity in major organs [73]. CPT (3 mg/kg) enhanced the antitumor efficacy of low-dose apatinib (60 mg/kg) combined with a programmed cell death protein-1 inhibitor (10 mg/kg) in a BALB/c mouse model of hepatocellular carcinoma. The combination reduced tumor size and further suppressed Nrf2 and p62 expression without affecting body weight or liver and kidney function [74]. Irinotecan, a semisynthetic derivative of CPT and topoisomerase I inhibitor, combined with axitinib (100 mg/kg, i.p., weekly and 25 mg/kg, p.o., twice daily, respectively) demonstrated strong synergistic antiproliferative and proapoptotic effects in Capan-1 human pancreatic cancer xenografts in nude mice, significantly reducing tumor volume, inhibiting neovascularization, and increasing tumor apoptosis [75].

In human clinical evaluation, an interventional trial (NCT01612546) found that injection (i.v.) of polymeric CPT produced adverse events (≥30% of patients), including fatigue (80%), hypertension (60%), nausea and abdominal pain (50%), and peripheral sensory neuropathy (40%). Serious adverse events were observed in 20% of patients, including cardiac chest pain (10%), anemia (10%), abdominal pain (10%), and gastric hemorrhage (10%). Another trial (NCT00170625) found that the CPT-topotecan regimen produced substantial toxicity, with 46% of patients experiencing serious adverse events, with 30.8% mortality, and hematologic toxicities, including thrombocytopenia (30.8%) and anemia (11.5%). Another interventional trial (NCT03531827) evaluating a CPT conjugate in combination with enzalutamide in prostate cancer patients (n = 4) was terminated early due to toxicity, with 75% of patients developing serious adverse events, including lower gastrointestinal hemorrhage, syncope, cystitis, and one treatment-related death. All patients experienced nonserious adverse events, most frequently anemia (75%), nausea (75%), lymphocyte reduction (50%), and hematuria (50%).

From a toxicity standpoint, the maximum tolerated dose of irinotecan in BALB/c mice was 240mg/kg (single i.p. dose) without weight loss [76], while a dose of 270mg/kg caused significant gut toxicity and pain in BALB/c mice, which were markedly reduced in Tlr4 knockout mice [77]. In Swiss mice, a dose of 75 mg/kg (i.p., for 4 days) induced severe intestinal mucositis with marked histopathological changes in the small intestine and colon [78]. From a clinical perspective, an interventional trial (NCT00469898) evaluated lung cancer patients (n = 50) treated with CPT and irinotecan. Serious adverse events were reported in 52% of patients, including nausea (16%), vomiting (10%), decreased neutrophil count (12%), decreased platelet count (10%), fatigue (8%), and diarrhea (6%), with 6% mortality. In a different trial (NCT00042939), pancreatic cancer patients (n = 94) were treated with irinotecan and docetaxel, with or without cetuximab. Serious adverse events occurred in 76%–80% of patients, including diarrhea (30%–47%), neutropenia (22%–29%), nausea (20%–30%), and febrile neutropenia (4%).

Apart from adverse events and toxicity instances, CPT and irinotecan exhibit formulation constraints. CPT possesses limited chemical stability in 0.9% sodium chloride infusion solutions. While refrigerated solutions remain stable for at least 7 days. Stability at room temperature varies from 3 to 7 days, depending on concentration, with higher concentrations being more stable (4.0mg/ml, 7 days) than low concentrations (0.5mg/ml, 3 days), respectively [79]. Irinotecan is found to be highly unstable in milli-Q water (pH of 6.3) due to the presence of reactive groups in its chemical structure, which favor hydrolytic reactions [80]. Irinotecan is also photolabile; under light exposure in saline solutions, the lactone ring degrades, producing multiple breakdown products [81].

2.4. PPT

PPT is an important naturally derived aryltetralin lignan obtained from P. peltatum and P. emodi [82]. The clinical utilization of PPT derivatives has historically centered on the treatment of aggressive solid tumours and hematologic malignancies. Etoposide (Vepesid^®, 20 mg/ml) was approved in 1983 (NDA 018768; Corden Pharma) for intravenous first-line combination therapy of small cell lung cancer and for refractory testicular tumors following surgery or radiotherapy [83]. The therapeutic versatility of etoposide expanded with the introduction of oral Vepesid^® capsules in December 1986 (NDA 019557, OneSource Specialty). Available in 50 and 100 mg strengths, the oral formulation provided a more convenient dosing option for lung cancer patients while maintaining similar combination-therapy indications [84]. To address solubility and administration challenges, the prodrug Etopophos^® (etoposide phosphate) was subsequently approved under NDA 020457 (1996) [85], and NDA 020906 (1998) [86], by Cheplapharm/Bristol Myers Squibb in intravenous strengths of 100, 500, and 1000 mg base/vial, offering equivalent clinical indications such as first-line small cell lung cancer therapy with cisplatin and treatment of refractory testicular tumours [85,86]. Complementing etoposide-based products, Vumon^® (teniposide 10 mg/ml, i.v.) gained FDA approval in July 1992 (NDA 020119, HQ Specialty Pharma) for use in hematologic malignancies, particularly leukemia [87].

PPT inhibits microtubule assembly in the mitotic period, thereby preventing cell division during the metaphase of mitosis, resulting in anticancer action [82]. The deoxy-form exhibited significant anticancer activity in MDA-MB-231 human breast cancer xenografts in nude mice. Intravenous doses of 5, 10, and 20 mg/kg reduced relative tumor volumes to 42.87%, 34.04%, and 9.63% of controls, respectively, with the 20 mg/kg dose outperforming etoposide and docetaxel [88]. Etoposide and teniposide are the derivatives of PPT, which do not inhibit microtubule assembly but instead act by interacting with DNA and inhibiting DNA topoisomerase [82]. A Phase I trial evaluated cediranib (20 mg/day) in combination with etoposide (100 mg/m², days 1–3) and cisplatin (80 mg/m², day 1) in patients with extensive-stage metastatic lung neuroendocrine cancer (n = 18). Common toxicities included nausea, vomiting, neutropenia, and diarrhea, with grade 1–2 hypertension in 44% and grade 3 hemoptysis in 11%. The regimen achieved a 67% objective response rate and a median progression-free survival of 7.9 months, demonstrating promising clinical activity with manageable toxicity [89]. Teniposide (10mg/kg) exhibited potent anticancer activity against in vitro, 3D cell culture, organoid models, and in vivo in Lewis lung carcinoma-bearing mice. Treatment downregulated apurinic/apyrimidinic endonuclease, a DNA repair enzyme overexpressed in lung cancer [90].

From a clinical perspective, etoposide, in combination with carboplatin and carfilzomib, showed good anticancer activity in previously untreated patients with extensive-stage small-cell lung cancer, with all enrolled participants (n = 32) demonstrating disease control during the treatment period of the Phase 1b study (NCT01987232). A clinical trial (NCT00004916) evaluated the teniposide activity in combination with ifosfamide and PTX in relapsed Non-Hodgkin’s Lymphoma. Two clinical trials (NCT06758700 and NCT07074470) are recruiting to study the effects of teniposide for extensive-stage small cell lung cancer and a combination regimen with monoclonal antibody and selinixor for patients with relapsed or refractory nervous system lymphoma.

PPT showed strong cytotoxicity toward both tumour (4T1) and normal (3T3) cells. In Sprague–Dawley rats, biodistribution studies in 4T1 tumour-bearing mice showed higher tumour accumulation for prodrug NPs versus PPT solution. The maximum tolerated dose was 10 mg/kg for PPT, with higher doses causing severe hemolysis and systemic toxicity. In vivo, prodrug NPs displayed strong antitumour activity without organ toxicity, improved safety, and therapeutic performance over free PPT [91]. PPT (1 nM) caused spindle defects and DNA damage-induced apoptosis in mouse fertilized oocytes and early embryos [92]. Etoposide (10, 15, or 20 mg/kg) showed clastogenic toxicity to dividing spermatogonia within 24 hours, in male Swiss albino mice. The cytogenotoxic effects persisted and were transmitted through the male germline with elevated abnormal sperm frequencies even at week 8 post-treatment [93]. Teniposide exhibited both clastogenic and aneugenic effects in somatic and germ cells of male mice. It induced micronuclei formation in erythrocytes, caused a ~48 hours meiotic delay, and produced disomic and diploid sperms, particularly affecting the second meiotic division [94].

From formulation perspectives, PPT as a precursor molecule for semi-synthetic derivatives also faces significant physicochemical limitations. Polymorph screening of PPT identified two stable anhydrous forms (Form I and II), a metastable hydrate, and an unstable amorphous form. Form II possesses the best solubility, while the amorphous form dissolves the fastest but is highly unstable [95]. In S180 tumour-bearing Kunming mice, a single dose of free PPT (5 mg/kg, i.p.) showed modest tumour inhibition (38.9%) and caused reduced body weight gain, indicating toxicity, with higher doses being lethal in preliminary tests. Its pharmacokinetics showed a T_max of 1.23 ± 0.09hours, a C_max of 3.87 ± 0.67μg/ml, and a half-life of 29.93 ± 4.72hours [96].

2.5. RES

RES is a phenolic compound found in grapes, peanuts, blueberries, and blackberries, and has been extensively studied for its anticancer properties. Notably, it exerts its effects by upregulating pro-apoptotic proteins such as p53 and BAX while downregulating cancer-associated pathways and mediators, including NF-κB, AP-1, HIF-1α, matrix metalloproteinases, Bcl-2, COX-2, cytokines, and cyclin-dependent kinases [97]. A Phase 1 randomized, double-blind, placebo-controlled trial enrolled nine patients with colorectal cancer and liver metastases to evaluate oral micronized RES (SRT 501, 5 g/day) or placebo for 10–21 days. Published findings by Howells et al. [98] demonstrated that SRT501 was well tolerated in this patient population, with mean plasma levels reaching 1,942 ± 1,422 ng/ml after a single dose, approximately 3.6-fold higher than those reported for equivalent doses of nonmicronized RES. Pharmacodynamic analysis showed a significant 39% increase in cleaved caspase-3, an apoptosis marker, in malignant liver tissue from SRT501-treated patients compared with placebo [98]. From a clinical perspective, multiple studies have evaluated the anticancer potential of RES across different tumour types, including breast cancer (NCT03482401), gastrointestinal cancers (NCT01476592), colon cancer (NCT00256334), colorectal cancer (NCT00433576), and multiple myeloma (NCT00920556).

