Background

Currently, various malignancies have greatly influenced and invaded human life, with 14 million new cancer patients annually worldwide, posing a global threat (Jaymand, et al. 2021). The global reluctance towards cancer has promoted researchers from different fields of science and technology to unfold more efficient diagnostic and therapeutic approaches (Xin et al. 2017). Significant solicitude has been dedicated to the combination of nuclear medicine and nanomaterials to transfigure the conventional methods of cancer diagnosis and treatment (Silindir-Gunay and Ozer 2017; Smith and Gambhir 2017). Graphene oxide (GO) is a promising carbon family nanomaterial with rational multifunctional applications owing to its realistic physicochemical properties such as honeycomb-lattice with sp2 hybridized carbon in two-dimensional geometry, the largest surface area for a nanoscale material and availability of various surface oxygen groups (hydroxyl, epoxides, and carboxylic groups) (Jaymand, et al. 2021; Kostas Kostarelos and Novoselov 2014; Pan et al. 2012; Smith and Gambhir 2017; Y. Yang, et al. 2012). The biocompatibility analysis of graphene-based materials is an indispensable prerequisite for in vivo biomedical applications. Hence, various studies had been explored the in vitro and in vivo cyto- and bio-compatibility declaring that graphene toxicity is highly dependent on the physiochemical properties (Lalwani et al. 2016; Yoo et al. 2015; B. Zhang et al. 2016). The size architectures, size and morphology, might affect the cellular uptake while functional groups functionalization might change the interactions with micronutrients and proteins (De Marzi, et al. 2014; H. Zhang, et al. 2013). The starting materials and production methods could evolve metallic impurities in the final product (Wong, et al. 2014). The post-synthetic treatment for providing aqueous dispersion might also influence the toxicity (Mullick Chowdhury et al. 2014). Regarding the in vivo toxicology and pharmacokinetics of graphene-based materials; Yang et al. explored the biodistribution behavior of PEGylated [125I]iodo-labeled nanographene sheets post-intravenous injection (IV) in long-term manner demonstrating reduced radioactivity levels in most organs and tissues by time (K. Yang, et al. 2011). They also explored the blood biochemistry and hematology studies declaring non-significant toxicity for 3 months. In the past decades, chelators-based radioisotopes labeling of GO as nano-theranostics had been demonstrated but showed some predestined challenges in regard to changing the hydrophilicity, surface charge and size of the tracer (Cornelissen, et al. 2013; Hong et al. 2012a, b; Kostas Kostarelos and Novoselov 2014). Intrinsic, non-chelation, radiolabeling methods were recently employed where the radioisotopes can directly tagged-into the nanoparticles surface preserving the native physicochemical as well as in vivo biological behavior (Goel et al. 2014; Shi, et al. 2017; Xiaolian Sun et al. 2015). In this regard, the small thickened GO radiotracers can facilely extravasate though the leaky vasculature’s body compartments (tumor and infarcts) owing to the enhanced permeability and retention (EPR) phenomena, passive targeting (Jasim, et al. 2021; Jaymand, et al. 2021). On the other hand, biological ligands can directly functionalize the oxygen-rich functional groups of GO surface for potentiating targeted drug delivery-based active targeting approach (Mohanta, et al. 2021; Zhao and Liu 2014). Folic acid (FA), a biological ligand associated with several human folate-receptors (FRs) overexpressed in many cancers, has received significant interest as a targeting candidate for cancer theranostics due to its low cost, high stability and facilely conjugated chemistry (Thapa, et al. 2016; Zhao and Liu 2014). Overall, GO-based nano-systems can facilitate the evolution of an ideological approach to scale up new technologies for tackling the detection limits of cancer’s early diagnosis and improving drug targeting as well as therapy approaches. In the present study, carboxyl-functionalized GO nanosheets (FGO), passively targeted nano-formulation, with superior physicochemical characteristics were developed using an improved and eco-friendly Hummers method. These nanosheets were exposed to amidation reaction to conjugate FA moiety compromising folate-targeting nano-formulation. The resulting two nano-formulations were appointed to chelator-free iodine-131 [131I] radiolabeling in which the optimization process was fully controlled. The in vivo studies of both nano-theranostics in tumor-bearing mice were performed to directly appraise different targeting approaches.