Dose (i.p.)-dependent toxicity in Swiss Albino mice is reported with an LD₅₀ of 1.18 g/kg for females and 1.07 g/kg for males. A dose of 0.312 g/kg is found as the safest, while ≤1.25 g/kg has no significant biochemical, hematological, and histopathological side effects [99]. Oral dose (25 mg) is well absorbed (≥70%), with a half-life of 9.2 hours, but very little unchanged drug (<5 ng/ml) is detectable due to rapid metabolism via sulfate and glucuronic acid conjugation and hydrogenation by intestinal microflora [100]. To address these limitations, various strategies have been proposed, including the synthesis of nano-delivery systems [101].

2.6. COL

COL is a tricyclic, lipid-soluble alkaloid extracted from plants of the genus Colchicum, including C. autumnale, and genera Gloriosa and Sandersonia. It works as a tubulin poison, binding to the COL site between the α- and β-tubulin subunits, disrupting microtubule polymerization, impairing mitotic spindle formation, and inducing cell-cycle arrest and apoptosis [102]. In MCF-7 and 4T1 breast cancer cells, COL dose-dependently reduced viability and proliferation at 0.1–1 and 10–400 µg/ml, respectively. COL markedly increased apoptosis, with early apoptotic cells reaching 14%–24% in MCF-7 (0.5–1 µg/ml) and significantly elevated in 4T1 (200–400 µg/ml). Mechanistically, COL upregulated p53 and BAX (1.6–6.3-fold), downregulated BCL-2 (0.18–0.74-fold), and increased the BAX/BCL-2 ratio up to 4.5-fold in MCF-7 and 10.8-fold in 4T1 [103]. In gastric carcinoma cell lines (AGS and NCI-N87), COL (2–10 ng/ml) dose-dependently inhibited proliferation and migration while inducing caspase-3–mediated mitochondrial apoptosis. In xenograft models, COL (0.05–0.1 mg/kg) significantly suppressed tumor growth via apoptosis without observable hepatic or renal toxicity or adverse biochemical changes [104]. In hepatocellular carcinoma (F28/KMUH, HCC24/KMUH, and HCC38/KMUH) cell lines, treatment with low and high doses (2 and 6 ng/ml) significantly (p < 0.05) inhibited proliferation in all tested cell lines with dose-dependent upregulation of AKAP12 and TGFB2. In vivo, COL administered to BALB/c-nu at 0.07 mg/kg/day for 14 days reduced tumour growth, slowed progression, and increased tumour necrosis compared with controls (all p < 0.05) [105].

In human clinical evaluation, a Phase 2 clinical trial (NCT01935700) in 15 patients with metastatic hepatocellular carcinoma demonstrated that COL (1 mg three times daily for 4 days with a 3-day rest) produced clinically acceptable anticancer activity [105]. In vitro, COL at 2–6 ng/ml significantly inhibited cell proliferation in a dose-dependent manner, with 6 ng/ml showing effects comparable to 1 μg/ml epirubicin, partly through upregulation of anti-proliferative (AKAP12, TGFB2) and pro-apoptotic (MX1) genes. In vivo, treated mice showed reduced tumour growth and increased necrosis [105]. Phase 1 studies (NCT01001052 and NCT01017003) in healthy volunteers showed predictable pharmacokinetics, similar drug exposure in young and elderly subjects, and mainly mild adverse events upon administration of a single oral dose (p.o., 0.6 mg).

Although initially explored as an anticancer agent, COL is limited by high toxicity and low tumor selectivity, resulting in damage to normal cells. For instance, in amniotic fluid and chorionic villus cells, COL (0.15–2.4 µg/ml) induced dose- and time-dependent cytotoxicity, inhibited proliferation, and triggered early and late apoptosis. Short-term exposure at a standard cytogenetic dose (0.15 µg/ml for 3 hours) caused G2/M arrest and metaphase accumulation, while G-banding analysis confirmed COL-induced polyploidy, with increased tetraploid cells, particularly in amniotic fluid cells [106]. COL induces significant developmental and renal toxicity, as demonstrated in zebrafish models where chronic exposure (0.15–1.5 mg/l, 4 weeks) caused podocyte disruption, renal tubular abnormalities, reduced cell density, and elevated oxidative stress kinase activity. Acute embryonic exposure (15–25 mg/l, 72 hours) resulted in high mortality, cardiac dysfunction, growth retardation, and renal edema, effects partially reversed by astaxanthin co-treatment [107]. A multiomics study further revealed pronounced cytotoxicity in HUVECs and HeLa cells within 24 hours, characterized by cell rounding, detachment, disrupted mitotic regulation, and microtubule instability [108]. In macrophages (THP-1), free COL caused severe ultrastructural damage, whereas encapsulation in polymeric NPs preserved cellular morphology. COL-NPs maintained metabolic activity in L929 fibroblasts and HUVECs over 24–72 hours, while free COL induced significant cytotoxicity. In C57BL/6 mice, COL-NPs preserved survival, body weight, and organ integrity, with no evidence of hepato-, nephro-, or cardiotoxicity, unlike free COL, which caused rapid mortality and weight loss despite similar biodistribution to heart and liver [109].

2.7. CUR

CUR is a polyphenolic compound isolated from the rhizomes of C. longa. CUR has been shown to interact with various biological targets, including proteins that play roles in antioxidant response, apoptosis, cell-cycle regulation, and cancer progression [110]. In breast cancer cell lines (MCF-7 and MDA-MB-231), CUR (2–40 μM) induced concentration- and time-dependent cytotoxicity. MDA-MB-231 cells were highly sensitive, with a 6–40 μM dose markedly reducing viability and clonogenicity, accompanied by upregulation of p21 and p27 and downregulation of SKP2. In contrast, MCF-7 cells showed relative resistance, with significant effects only at 30–40 μM after 12–24 hours. The observed responses correlated with differential modulation of PI3K/Akt signaling and Foxo1/Foxo3a phosphorylation [110]. In gastric cancer cell lines (AGS and HGC-27), CUR (10 and 20 μM) dose-dependently suppressed tumor progression by inducing autophagy-mediated ferroptosis via PI3K/AKT/mTOR inactivation, with upregulation of ATG5, ATG7, Beclin 1, and LC3B and significant reductions in p-PI3K, p-AKT, and p-mTOR (P < 0.001) [111]. In liver cancer cell lines (HepG2, Huh7, and MHCC97H), CUR (1.2–9.6µg/ml) showed dose-dependent anticancer effects at 24–48 hours. In HepG2 xenografts, intragastric CUR (120–240 mg/kg/day for 15 days) markedly inhibited tumor growth, reduced tumor weight, decreased CD11b⁺Gr1⁺ myeloid-derived suppressor cells and TLR4/NF-κB signaling (p < 0.05), and suppressed angiogenesis via downregulation of vascular endothelial growth factor (VEGF), CD31, and α-smooth muscle actin [112].

In A549 lung cancer cells, CUR (1.56–50 µg/ml) induced dose- and time-dependent cytotoxicity at 24–72 hours, causing S- and G2/M-phase arrest and p53-independent apoptosis. Treatment significantly upregulated Bax while downregulating Bcl-2, pERK1/2, p53, and p21 (p < 0.05). Combined treatment of CUR (0.39–12.5 µg/ml) with gemcitabine (0.68–1.30 µM) modestly reduced cell viability, with limited synergy [113]. In an immunocompetent orthotopic Tu-2449 glioma mouse model, dietary CUR (0.05% w/w) suppressed intracranial tumor growth and proliferation, resulting in long-term tumor-free survival in 15% of treated animals, whereas all controls died within 35 days. Reduced nuclear pY-STAT3 and Ki67 staining confirmed in vivo inhibition of STAT3 signaling and tumor proliferation [114]. In patient-derived glioblastoma stem cells, CUR reduced cell viability dose-dependently (IC₅₀ ≈ 25µM). Sub-toxic doses (2.5µM) decreased proliferation, sphere formation, and colony formation via ROS generation, activated MAPK signaling, and downregulated STAT3 and IAP family proteins [115]. Similarly, other in vivo studies have reported comparable findings in models of chronic myeloid leukemia [116] and pancreatic cancer [117]. In HT29 colorectal tumor-bearing mice, a turmeric ethanolic extract containing CUR (18.7% w/w) and ar-turmerone (5.3%) achieved the highest plasma CUR levels, indicating superior bioavailability. When combined with bevacizumab, the extract significantly inhibited tumor growth, outperforming CUR plus bevacizumab and bevacizumab alone, with efficacy comparable to 5-FU + leucovorin + oxaliplatin (FOLFOX) plus bevacizumab and no observable toxicity [118]. In both androgen-dependent LNCaP and metastatic C4-2B prostate cancer cells, CUR (10 μM) inhibited cell proliferation, suppressed PI3K/Akt/mTOR, and FOXM1 signaling pathways. Treatment also activated PTEN-dependent cell cycle arrest and apoptosis, down-regulated MYC, all resulting in the anti-proliferative and chemo-preventive effects in prostate cancer [119].

From a clinical perspective, multiple studies have investigated the effects of oral CUR in cancer patients. In a placebo-controlled, double-blind trial (NCT03211104), prostate cancer patients (n = 107) received 1,440mg/day for 6 months during intermittent androgen deprivation therapy. A Phase 1 study (NCT03980509) evaluated the effects of CUR on apoptosis and proliferation in primary breast tumours in 22 patients. Another Phase 1 trial (NCT01201694) assessed the safety and maximum tolerable dose of water-soluble CUR in advanced cancer patients (n = 28), starting at 100mg twice daily with dose escalation. A pilot study (NCT06080841) is testing 1–6g/day oral CUR, with or without piperine, in 30 locally advanced cervical cancer patients to examine p53 expression and apoptosis. No results have been posted for these four studies as of October 2025. Regarding safety, a Phase 1/2 open-label study (NCT00094445) in advanced pancreatic cancer patients (n = 50) found 6-month survival in 34.1%. Serious adverse events occurred in 20.5% (cardiac, renal, neurological, musculoskeletal), while 13.6% reported nonserious events, mainly gastrointestinal symptoms, fatigue, depression, and edema.

The clinical application of CUR might be hindered by adverse events and low bioavailability due to poor solubility. CUR exhibits extremely low aqueous solubility, with measured values of only 6.24 ± 0.49 mg/l (at 70°C), 11.56 ± 0.67 mg/l (at 80°C), and 19.62 ± 0.84 mg/l (at 90°C) [120]. Consistent with its poor solubility profile, oral CUR administration in healthy volunteers (0.5–12 g, p.o.) produced negligible serum levels at doses below 8 g, and even at 10–12 g resulted in very low systemic exposure, with peak plasma concentrations of only 29.7–51.5 ng/ml occurring 1–4 hours post-ingestion [121]. In two studies on chemotherapy-refractory colorectal cancer patients (n = 15 each), oral CUR (0.4–3.6g/day) showed very low systemic bioavailability, with fecal concentrations ranging from 64 to 1,054nmol/g, plasma CUR up to 11.1nmol/l, and urinary CUR, sulfate, and glucuronide levels between 0.1 and 510nmol/l depending on the metabolite [122,123].

2.8. QUE

QUE is a flavonoid abundantly found in berries, onions, and leafy vegetables, has been linked to significant anticancer properties [124]. A multitude of preclinical research, conducted both in vitro and in vivo, have shown encouraging results. The anticancer properties of QUE result from various mechanisms, including cell cycle arrest through the modulation of cyclin D1 and p53 pathways, induction of apoptosis via the upregulation of pro-apoptotic proteins and inhibition of anti-apoptotic factors, and stimulation of autophagy. Moreover, it impedes cell growth, angiogenesis, and metastasis [125,126]. Clinical evaluation in humans shown an inverse relationship between elevated dietary QUE consumption and the incidence of gastric adenocarcinoma [127]. A separate clinical study evaluated its efficacy in preventing and controlling chemotherapy-induced oral mucositis, indicating a significant decrease in mucositis occurrence among patients treated with QUE [128]. Ongoing clinical trials are being conducted to evaluate the effectiveness of the compound in treating various types of cancer, including prostate cancer (NCT01912820), squamous cell carcinoma (NCT03476330), and lung cancer (NCT04267874).

Despite promising pharmacological potential, QUE suffers from major formulation and pharmacokinetic limitations, including extremely low water solubility, high first-pass metabolism, and limited oral bioavailability. At 25°C, its solubility is 0.00215 g/l (anhydrous) and 0.00263 g/l (dihydrate), increasing only at high temperatures (0.665 and 1.49 g/l at 140°C, respectively) [129]. Consequently, human studies demonstrate low and variable oral bioavailability strongly influenced by glycosylation, with only glucuronidated metabolites detected in plasma, indicating extensive first-pass metabolism and absence of free aglycone. In a four-way crossover study, QUE glucosides were rapidly absorbed (T_max ≈ 0.7 hours; C_max ≈ 2.1–2.3 µg/ml) and eliminated with a half-life of ~11 hours [130]. QUE aglycone also exhibits formulation-dependent bioavailability. Standard preparations yield low serum levels, whereas lipid-based formulations (LipoMicel^®) increased Cmax 7-fold (500 mg) to 15-fold (1,000 mg), with even a half-dose achieving ~3-fold higher exposure. However, circulating QUE remained predominantly methylated, sulfated, and glutathione-conjugated, reflecting rapid metabolism. Although lipid-based systems extended detectable plasma levels up to 72 hours, poor intrinsic bioavailability and extensive conjugation remain major limitations [131]. Similar findings were observed in dogs, where only ~4% of orally administered QUE (10 mg/kg) reached systemic circulation, and >80% of circulating flavonols were conjugated metabolites, again highlighting substantial first-pass metabolism and limited oral bioavailability [132].

2.9. ADG

ADG is one of the most highly studied phytochemicals isolated from A. paniculata, a natural herbal medicine with pharmacological properties including anti-inflammatory, anti-oxidant, and anti-cancer properties [133]. In 1911, Gorter was the first to isolate it as a crystalline substance, identifying it as a diterpene lactone [134]. ADG induced apoptosis in human breast adenocarcinoma (MCF-7) and human triple-negative (MDA-MB-231) breast cancer cells by suppressing Bcl-2 expression while upregulating Bax at both the mRNA and protein levels. In MCF-7 cells, an ER-positive breast cancer model, it exhibited antiproliferative effects by downregulating ERα, PI3K, and mTOR signaling pathways. Similarly, in MDA-MB-231 cells, it inhibited proliferation primarily through apoptosis induction [135]. Treatment with ADG markedly enhanced the activation of cytochrome c and initiated the caspase-dependent apoptotic signalling pathway. Additionally, ADG was observed to inhibit the binding of the transactivators CREB2, C-Fos, and NF-κB, as well as to block the recruitment of coactivator p300 to the COX-2 promoter in MCF-7 breast cancer cell lines [136]. In nasopharyngeal carcinoma, ADG has been shown to lower the levels of EGFR and survivin involved in cancer cell growth, thus inhibiting tumour proliferation [137]. In pancreatic and liver cancer cells, ADG has been observed to promote the breakdown of a mutated protein, leading to cell cycle arrest [138].

Despite promising activities, conventional dosage forms of ADG show high lipophilicity (log p = 2.63) [139]¸ and poor palatability. A study by Ren et al. [140] showed that the intrinsic solubility of ADG in water at 25°C was 212 mmol/l (74 mg/ml). The same study also revealed that after giving a single oral dose of 25 mg/kg of the ADG in rats, it was found that the T_max was 67.67 minutes, C_max was 2.07 mg/l, and the AUC_0–1 was 482.01 mg/l/min, which showed that ADG has a poor bioavailability [140]. From a pharmacokinetics perspective, ADG is rapidly absorbed and metabolized (rat model). A concentration of 1 µM (0.35 µg/ml) in plasma was obtained within 30 minutes after administration of 50 mg/kg, and bioavailability was 1.19% [141]. ADG was measured in plasma and various tissues after administration (p.o., 100 mg/kg/day for 4 weeks), with the highest concentrations in the kidney, followed by the liver, spleen, and brain. The maximum concentration was 115.81 ng/ml at 0.75 hours, and the half-life elimination was 2.45 hours [142]. After 200 mg oral consumption, ADG was measured in plasma, showing a maximum concentration of 58.62 ng/ml at 1.6 hours and an elimination half-life of 10.5 hours [143]. Improving the bioavailability of ADG remains a significant challenge in advancing its clinical applications.

2.10. Gingerol

Gingerol is a phenolic compound with notable anticancer properties, derived from the fresh rhizome of Z. officinale. Gingerol exerts its anticancer effects through multiple mechanisms, including suppression of inflammation via NF-κB and COX-2 inhibition, induction of phase II detoxification enzymes through Nrf2 activation, tubulin interaction–induced genomic instability, and gene expression modulation favoring pro-apoptotic over anti-apoptotic pathways [15]. These effects have been validated in animal models of breast cancer as well as in several in vitro models, including neuroblastoma, sarcoma, human myeloid leukemia, and Caco-2 breast cancer cells [144–147]. Recent studies indicate that gingerol may enhance the efficacy of cisplatin [148] and DOX [147] when used in combination.

Several clinical trials have investigated the potential of ginger root extracts, particularly those rich in gingerol, for cancer prevention. In one pilot study, individuals at higher risk for colon cancer were given 2 g of ginger, standardized to contain 5% gingerol. The findings indicated that ginger supplementation could be helpful in preventing the onset of cancer [149]. In a pilot study of newly diagnosed cancer patients, supplementation with ginger extract standardized to 20 mg/day of 6-gingerol, initiated three days before chemotherapy and continued through the fourth cycle, improved antioxidant status and reduced oxidative stress markers, suggesting a protective role against chemotherapy-induced toxicity [150]. A Phase 2 randomized trial also found that 6-gingerol significantly increased the overall response to chemotherapy-induced nausea and vomiting, while enhancing appetite and quality of life in cancer patients undergoing chemotherapy [151]. A food supplement containing standardized gingerol, menthol, and limonene improved symptom severity in irritable bowel syndrome patients (n = 56). The supplement was well tolerated, causing no notable side effects, and gut microbiota composition analyzed via 16S rRNA gene sequencing showed no significant changes [152].

Gingerol, particularly 6-gingerol, is the most abundant and studied [153] gingerol component is significantly constrained by poor biopharmaceutical properties, including low solubility, limited permeability, extensive first-pass metabolism, and consequently low systemic bioavailability. 6-gingerol is insoluble in water and dissolves only in organic solvents such as ethanol, benzene, ether, chloroform, and methanol. In aqueous media, its solubility remains very limited, being reported only as >100 µM (≈ 0.029 mg/ml) in pH 2 (hydrochloride buffer), pH 4 (citrate buffer), and pH 7.4 (phosphate buffer) [154], with an experimentally measured solubility of just 0.41 mg/ml in 0.1 N hydrochloric acid (pH 1.2) [155]. Its predicted log P of 2.48–3.62 and provisional Biopharmaceutical Classification System Class I classification indicate low solubility and low permeability [156]. In the rat model, 6-gingerol undergoes extensive phase II metabolism, primarily forming glucuronide and sulfate conjugates in the intestinal mucosa and, to a lesser extent, the liver [157]. As a result, circulating components after oral ingestion are predominantly conjugated metabolites, with free aglycones appearing only at very low serum levels, as demonstrated in mice given 250 mg/kg ginger extract [154]. Metabolic studies confirm that hepatic uridine diphosphate-glucuronosyltransferases rapidly convert 6-gingerol into glucuronide conjugates, with ~50% excreted via bile and only 2%–3% eliminated in urine as the free compound [157,158], highlighting substantial first-pass metabolism. Pharmacokinetic data further support these constraints; in rats, the oral bioavailability is extremely low (~0.17%) following a 30 mg/kg dose [159]. In humans, an escalating-dose study (100 mg–2.0 g ginger extract, 5% total gingerols) showed rapid absorption (T_max 55–65.6 minutes) and short elimination half-lives (75–120 minutes), with no detectable free gingerol in plasma; only conjugated metabolites were present [160].