Results and discussion

The synthetic procedure of CGO is depicted schematically in three steps (Fig. 1) and demonstrated as follows: synthesis of GO nanosheets, carboxyl-functionalization of GO nanosheets and conjugation of FGO with FA via amide bond formation.

Fig. 1
figure 1

Schematic illustration of the synthetic procedure of folic acid conjugated graphene oxide (CGO)

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Synthesis and functionalization of graphene oxide nanosheets

Improved Hummers method, an eco-friendly procedure, was utilized to synthesis graphene oxide nanosheets in which sodium nitrate was excluded during the preparation preventing the formation of harmful gases (NO2/N2O4) (J. Chen et al. 2013; K. Yang et al. 2013). This method relies on the oxidizing capability of H2SO4 solution containing KMnO4 to convert graphite to GO. The obtained yellow-colored slurry during the synthesis and the yellowish-brown color after exfoliation indicate the successful oxidation of graphite and the formation of small sized GO (J. Chen, et al. 2013; Velasco-Soto, et al. 2015; K. Yang, et al. 2013). The successful formation of GO was confirmed by the UV–visible spectrum analysis (Fig. 2A). It declared absorption peaks at 235 and 305 nm related to π–π* and n–π* transitions of C=C and C=O bonds, respectively (Noroozi et al. 2016; Romero et al. 2017). It is worth-noting that GO are rich in epoxy and hydroxyl groups that need to be converted to carboxyl groups for providing further functionalization/conjugation as well as improving the water solubility (Xiaoming Sun et al. 2008; L. Zhang et al. 2010). In our study, the carboxylation of graphene oxide was performed utilizing chloroacetic acid in highly basic conditions which indicated by the color change from yellowish-brown to brownish-black attributed to the partial reduction (Song and Xu 2013; Zhao and Liu 2014).

Fig. 2
figure 2

UV–vis spectrum of A carboxyl-functionalized graphene oxide (FGO) B folic acid conjugated graphene oxide (CGO)

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Synthesis of folate conjugated graphene oxide nanosheets (CGO)

Upon carboxylation, the affordable carboxylic groups on the GO edges could be utilized for the chemical conjugation with folic acid via amide bond formation. The amidation reaction-based EDC/NHS chemistry is a very popular procedure for the synthesis of high yield conjugates (Hermanson 2013). EDC/NHS agents have the ability to activate the carboxylic groups on the GO for straightforward reaction with the amine group of folic acid forming amide linkage nano-conjugate (Fig. 3). The successful conjugation was checked by UV–visible analysis demonstrating the appearance of a new peak at 385 nm in addition to the other two peaks related to GO (Fig. 2B). This new peak may be attributed to the pterin ring in FA (Kato, et al. 2004; Zhao & Liu 2014).

Fig. 3
figure 3

Schematic illustration of the amide bond formation between FGO and FA utilizing EDC/NHS chemistry

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Characterization of FGO and CGO

For the morphological examination, graphene nanosheets showed a crystalline nanometric texture while conjugated graphene nanosheets showed jaggy-like texture (Fig. 4). The lateral width of CGO was 10.5–12.5 nm as one side dimension (Fig. 5). Both FGO and CGO had shown high stability nature that was confirmed with their surface zeta potential charge − 41 mV and − 34 mV, respectively.

Fig. 4
figure 4

SEM images of a FGO and b CGO

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Fig. 5
figure 5

SEM images of CGO lateral width

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FT-IR was performed to study the surface functional groups and to confirm the conjugation between FGO with folic acid. 1695.62 cm−1 in CGO confirming amide carbonyl group and 1020.08 cm−1 in CGO confirming C–N stretching. The broad band at 3438.10 cm−1 in CGO confirms carboxylic group (Fig. 6).