3.1. Background

Graphene is a two-dimensional material made of sp²-hybridized carbon atoms arranged in a honeycomb lattice. Since its discovery in 2004, graphene has become a major focus in nanomaterials research, a breakthrough that led to the 2010 Nobel Prize in Physics awarded to Andre Geim and Konstantin Novoselov [161]. Its parent material, graphite, is composed of stacked one-atom-thick graphene layers arranged in a hexagonal structure. Graphite occurs naturally in forms such as flake and amorphous graphite and can also be produced synthetically using approaches like carbon graphitization, chemical vapor deposition, or carbide decomposition [162].

GO is an oxidized form of graphene enriched with functional groups, including carbonyl (=O), hydroxyl (-OH), ether (-O-), and carboxyl (-COOH) groups, which are present on both sides of the layer and along the edges of the plane [163]. GO is typically synthesized by oxidizing graphite into graphite oxide, followed by exfoliation. Its oxygenated framework renders it hydrophilic and dispersible in aqueous and organic solvents, a property that sharply contrasts with pristine graphene [164]. rGO is obtained when GO undergoes chemical, thermal, or green reduction to partially remove oxygen functionalities. Although its conductivity does not fully match pristine graphene, rGO exhibits significantly improved electrical, thermal, and chemical performance relative to GO, making it more suitable for electronic and biomedical applications [165]. GQDs represent ultrasmall (<20 nm) fragments of graphene that possess size-dependent optical properties and high surface-to-volume ratios. Their strong intrinsic luminescence and higher photostability compared with organic dyes allow sensitive bioimaging and disease diagnostics, particularly in cancer [166,167]. While pure graphene is a zero-band-gap semiconductor, introducing oxygen-rich domains into GQDs generates quantum confinement and tunable emission characteristics [168]. GQDs can be synthesized using either top-down approaches (oxidative cutting of GO, chemical ablation, plasma treatment) or bottom-up approaches (pyrolysis of sugars, citric acid, polythiophenes) [169].

3.2. Synthesis and phytochemical loading

Graphite is a thermodynamically stable carbon material, and its conversion into graphene derivatives such as GO requires highly concentrated acids and strong oxidizing agents (Table 1). Nearly all modern GO synthesis protocols are derived from three classical oxidation routes, including Brodie’s method (1855) [170], Staudenmaier’s method (1898) [171], and Hummer’s method (1958) [172]. These wet-chemical oxidation approaches insert hydroxyl, epoxy, carbonyl, and carboxyl groups across the graphene lattice, resulting in highly hydrophilic sheets with strong binding affinity toward various drug molecules. Figure 2a illustrates a modified Hummers’ workflow, and Figure 2b summarizes chemical, thermal, radiation-mediated, and green reduction strategies used to convert GO into rGO.

Figure 2. (a) Stepwise synthesis scheme of GO by modified Hummers’ method. Initially, graphite powder is mixed with concentrated sulfuric acid (H2SO4) under stirring. Potassium permanganate (KMnO4) is gradually added as a strong oxidizing agent, leading to a color change that reflects the progression of oxidation. Over time, the graphite structure becomes oxidized, introducing various oxygen-containing functional groups (e.g., –OH, –COOH, and –C=O), resulting in the change of graphite layers into GO. The color transitions from black to brownish-yellow indicate successful oxidation. The final product, GO, is obtained after heating, dilution, and washing steps. (b) shows various methods for reducing GO to rGO, including chemical, thermal, radiation-mediated, and green reduction approaches. [Click here to view]

Table 1. Synthesis of GO, using different carbon sources, oxidizing agents and temperature.

The abundant oxygen-containing groups on GO and rGO play a central role in their interaction with phytochemicals, particularly polyphenols such as CUR, QUE, and juglone. Binding occurs mainly through π–π stacking between aromatic rings and sp² carbon domains, hydrogen bonding with hydroxyl and carboxyl groups, and hydrophobic interactions with graphitic regions. CUR, for example, shows high affinity for GO/rGO via combined π–π stacking, hydrogen bonding involving its phenolic and β-diketone groups, and hydrophobic insertion into graphitic domains [173]. GO synthesized via the modified Hummers’ method produces thin, lamellar nanosheets enriched with oxygenated groups that facilitate these interactions. Loading of QUE (2.5%–5% w/v) and juglone (5%–10% w/v) occurs through noncovalent forces, predominantly π–π stacking and hydrogen bonding between polyphenolic functional groups and the GO surface [174]. In another study, GO nanosheets, when grafted with hyperbranched polyglycerol, enable a very efficient QUE loading through π–π stacking, hydrogen bonding with numerous hydroxyl groups, and hydrophobic entrapment within the polymer cavities. This system achieved exceptionally high loading capacity (185% w/w) and encapsulation efficiency (93%) [175].

PTX is highly nonpolar, preferentially associates with hydrophobic graphitic regions of GO or partially rGO through strong π–π stacking with regenerated sp² carbon lattice [176]. Similarly, CPT is incorporated via noncovalent interactions, achieving a loading efficiency of 45% within the GO matrix [177]. CUR and DOX interact strongly with GO-COOH via π–π stacking, hydrogen bonding, and partial ionic interactions. Under mildly acidic conditions (pH 5.5–6), which minimize GO aggregation and maximize binding site availability, high loading efficiencies were achieved for CUR (79.8%) and DOX (90.4%). Co-loading further enhanced incorporation to 81.2% and 91.8%, respectively, confirming stable and cooperative binding within the GO-COOH framework [178]. Vinblastine loading onto GQDs also relied on π–π stacking and n–π* interactions, achieving a loading efficiency of 95.2% [179]. Plant extract and essential oil similarly bind through hydrophobic and π–π interactions. For example, Juniperus squamata root essential oil was loaded onto polyvinylpyrrolidone (PVP)-grafted GO via physical adsorption involving π–π interactions, hydrogen bonding, and hydrocarbon affinity. PVP functionalization enhanced loading efficiency to 17.4% (w/w), compared to 13.6% for unmodified GO [180].

Phytochemical loading can be further enhanced by surface functionalization of GO. GO synthesized by the Hummers’ method is rich in epoxide, hydroxyl, and carboxyl groups, providing reactive sites for polymer conjugation. Functionalization with chitosan and FA enables strong electrostatic, hydrogen-bonding, and π–π interactions with phytochemicals. In this system, 6-gingerol binds via hydrogen bonding with chitosan amine groups, hydrophobic interactions with GO sp² domains, and π–π stacking with the GO basal plane, achieving an encapsulation efficiency of 86.2% (w/w) [181]. Proanthocyanidin (grape seed extract), when loaded onto chitosan functionalized GO, primarily through π–π stacking, hydrogen bonding, and hydrophilic interactions with both GO and chitosan, achieves a loading content of ~20% (w/w) [182]. In another system, GO synthesized by the Hummers’ method was incorporated with CUR through gradual dropwise addition. CUR adsorbed onto GO via π–π stacking and hydrogen bonding before being integrated into collagen scaffolds, reaching a maximum loading content of 31.54% (w/w) at 0.7 mg/ml CUR [183]. GO functionalized with PVP introduces abundant carbonyl and methylene groups, resulting in efficient loading of QUE and gefitinib through π–π stacking with residual sp² regions and hydrogen bonding with polar PVP segments, resulting in loading efficiency of ~20% for QUE and ~46% for gefitinib [184]. It is worth mentioning that different research groups [172,185–197], have reported the synthesis of GO using various carbon sources, oxidizing agents, and temperature conditions (Table 1). These synthesis parameters significantly influence the structural properties of GO and its suitability for subsequent phytochemical(s) loading.

3.3. Functionalization of Graphene for targeted and controlled drug delivery

Surface functionalization improves the physicochemical properties, biocompatibility, and targeting ability of graphene-based nanomaterials. Chemical modification with polymers (e.g., polyethylene glycol, Pluronic, polyethyleneimine), macromolecules (e.g., albumin, phospholipids), or biological ligands (e.g., FA, aptamers, peptides, glucosamine, antibodies) enhances colloidal stability, biocompatibility, and targeted drug delivery. Major functionalization strategies employed for GO- and rGO-based nanocomposites are shown in Figure 3.

Figure 3. (a) Surface functionalization strategies for GO and rGO nanocomposites, including covalent attachment of polymers, macromolecules, or targeting ligands. (b) Examples of preclinical studies on the synthesis of nanocomposites, such as TiO2/GO [209] , Fe²+-modified GO [210] , and hydrogel-based systems [211] . [Click here to view]

CUR-loaded polyethylene glycol (PEG)–GO nanocarriers exhibited good colloidal stability and reduced phagocytic uptake. Drug release studies demonstrated that CUR release reached 50% at pH 5.5 and 60% at pH 7.4 after 96 hours, suggesting enhanced release in the tumour microenvironment [198]. GO functionalized with a polyethyleneimine–sericin polypeptide enabled pH-responsive, tumour-selective CUR delivery via a charge-reversal mechanism. Amide linkages remained negatively charged at physiological pH (~7.4) for prolonged circulation, partially hydrolyzed at tumor-like pH (~6.5), and fully cleaved under endosomal/lysosomal conditions (~5.5), enhancing cellular uptake and nuclear delivery. Drug release was 34% faster under acidic conditions within the first 12 hours, with cumulative release over 192 hours of 25.4%, 26.1%, and 31.5% at pH 7.4, 6.5, and 5.5, respectively. The nanocarrier was highly biocompatible, maintaining >95% C2C12 cell viability at 20–500 µg/ml, and markedly improved uptake in vitro, reducing IC₅₀ values 2- and 7-fold in SkBr3 and PC-3 cells compared with free CUR [199]. Pluronic-functionalized rGO co-delivering CUR and PTX, exhibited strong synergistic anticancer activity, with IC₅₀ values of 13.24 µg/ml (A549) and 1.45 µg/ml (MDA-MB-231 cells), with a 4–6-fold more potency than free drugs. The formulation increased intracellular ROS >2-fold, induced mitochondrial depolarization, and markedly enhanced apoptosis, while maintaining high biocompatibility in normal MRC-5 cells (>80% viability at equivalent concentrations) [200].

RES-loaded folate-terminated PEG-phospholipid-coated rGO nanosystems exhibited cargo-protection and active tumor targeting. Folate functionalization enabled receptor-mediated uptake in MCF-7 cells, achieving efficient, selective internalization with excellent biocompatibility. The system protected RES from light-induced degradation and, under 808nm NIR irradiation, produced a potent chemo-photothermal effect, effectively eradicating tumors after a single intratumoral dose in vivo [201]. Glucosamine-functionalized GQDs (20–30 nm) significantly improved the physicochemical behavior, biocompatibility, and targeting performance of CUR. The photoluminescent GQDs were stable and exhibited pH-responsive sustained release, with 37% release at pH 5.5 versus 17% at pH 7.4 over 150 hours. Glucosamine-mediated receptor uptake enabled active targeting of MCF-7 cells, with time-dependent internalization confirmed by fluorescence imaging and flow cytometry, showing 23% uptake for functionalized versus 16.8% for nonfunctionalized GQDs after 24 hours [202].

Carboxylated GO (~80 nm), when covalently functionalized with PEG-amine (linear PEG of 2, 10, and 20 kDa, and branched four-arm PEG), improved its physicochemical performance. The resulting PEG-GO system, when loaded with PTX, exhibited sustained, burst-free release at pH 7.4 (<40% over 48 hours), while acidic pH 6.0 triggered accelerated drug liberation, with cumulative release increasing to ~60%–90% at 48 hours, and a rapid ~50% release within 1 hours for 4-arm PEG formulations. In A549 lung cancer cells, PTX loaded system showed time-dependent cellular uptake and cytotoxicity, exceeding free PTX at 24 and 48 hours, causing ~60% and ~75% cell death, respectively, at 25 µg/ml PTX. Apoptosis induction was also enhanced, reaching ~27% at 48 hours, compared to ~18% for free PTX [203]. An aptamer-conjugated superparamagnetic GO nanocarrier loaded with PTX enabled selective breast cancer targeting with pronounced pH-responsive release. Cumulative PTX release was 67.6% at pH 2.0, 38.4% at pH 5.5, and 17.9% at pH 7.4, demonstrating tumor- and endosome-selective drug liberation. The system was biocompatible, maintaining >80% viability in L-929 fibroblasts (6.125–100 µg/ml), while inducing dose-dependent cytotoxicity in MCF-7 cells. Flow cytometry confirmed aptamer-mediated targeting, showing clear fluorescence shifts in MCF-7 but not in L-929 cells [204]. In another work, PTX-loaded GO was coated with human serum albumin, PEGylated for stability, and conjugated with anti-VEGF antibodies for active tumor targeting and photothermal-triggered chemotherapy. Under near-infrared (NIR) irradiation (808 nm, 1 Watt/cm², 5 minutes), temperatures rose ~22°C, inducing NP swelling and on-demand PTX release. Drug release increased from 6.2% at pH 7.4 without irradiation to 20.1% after a single NIR cycle, reaching 81.2% cumulatively after four cycles versus 11.2% without NIR. Antibody functionalization enhanced cellular uptake (49.6 ± 4.1% vs. 13.6 ± 3.9% for nontargeted carriers) via receptor-mediated endocytosis. Combined NIR treatment induced 91.2% apoptosis, compared with 42.8% for the nanocarrier alone and 32.6% for free PTX. In vivo, NIR-assisted therapy achieved complete tumor suppression and 100% survival over 100 days without hemolysis, organ toxicity, or abnormal blood biochemistry [205].

Ma et al. [206] developed a pH-sensitive DOX-delivery system using rGO (Fig. 4). Drug release from rGO was slower and pH-dependent, with 35% released at pH 5.0 versus 24% at pH 7.4 over 144hours, while free DOX was nearly completely released at pH 5.0 within 12hours. The formulation exhibited minimal hemolysis (<1% across 5–500mg/ml), with slight increases at higher concentrations. In A549 cells, rGO–DOX showed stronger red fluorescence after 4hours than free DOX, indicating more efficient endocytosis-mediated uptake. Cytotoxicity assays in A549 and MCF-7 cells showed dose- and time-dependent effects; at ≤1mg/ml after 24hours, inhibition was similar, whereas at higher doses, free DOX was more cytotoxic (e.g., 61.0% vs. 49.3% in MCF-7 at 20mg/ml) [206]. Microwave-assisted silane functionalization of GO with 3-aminopropyltrimethoxysilane imparted strong pH-responsive release behavior, with rapid DOX liberation at acidic pH (5.4), reaching 87.1% within 0.5 hours and ~99%–100% within 6–7 hours, while release at physiological pH (7.4) was markedly suppressed (11.3% at 0.5 hours and 94.3% only after 72 hours) [207]. PEGylated GO surface-functionalized with fibroblast activation protein–targeting peptides and loaded with DOX created a dual pH- and photothermal-responsive nanoplatform for oral squamous cell carcinoma. PEG and peptide modification improved colloidal stability, circulation time, and tumor targeting, confirmed by enhanced cellular uptake and tumor-specific fluorescence. Drug release was stimulus-responsive, with ~34% at pH 7.4 and 67% at pH 5.5 within 72hours, increasing to 48% and 78% under NIR irradiation. The nanocarrier was highly biocompatible (>90% HOK cell viability), while synergistic chemo-photothermal therapy reduced CAL-27 cell viability to ~42% in vitro and achieved near-complete tumor inhibition in vivo without systemic toxicity [208].

Figure 4. (DOX-loaded rGO with pH-dependent behavior. In the first step, GO was treated with riboflavin-5′-phosphate sodium salt dihydrate, which acts as a reducing agent, resulting in the formation of rGO. Subsequently, DOX was loaded via sonication π–π interactions. Upon cellular uptake, the nanosystem is enclosed within endosomes, which are hydrolyzed upon fusion with lysosomes, leading to drug release and induction of cellular apoptosis. A preclinical study demonstrating this mechanism is reported in reference [206]. [Click here to view]

4.1. GO

GO has been used as a versatile nanocarrier for delivering various phytochemical anticancer agents. These include: apigenin and PTX [212], berberine [213], CPT [214], CUR [215], gingerol [181], irinotecan [216], PPT [217], QUE [218], QUE and gefitinib [184], QUE and lurbinectedin [219], vincristine [220], J. squamata (root essential oil) [180], and Moringa peregrina (leaf extract) [221].

Pal et al. [212] investigated the synergistic effects of PTX combined with apigenin-loaded onto GO in SKOV-3 ovarian cancer cells. Treatment showed enhanced anti-proliferative activity compared to free PTX (5 nM) or apigenin-loaded GO (10μM) alone, with reduced IC₅₀ values. Study results showed that the combination suppressed SOD activity, increased ROS accumulation, induced mitochondrial depolarization, DNA damage, and cell cycle arrest, leading to apoptosis. Furthermore, molecular analyses confirmed upregulation of caspase-3 and Bax and downregulation of Bcl-2 [212]. Yu et al. [213] evaluated the electric-sensitive release and redox behavior of berberine-loaded PEGylated and non-PEGylated GO nanocomposites. At pH 7.4, cumulative release over 48hours was ~19% for GO and ~27% for PEG-GO, increasing to ~66% and ~64% at pH 5.8. Under voltage stimulation, GO exhibited burst release (~17% at 0.2V and ~37% at 0.1V within 2hours), whereas PEG-GO released more slowly (~11% at 0.2V and ~22% at 0.1V). GO also showed higher sensitivity under combined stimuli, releasing ~46% at pH 6.2 with 0.1V in 4hours, about double the release without voltage, while PEG-GO showed minimal change under the same conditions [213].

Similarly, GO, chitosan, and magnetite (Fe₃O₄) NPs, along with their nanocomposites, were engineered to enhance the loading and release efficiency of CPT. Loading efficiencies were 70% (chitosan), 81% (chitosan–magnetite), 58% (chitosan–GO), and 74% (chitosan–GO/Fe₃O₄). Drug release was pH-dependent, with 87%, 80%, 88%, and 90% released at pH 5.0, and 84%, 72%, 89%, and 87% at pH 7.4, respectively. CPT-loaded nanosystem achieved maximal cytotoxicity against HepG2 and SMMC-7721 cells at 5 and 12.5µM, respectively [214]. Einafshar et al. [216] developed cyclodextrin (CD)-functionalized GO nanocomposites as a multifunctional platform for the delivery of SN38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of irinotecan). The researchers developed α-, β-, and γ-CD-coated nano-GO coordinated with superparamagnetic iron oxide (Fe₃O₄) NPs to improve stability, biocompatibility, and targeting potential. SN38 was physically loaded with drug loading efficiencies of 21% (α-CD), 13% (β-CD), and 22% (γ-CD). Drug release was higher at pH 7.4 than at pH 5.5, consistent with SN38 solubility. In vitro, all CD-GO-Fe₃O₄-SN38 nanoconjugates enhanced cytotoxicity against HT-29 colorectal cancer cells compared to free SN38, with β-CD-GO-Fe₃O₄-SN38 showing the greatest effect (~48% higher inhibition) under combined photothermal and chemotherapeutic treatment [216]. Ramazani et al. [215] reported the synthesis of GO and Au/GO nanocomposites via Hummer’s method and evaluated their use as nanocarriers for CUR delivery. At physiological pH (7.4), CUR exhibited slow and controlled release from GO and Au/GO nanocomposites, reaching ~11% and ~9%, respectively, after 48 hours, whereas under acidic conditions, the release rate was markedly higher over the same period. Cytotoxicity studies on MCF-7 breast cancer cells and HEK293 normal cells showed that the CUR-loaded Au/GO nanocomposite (6–10 nm) exhibited selective anticancer activity without harming healthy cells after 48–72 hours. Free CUR was cytotoxic to both cell types, whereas Au/GO/CUR showed higher growth inhibition in MCF-7, with IC₅₀ decreasing to 30µg/ml at 72hours, while IC₅₀ in HEK293 remained nearly constant. The nanocomposite exhibited minimal toxicity in brine shrimp larvae (LC₅₀=657.35µg/ml) and negligible red blood cell disruption, demonstrating pH-responsive, targeted anticancer activity with low off-target effects [215].