Fig. 6
figure 6

FT-IR analysis of folic acid, FGO and CGO

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In vitro cytotoxicity assay

It is crucial to determine whether the developed two nanosheets have a harmful effect on the normal cells. Figure 7 demonstrates the cytotoxic profile of FGO and CGO nanosheets against WI-38 cell line. It declared that the cells exposed to 25 µg/ml of FGO and CGO exhibited 96.94 and 100% viability level which decreased to 78.43 and 91.43% by increasing the concentration to 100 µg/ml, respectively. These results ensure that FGO as well as CGO are comparatively non-toxic to normal cells as no significant toxicity was observed, even at the highest tested concentration (100 µg/ml).This cyto-compatible behavior may be attributed to the nano-sized construction as well as carboxyl functionalization where GO toxicity extremely depends on its size and hydrophobicity (Burdanova et al. 2021; Muthoosamy et al. 2014; Sasidharan, et al. 2011). These results exhibited a reasonable cyto-compatibility profile that prompted further in vivo experiments. Our results are in accordance with most of the current literatures demonstrating the great biocompatibility behavior of graphene quantum dots (GQDs) (Perini et al. 2020, 2021, 2022). Shang et al. reported that GQDs showed high biocompatibility on neural stem cells even at relatively high concentrations of nanoparticles (250 µg/ml) (Shang, et al. 2014). Fasbender et al. also reported high viability of human red blood cells even after administration of GQDs at 500 µg/ml (Fasbender, et al. 2017). Moreover, Nurunnabi et al. highlighted low cytotoxicity of carboxylated GQDs at concentrations ranging from 0 to 250 µg/ml (Nurunnabi, et al. 2013). Another study investigated the effect on A549 and C6 cell lines of three different surface chemistries: carboxylation, amination and functionalization with dimethylformamide. In all cases, they measured a slight reduction in cell viability as well as a very low mortality (Yuan, et al. 2014).

Fig. 7
figure 7

Cell viability assay of FGO and CGO against WI-38 normal cells cultured 37 °C for 48 h

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Radiosynthesis of radioiodinated functionalized graphene nanosheets (IFGO) and folate conjugated graphene oxide nanosheets (ICGO)

The radiolabeling of both tracers was achieved using intrinsic radioiodination approach where the radionuclide is directly attached to the surface of the nanoparticles without the need of any chelators. The radioiodination of FGO and CGO reach their highest RCP by optimization of all parameters affecting the radioiodination process. The electrophilic substitution of iodine-131 was carried out in the presence of chloramine-T as an oxidizing agent. Iodide ion was converted to iodonium ion by the aid of Chloramine-T (Fayez et al. 2020) which has a significant impact on the radioiodination process, also it permits the occurrence of the electrophilic substitution process. Figures 8a and 9a show the optimum amount of 200 µg from CAT which create the highest RCP of 96.58% and 94.11% for [131I]I-FGO and [131I]I-CGO, respectively. Insufficient oxidation of I-131 and undesirable oxidative byproduct may be due to low or high amounts of CAT, respectively (Selim et al. 2021; Swidan et al. 2014). RCP also depends greatly on the pH values of the reaction mixture. The RCP was increased by increasing pH from acidic values to nearly neutral values (pH 8) and decreases by increasing pH values more than 8 as shown in Figs. 8b and 9b for [131I]I-FGO and [131I]I-CGO, respectively, this decrease in RCP may be due to iodate (IO3−) and hypoiodite (IO−) ion formation (Saha 2012; M. Swidan et al. 2015). Figures 8c and 9c illustrate the effect of substrate amount (FGO and CGO) on the RCP, % where the highest RCP was gained at 500 µL substrate for both reaction which can capture all iodonium ion from the reaction mixture and there are no significant changes in RCP during increasing substrate amounts. The speed of this reaction and stability of the formed radioiodinated compound is shown in Figs. 8d and 9d for [131I]I-FGO and [131I]I-CGO, respectively. The reaction was completed after 30 min and their RCP were stable for 24 h. Overall, it is worth-noting that the direct introduction of 131I radionuclide to the surface of GO (intrinsic radioiodination) did not alter the physicochemical properties, size and charge characteristics, of the native preparation based on the re-characterization analysis beyond the full radioactive decay (~ 10 half-lives). This superior intrinsic radioiodination might introduce the priority of our developed radiotracers over the previously published ones synthesized by the chelator-based radiolabeling (Cornelissen, et al. 2013; Hong et al. 2012a, b; D. Yang, et al. 2016). For example, Cornelissen et al. demonstrated the radiolabeling of GO with indium-111 [111In] through diethylenetriaminepentaacetic acid (DTPA) chelating agent strategy revealing dramatic increase in the thickness of the GO sheets (Cornelissen, et al. 2013).