Al-Janabi et al. [181] developed 6-gingerol-loaded GO and modified with chitosan–FA to enhance anticancer efficacy. The resulting nanocomposites (~73 nm, 81.7% encapsulation) showed strong antioxidant activity and significant cytotoxicity, with gastric cancer cells being most sensitive (IC₅₀ ~ 27 µM). Treatment reduced angiogenesis in chick chorioallantoic membrane assays, supported by downregulation of VEGF and VEGF-receptor, and decreased antioxidant gene superoxide dismutase expression, indicating a pro-oxidant mechanism in cancer cells [181]. Zhu et al. [217] reported the synthesis and evaluation of PEG-functionalized GO nanocarrier for the delivery of PPT (~10% loading) to improve stability in physiological solutions. Treatment showed significantly higher cytotoxicity than free drug after 48–72 hours, particularly at 20–40 nM, due to sustained release and enhanced cellular uptake. Fluorescein isothiocyanate-labeling studies confirmed that PEG-functionalized GO entered cells in a time-dependent manner, while apoptosis assays revealed substantial cell death comparable to higher doses (60 nM) of free drug [217].

Three studies mentioned the loading of QUE [218], QUE and gefitinib [184], QUE and lurbinectedin [219], into GO. Specifically, Najafabadi et al. [218] reported gelatin–PVP-coated pH-sensitive GO nanocomposites for QUE delivery. The resulting nanosystem achieved an encapsulation efficiency (87.5%) and drug loading (45%), with a good colloidal stability. Drug release was pH-dependent, with 24hours release of 34% at pH 7.4 and 60% at pH 5.4, and cumulative release after 96hours reaching 91% and 95.5%, respectively, following Higuchi kinetics. The dual nanoemulsion improved sustained release and long-term stability. In MCF-7 cells, the formulation induced 53.1% cell death (36.5% apoptotic) while showing negligible toxicity toward normal L929 cells; cell viability after 24hours was 67% versus 75% for free QUE, confirming enhanced anticancer efficacy [218]. A similar nature study by Tiwari et al. [184] investigated the co-delivery of QUE and gefitinib using PVP-coated GO nanocomposites and compared it to single-drug-loaded and free drug systems in PA-1 ovarian cancer and IOSE-364 epithelial cells. The GO-loaded nanocomposite (QUE-gefitinib combined) complex showed superior cytotoxicity against PA-1 cells, greater than single-drug systems or free drugs, while being less toxic to IOSE-364 cells. The formulation also exhibited favorable release behavior, enhanced solubility, and effective targeting [184]. Recently, Liao et al. [219] encapsulated QUE and lurbinectedin onto GO (<50 nm), and their anticancer effects were evaluated against A549 and PC9 lung cancer cells. The formulation induced strong cytotoxicity, morphological changes, upregulated p53, Bax, and Caspase-3, and downregulated Bcl-2. Treatment reduced metastasis via MMP-2–mediated collagen IV cleavage and inhibited wound healing. Compared to free or single-drug, GO-loaded QUE and lurbinectedin showed higher loading efficiency (45%), pH-responsive release, enhanced mucoadhesion, and significantly stronger anticancer activity (p219].

Atay et al. [220] reported that GO acts as both a carrier for the chemotherapeutic drug vincristine and a photothermal agent that generates heat when exposed to NIR irradiation (808 nm). To test this, primary mammary tumour cells from a dog were cultured and treated with the formulation, then exposed to NIR laser irradiation. Treatment resulted in a significant reduction in tumour cell viability (90%), less than that of untreated control cells (p < 0.0001) [220]. Madeo et al. [222] developed GO, zinc oxide (ZnO) NPs, Zn-GO hybrid nanocomposite for the delivery of PTX. Drug release studies showed maximum release of 34% for GO and 50% for Zn-GO. In MDA-MB-231 cells, PTX-loaded Zn-GO exhibited significantly higher cytotoxicity than free PTX (20.4% vs. 45.8% viability at 0.110 µg/ml, p < 0.01). ZnO-loaded nanosystem had minimal toxicity, while the GO-nanosystem displayed stronger, concentration-dependent effects [222].

4.2. rGO

rGO has also been explored for drug loading, including A. paniculata (leaf extract) [223], Camellia sinensis (leaf extract) [224], Citrullus colocynthis (leaf extract) [225], CUR [32,33], CUR and PTX [200], irinotecan and docetaxel [226], J. squamata (root essential oil) [180], M. peregrina (extract) [221], RES [227], and PTX [176], respectively.

Kadiyala et al. [223] reported a green, one-pot solvothermal synthesis of zirconium oxide (ZrO₂) NPs uniformly dispersed on rGO using A. paniculata leaf extract, forming ZrO₂/rGO nanocomposites. The anticancer potential of these NPs was evaluated against human lung (A549) and colon (HCT116) cancer cell lines, with normal human mesenchymal stem cells (hMSCs) as controls. Treatment exhibited dose-dependent cytotoxicity, significantly reducing cell viability at 10 ppm (up to 96%–98% for cancer cells), while the plant extract alone maintained high viability (~95%). Flow cytometry confirmed apoptosis and ROS generation, higher in HCT116 than A549 cells. Cytotoxicity was dose- and composition-dependent, with lower ZrO₂ doping (0.01M) inducing stronger ROS and cell death than higher doping (0.1M), comparable to cisplatin [223]. Fatima et al. [224] synthesized rGO (3.92 nm) from GO using C. sinensis as a reducing agent. The rGO showed strong antioxidant activity (DPPH 85.98%, ABTS 88.87%) and total phenolic content of 842µg/g. Hemolysis was low at pH 7.4 (4.92%) but higher at pH 5.6 (11.15%), indicating safety under physiological conditions. Against A549 cells, rGO alone caused 68.1% cell death at 520µg/ml, while DOX and DOX + rGO induced 76.5% and 96.2%, respectively, via oxidative stress without harming healthy cells [224]. Zhu et al. [225] synthesized rGO from GO using C. colocynthis leaf extract. Cytotoxicity studies showed that GO was more effective in reducing DU145 cell survival at both low and high concentrations. At 4 µg/ml, GO reduced viability to 75% compared to 86% with rGO, while at 80 µg/ml, viability further decreased to 10% with GO versus 20% with rGO [225]. Lin et al. [176] synthesized rGO using E. milii leaf extract and loaded PTX. In A549 lung cancer cells, treatment at 200µg/ml reduced viability to 29%, and 500µg/ml further decreased it to 10%, demonstrating strong dose-dependent cytotoxicity. Lower concentrations of rGO alone (1–100µg/ml) showed minimal morphological changes and maintained high cell viability, confirming the biocompatibility of the carrier. Fluorescence microscopy indicated substantial cell death only at higher PTX-loaded rGO concentrations [176].

CUR-loaded rGO (~42 nm) showed strong photothermal activity, and a sustained release profile (17%–22% at 24 hours; 27%–35% at 48 hours), compared to the complete release of free CUR within 24 hours. Treatment with the formulation achieved higher cytotoxicity against MCF-7 and A549 cells, especially under irradiation (90% inhibition at 40 µg/ml), confirming a synergistic effect of chemotherapy and photothermal therapy. The IC₅₀ values for MCF-7 were significantly reduced for rGO (7.72 µM in dark; 12.74 µM under irradiation) compared to free CUR (10.86 and 19.8 µM), respectively [32]. In a similar nature study, Kazemi et al. [33] prepared chitosan-magnetite (Fe₃O₄) rGO nanocomposites via a water-in-oil emulsification method for targeted delivery of CUR against MCF-7 breast cancer cells. Drug release studies revealed a faster release of CUR in acidic medium. Treatment exhibited the highest cytotoxicity at 0.1 µg/ml, inducing apoptosis as confirmed by flow cytometry [33]. Another study functionalized rGO with the amphiphilic polymer PF-127, which enabled highly efficient co-delivery of CUR and PTX. The resulting rGO (140 nm) showed high loading capacity (CUR ≈ 97%, and PTX ≈ 98%). Drug release was biphasic, rapid in the first 8hours and sustained thereafter, with enhanced release at acidic pH (4.0), favoring tumour targeting. The formulation exhibited potent synergistic anticancer effects (CI < 1), increasing ROS, mitochondrial depolarization, and apoptosis in A549 and MDA-MB-231 cells. IC₅₀ values for rGO (CUR + PTX) were 13.24µg/ml (A549) and 1.45µg/ml (MDA-MB-231), while rGO (PTX) alone showed IC₅₀ of 0.0077µg/ml (A549; ~25-fold lower than free PTX) and 0.0148µg/ml (MDA-MB-231; ~8-fold lower) [200]. A folate-conjugated rGO nanocarrier was developed for the co-delivery of irinotecan and docetaxel (250 μg/ml each), achieving very high drug loading (>100%). Drug release was pH-dependent, with significantly faster release at acidic pH (5.0) compared to neutral (7.4), favoring tumour-specific delivery. In folate receptor-positive MCF-7 cells, it enhanced cytotoxicity, apoptosis (upregulating p53, p21, Bax, cleaved caspase-3; downregulating Bcl-2), and synergistic effects (CI = 0.23), while uptake was FA-mediated. NIR irradiation (808 nm) further amplified apoptosis via photothermal heating [226].

Melo et al. [227] developed injectable chitosan-based hydrogels co-loaded with dopamine-rGO (66.7µg/ml) and RES (35µg/ml) for chemo-photothermal therapy of breast cancer. The hydrogels were cytocompatible, showing normal human dermal fibroblasts viability of 92% and 84% after 24 and 48hours. In vitro, dopamine-rGO hydrogel alone had minimal effect on MCF-7 cells (85% viability), while dopamine-rGO hydrogel + NIR (808nm, 1.7Watt/cm², 10minutes) reduced viability to 72%, indicating insufficient photothermal effect alone. RES + dopamine-rGO hydrogel decreased MCF-7 viability to 75%, whereas free RES (35µg/ml) reduced it to 61%, suggesting controlled release by the hydrogel. The combined chemo-photothermal treatment (RES + dopamine-rGO hydrogel + NIR) drastically lowered viability to 31%, confirmed by Calcein-AM/PI staining, demonstrating strong synergistic anticancer efficacy [227].

4.3. GQDs

In the past decade, GQDs have received tremendous attention, due to their outstanding advantages such as low toxicity, low cost, excellent biocompatibility, resistance to photobleaching, and ease of fabrication [228]. Cellular studies revealed that smaller GQDs have weaker DNA dye quenching ability but higher cytotoxicity compared to as-synthesized mixtures, highlighting the importance of precise size control [229]. A study reported a multifunctional nanocomposite combining GOQDs, upconversion NPs (UCNPs), and hypocrellin A (HA) for cancer imaging, drug delivery, and photodynamic therapy (PDT). UCNPs were synthesized and PEGylated for biocompatibility and imaging via up-conversion luminescence (UCL). In vitro studies with HeLa cells showed that HA/GOQD/UCNPs treatment plus 460 nm irradiation reduced viability to ~52% compared to 92% without irradiation, demonstrating dose-dependent PDT-induced cytotoxicity via singlet oxygen generation. The nanocomposite also enabled efficient cellular uptake and UCL imaging under 980 nm excitation [230]. Zhang et al. [231] optimized solvothermal synthesis of GQDs by adjusting GO/N,N-dimethylformamide ratio, filling, and reaction time, achieving a fluorescence quantum yield of 17.4% (10mg/ml GO, 80% filling, 12hours). GQDs showed higher biocompatibility than cadmium telluride QDs (>90% vs. <60% viability at 200µg/ml) in Cal-27 oral squamous carcinoma cells, with bright intracellular green fluorescence confirming bioimaging potential [231]. In another work, Rajender et al. [232] reported the synthesis of edge-controlled, highly fluorescent 1–4 layer- GQDs (5–8 nm) with a high density of armchair edges and oxygen defects exhibited a photoluminescence quantum yield of ~32%, with time-resolved photoluminescence indicating charge transfer to the surrounding medium. Confocal imaging showed bright intracellular fluorescence in A-375 melanoma and human cervical adenocarcinoma (HeLa) cancer cells, with cell viability significantly higher in A-375 cells (at 44.4 µg/ml), respectively [232].

While the majority of the mentioned studies used GQDs for cancer cell imaging and photodynamic therapy [230–232], only a few studies have demonstrated their potential in drug loading for phytochemicals. For instance, vinblastine-loaded GQDs were prepared at weight ratios of 1:1, 1:3, and 1:5, yielding particle sizes of 50–70nm, 100–150nm, and ~500nm, respectively, versus 10–50nm for bare GQDs. Cytotoxicity assays on multiple cancer cell lines, including HeLa, human gastric carcinoma (HGC-27), adenocarcinomic human alveolar basal epithelial cells (A549), MCF-7, and human Grade IV glioblastoma cell line (CCF-STTG1) alongside the normal Vero cell line, demonstrated that GQDs enhanced the cytotoxicity of vinblastine toward cancer cells while reducing effects on normal cells. The 1:5 GQD–vinblastine composite demonstrated the highest tumour suppression. In vivo, treatment significantly reduced tumour growth without systemic toxicity, and histopathological evaluation of liver tissues showed no graphene accumulation or structural damage, confirming efficient clearance and biocompatibility [179]. In another work, tryptophan-conjugated GQDs were developed as nanocarriers for CUR to improve drug loading and delivery to MCF-7 breast cancer cells. Tryptophan conjugation increased CUR loading by 23% compared to bare GQDs. CUR-loaded conjugated GQDs showed pH-sensitive release at pH 5.5 and 7.4, following a pseudo-second-order kinetic model. In vitro cytotoxicity studies confirmed that bare GQDs and conjugated-GQDs were nontoxic, while CUR-loaded conjugated GQDs effectively killed MCF-7 cells [233].

Ghafary et al. [234] reported the development of functionalized (MiRGD peptide) GQDs as targeted nanocarriers for DOX and CUR delivery. GQDs (~10nm) exhibited excitation-independent fluorescence (max emission 450nm) and surface amino/hydroxyl functional groups. MiRGD peptides were expressed in Escherichia coli, purified, and complexed with DOX or CUR, forming complexes, which were further loaded onto GQDs to produce GQDs nanocomposites (~50nm for DOX, ~100nm for CUR). Encapsulation efficiency increased to 78% for DOX and 70% for CUR in GQD-loaded complexes, compared to 70% and 43% in peptide-only complexes. Drug release was pH-sensitive, with 77% of DOX and 73% of CUR released at pH5.5 versus 47% and 21% at pH7.4 over 72hours. Functionalized GQDs showed 2–4× higher uptake in αv integrin-positive HUVEC cells than free drugs, with minimal uptake in HFF cells. In 4T1 tumour-bearing mice, nanocomposites preferentially accumulated in tumours (~2.5× higher fluorescence than surrounding organs) with trace distribution (<10% of tumour signal) in liver, lungs, spleen, kidneys, and heart [234].

Liang et al. [235] reported the development of CPT-loaded polymeric micelles with silver (Ag) NPs were made using GQDs as photocatalysts. The polymer formed micelles (40–110nm), which helped produce AgNPs in place (2.5–20.6nm) and carry CPT. Micelles released up to 80% of CPT over 5 days under acidic, tumour-like conditions at 37°C. In vitro, 150μg/ml of the formulation treatment killed 75% of cancer cells within 48hours [235]. Hydrogel nanocarriers composed of GQDs, chitosan, and γ-alumina (~453nm) were loaded with QUE (encapsulation efficiency 87%, drug loading 46.75%), showing sustained pH-sensitive release (95% at pH5.4, 71% at pH7.4, 96hours) and improved cytotoxicity (49% vs. 35% cell death for free QUE) [236]. Nitrogen-doped GQDs (10–14nm, excitation 340nm) loaded with methotrexate via π–π stacking showed ~60% release at 9hours and complete release by 48hours (PBS, pH7.4, 37°C). Cytotoxicity was time-dependent: free methotrexate (1–4mM) was more toxic at 12hours, while GQD-loaded methotrexate exceeded toxicity at 24–48hours, with enhanced intracellular retention. GQDs alone (10–40mM) were biocompatible [237]. Sawy et al. [238] evaluated DOX loading on carbon nanomaterials (CNMs), including GO, GQDs (types 1 and 2), and multiwalled carbon nanotubes (c-SWCNTs and c-MWCNTs). DOX loading was highest on GO (2.85mg/mg), followed by GQDs-1 (1.6mg/mg) and GQDs-2 (1.14mg/mg), with sustained intracellular release. Unmodified CNMs showed minimal toxicity (>60%–100% viability at 200μg/ml), whereas DOX-loaded CNMs induced concentration- and time-dependent cytotoxicity (IC₅₀: DOX-GQDs1 0.74μg/ml, DOX-GQDs2 1.07μg/ml, DOX-GO 1.77μg/ml, DOX-c-SWCNTs 1.69μg/ml vs. free DOX 2.8μg/ml). Cellular uptake was higher for small CNMs (DOX-GQDs1: 122.6a.u. vs. free DOX 79.5a.u.), while c-MWCNTs showed poor uptake. Apoptosis and G0/G1 cell cycle arrest were confirmed in MCF-7 cells. In vivo, GQDs1 and GQDs2 (5–20mg/kg) were well tolerated with no mortality or significant hematological, biochemical, or histological changes [238]. Table 2 reresents a summary of graphene-based nanosystems for phytochemical delivery.

Table 2. Surface functionalized GO, and rGO loaded with anticancer drugs.

6.1. GO

GO exhibits a well-documented pattern of size-, dose-, and exposure-dependent toxicity that spans inflammatory, hematological, developmental, and organ-specific effects. For instance, GO (150–250 nm) administered at 0.5, 1.0, and 2.0 mg/ml in mouse skeletal muscle induced dose- and time-dependent inflammation and immune cell infiltration, while in human red blood cells, concentrations of 0.2, 2.0, and 20 mg/ml caused hemolysis and impaired coagulation [241]. GO sheets (lateral dimension 1–1.5 µm, and thickness 1.5–2.5 nm) administration (i.p., 2 and 5 mg/kg for 5 days) induced dose-dependent systemic toxicity. At low doses (2mg/kg), mild excitatory effects were noticed with increased locomotor activity (~74%). Serum biomarkers indicated organ stress, including reduced creatinine and dose-dependent hepatic dysfunction. At high doses (5mg/kg), a sharp increase was noticed in oxidative stress markers, including liver peroxidase activity and elevated malondialdehyde in the liver and brain. Organ analysis showed ~21% kidney index increase (1.48–1.49 vs. 1.22 controls), and liver water content increased from 60.48% (control) to 68.67% and 81.72% at 2 and 5mg/kg, indicating hepatic edema. Histopathology confirmed hepatocellular swelling and inflammation at 5mg/kg, with no overt neuronal damage [242]. Administration (p.o., 10, 20, or 40 mg/kg) of GO with sheet sizes around 1162 nm (TEM: 62.5 ± 51.42 nm) for either one day (acute) or five consecutive days (subacute) produced marked genotoxic and histopathological effects in mice. Both treatment durations induced a highly significant, dose-dependent increase in micronucleated polychromatic erythrocytes (p < 0.001), accompanied by a significant reduction in polychromatic and normochromatic erythrocyte ratio (p < 0.001), indicating bone marrow cytotoxicity. Histopathology revealed dose-dependent hepatic and neuronal damage, ranging from slight-to-moderate hepatocyte degeneration and necrosis at 10–20mg/kg to severe parenchymal necrosis at 40mg/kg, accompanied by inflammatory infiltration and necrotic brain lesions [243]. Similarly, exposure to pristine graphene (350 nm–6 µm) and GO (100 nm–2.5 µm) in zebrafish embryos demonstrated clear dose-dependent toxicity. Increasing concentrations of pristine graphene (50–100 µg/ml) and GO (20–100 µg/ml) significantly elevated developmental abnormalities, including tail deformities, pericardial edema, and higher nonhatching rates, primarily due to material agglomeration on the chorion surface [244].

Exposure to GO nanosheets (small and large, 5–100 μg/ml) caused dose- and time-dependent ocular toxicity in mice, with iris neovascularization, apoptosis of corneal epithelial cells, increased inflammatory (IL-6, IL-8, TNF-α), and oxidative stress (malondialdehyde) markers. In contrast, rGO nanosheets of similar size and dose showed no significant ocular damage, maintaining normal corneal morphology, cell viability, and antioxidant levels [245]. A study using zebrafish and RAW264.7 cells as in vivo and in vitro models evaluated the developmental neurotoxicity and immunotoxicity of GO. No acute developmental toxicity was observed at 0.01, 0.1, and 1 μg/ml GO over five days. However, exposure to 10 μg/ml GO led to decreased hatching rates, increased malformations, elevated heart rates, and hypoactivity in locomotor behavior. Significant increases in immune-related mRNA levels (IL-6, IL-8, TNFα, and IFN-γ) were observed under environmental exposure conditions, along with pro-inflammatory activation in macrophages [246]. Low-dose GO exposure (p.o., 0.01–1 mg/l, during pregnancy and lactation) induced multigenerational neurobehavioral alterations, including anxiety, altered swimming behavior, and liver atrophy, with some effects persisting into second-generation offspring in mice [247]. Administration (i.v., up to 1.25 mg/kg) to pregnant mice caused embryo toxicity with increased fetal resorption, stillbirths, and decreased birth weight. This toxicity was associated with disrupted maternal gut microbiota, which could be partially alleviated by fecal microbiota transplantation through restoration of microbial balance and modulation of fecal metabolites [248]. Functionalized GO also demonstrates long-term persistence. Administration (i.v., 4, 8, or 16 mg/kg) of poly sodium 4-styrenesulfonate functionalized-GO (300–700 nm, ~2 nm thickness) produced long-term organ retention and dose-dependent toxicity in BALB/c mice. Fluorescence imaging confirmed rapid systemic circulation (1–30minutes) and predominant liver localization, with persistent lung, liver, and spleen retention up to 6 months and visible black aggregates, especially at higher doses (8–16mg/kg). Serum biochemistry indicated acute hepatic injury at 16mg/kg, with elevated liver markers on day 1 that largely normalized by days 7–90, except for a mild alanine aminotransferase rise at day 90. Histopathological evaluation showed chronic inflammation, including macrophage accumulation in the lung, Kupffer cell activation in the liver, and spleen macrophage infiltration, with black aggregates observed up to 180 days [249].

6.2. rGO

rGO generally demonstrates a more favorable biocompatibility profile than GO; however, its toxicity remains strongly influenced by size and surface chemistry, with evidence of reproductive, neurological, and barrier-disruptive effects under specific exposure conditions. In Kunming mice, rGO exhibited markedly superior ocular safety compared to GO at all tested doses (25, 50, and 100μg/ml) over seven days regimen. No iris neovascularization, corneal opacity, stromal thickening, or inflammatory cell infiltration was observed, and vascular parameters remained comparable to controls. Histopathology confirmed intact corneal layers with normal epithelial, stromal, and endothelial morphology, while biochemical analyses showed no elevation of inflammatory cytokines (IL-6, IL-8, TNF-α) or oxidative stress marker (malondialdehyde) [245]. In C57BL/6 mice, oral administration of small (~88±31nm) and large (~472±249nm) rGO (60mg/kg/day for 5days) produced no changes in body weight, temperature, or organ-to-body-weight ratios. Behavioral assessments revealed only transient locomotor reduction on day 1 and short-term impairments in balance and neuromuscular coordination during the first 2–3 days, which normalized with training. Spatial learning and memory in the Morris water maze were unaffected in both short- (1–8days) and long-term (22–67days) evaluations. Muscle and liver glycogen, liver and kidney function, and hematological parameters remained normal, confirming the absence of systemic toxicity [250]. In a chronic intratracheal exposure study, C57BL/6J female mice received a single administration of rGO nanosheets (lateral size 1–2μm) at doses of 18, 54, or 162μg and were monitored for 90days. High-dose exposure induced pronounced cardiopulmonary stress, including significant airway inflammation, elevated pro-inflammatory cytokines (IL-1β and TNF-α), and marked suppression of endogenous antioxidant defenses (superoxide dismutase and glutathione peroxidase). Histopathology revealed tubular injury, disruption of blood–brain barrier integrity, and reduced sperm viability, indicating potential reproductive toxicity [251]. rGO nanosheets (70 nm lateral size) demonstrated size- and dose-dependent reproductive toxicity in female mice. Low (6.25 mg/kg) and intermediate (12.5 mg/kg) doses at late gestation (~20 days) caused complete abortion; high-dose (25 mg/kg) exposure resulted in maternal mortality in 7 of 8 mice [252]. Functionalization of rGO with dodecyl amine and trimethylammonium chloride was reported to be nongenotoxic, with controlled immunotoxicity. Although trimethylammonium functionalized rGO exhibited higher cytotoxicity and inflammatory responses [253].

Administration (i.p., 50 mg/kg) of PEG–functionalized rGO (small 27 nm, and large 50 nm) in BALB/c mice resulted in high and persistent accumulation in the liver and spleen due to efficient uptake by the reticuloendothelial system. Larger rGO-PEG showed nearly two-fold higher uptake than smaller particles at day 7 post-injection. Histology revealed pigment-like aggregates persisting up to 30days without significant inflammation or organ dysfunction. Following oral administration (100mg/kg), neither size variant exhibited detectable systemic absorption, and both were rapidly excreted, with no hepatic, renal, hematological, or histopathological abnormalities over 90days [254]. In vitro, rat astrocytes and brain endothelial cells exposed to non-PEGylated rGO (342±23.5nm) and PEGylated rGO (910±32.7nm) showed concentration-dependent toxicity. Non-PEGylated rGO caused minimal effects, whereas rGO-PEG at 100μg/ml markedly reduced cell viability. In vivo, administration (i.v.) of rGO-PEG disrupted blood–brain barrier integrity, downregulating occludin, β-catenin, laminin, and connexin-43, and elevating ROS levels by 270%, indicating that PEGylation increased particle size and toxicity toward astrocytes, endothelial cells, and barrier-associated proteins [255]. Pluronic-functionalized rGO administration (i.v.) at 10 mg/kg (Swiss Albino mice) and 5 or 10 mg/kg (pregnant Wistar rats) showed no observable adverse effects, absence of organ damage, inflammatory responses, or developmental toxicity. Biodistribution studies using confocal Raman mapping showed minimal organ accumulation, with only a small fraction crossing the placenta. Across all exposure conditions and doses, no systemic toxicity, developmental abnormalities, or neurobehavioral deficits were detected in dams or pups [256].

6.3. GQDs

GQDs present a toxicological landscape characterized by dose-dependent cytotoxicity, oxidative and behavioral disturbances, and functionalization-driven variation in biological safety. In vitro assessments showed that GQDs exhibited mild mutagenic activity in specific Salmonella strains, inducing mutations in TA97a at 100 µg/plate and in TA102 at 1,000 µg/plate, depending on metabolic activation. Cytotoxicity studies demonstrated a dose- and time-dependent reduction in cell viability, with 3T3BalbC fibroblasts more susceptible than HepG2 cells due to mitochondrial and membrane damage [257]. Aqueous dispersion of raw and amino-functionalized GQDs showed strictly dose-dependent toxicity in yeast (Saccharomyces cerevisiae) and H9c2 cardiomyoblast models. Raw GQDs (6.8nm), glutathione-functionalized GQDs (17.9nm), and thiourea-functionalized GQDs (36.4nm) caused no significant toxicity at concentrations up to 2%, whereas higher exposures (up to 25%) produced only inhibitory effects in yeast. Comparison of IC₅₀ values showed minimal toxicity differences among the GQD types, indicating that the slight toxicity observed originated mainly from noncarbonized citric acid precursors rather than the carbon-dot core. Functionalization generally reduced cytotoxicity, with thioacetamide-modified GQDs being slightly more toxic than thiourea- and glutathione-functionalized counterparts [258].

Long-term oral exposure (10, 20, or 40 mg/kg for 30 days) in mice induced dose-dependent anxiety-like behavior. In behavioral tests, locomotor activity and central-zone/open-arm exploration decreased, while step-through latency was reduced. In the hippocampus, oxidative stress was prominent with a decrease in catalase activity, an increase in MDA, and glutathione peroxidase activities rose significantly, and total antioxidant capacity declined across all doses. Histopathological evaluation confirmed neuronal damage in Cornu Ammonis 1 (hippocampus) and dentate gyrus, with cytoplasmic vacuolization, karyopyknosis, increased glial cells, and elevated abnormal neuron counts (p < 0.001) [259]. In zebrafish embryos, four GQD types (raw, GO-, carboxyl-, and aminated-GQDs) at tested doses of 50, 100, and 200 µg/ml exhibited concentration-dependent developmental toxicity. Aminated-GQDs caused developmental arrest at ≥100µg/ml, reducing survival, heartbeat, and increasing malformations. Transcriptomic analysis showed all GQDs affected ion channel regulation and spliceosome pathways, while aminated-GQDs uniquely upregulated protein C anticoagulant, complement/coagulation, cell adhesion, and MAPK apoptotic signaling [260]. In mice, intranasal exposure to nitrogen-doped or amino-functionalized GQDs (0.1–1mg/kg, every other day for 28days) mainly accumulated in the liver and kidneys. Amino-functionalized GQDs caused mild biochemical changes (alkaline phosphatase and transaminase, urea), indicating slight hepatic and renal stress. Histology showed minor alveolar dilation in lungs and renal vasodilation/congestion at 1mg/kg, with no significant liver lesions [261].