Fig. 8
figure 8

Variation of RCP of [131I]I-FGO as a function of variable parameters: a) CAT amount; b) pH; c) substrate amount; d) reaction time

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Fig. 9
figure 9

Variation of RCP of [131I]I-CGO as a function of variable parameters: a) CAT amount; b) pH; c) substrate amount; d) Reaction time

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Biological evaluation of radioiodinated functionalized graphene nanosheets (IFGO) in normal mice model

IFGO showed a typical biodistribution behavior as a nanomaterial (Fig. 10). It was washed out from blood in a good behavioral way with a decrease from 16.35 ± 1.54 to 3.21 ± 0.14%ID/g at 30 min and 150 min, respectively. Three main organs represented the targeted organs of nanoparticles; Liver, spleen and lungs. The highest uptake %ID/g of liver (13.68 ± 1.56), spleen (6.07 ± 1.01) and lungs (9.69 ± 1.21) was observed at 90 min, 60 min, 60 min, respectively. Hepatobiliary and renal pathways were the main excretory ways of IFGO as shown in the uptake %ID/g of liver, kidneys and intestine. Renal pathway was the fastest excretory route as kidneys showed 8.32 ± 1.83%ID/g at 30 min. All other body organs did not show any valuable accumulation of IFGO. The in vivo distribution behavior of IFGO in normal mice was matched with the biodistribution of almost carbon-based radiolabeled nanomaterials (Jaymand, et al. 2021; K Kostarelos et al. 2009). Wei et al. investigated the in vivo biodistribution of technetium-99m labeled oxidized multi-walled carbon nanotubes (99mTc labeled oMWCNTs) declaring that the radiotracer was mostly accumulated in liver, spleen and lungs (Wei, et al. 2012). Menezes et al. fabricated GQDs through a green chemistry approach and labeled them with 99mTc. They reported very limited off-target uptake by the mononuclear phagocytic system and the organ biodistribution in healthy animals (De Menezes, et al. 2019). Additionally, Yang et al. explored the long-term in vivo biodistribution of 125I-labeled nanographene sheets (NGS) functionalized with polyethylene glycol (PEG) revealing main accumulation in the reticuloendothelial system (RES) including liver and spleen after intravenous administration and can be gradually cleared, likely by both renal and fecal excretion (K. Yang, et al. 2011).

Fig. 10
figure 10

Normal biodistribution behavior of radioiodinated functionalized carbon nanosheets (IFGO) in normal mice model as a function of %ID/g organ

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In vivo comparative study between radioiodinated functionalized graphene nanosheets (IFGO) and radioiodinated conjugated graphene nanosheets (ICGO) in tumor-bearing mice model

Figure 11 confirms efficient selectivity of ICGO to target tumor tissues in huge advance than IFGO. By deep analysis of the uptake %ID/g of the main important organs, liver, spleen, lungs and tumor, it is so clear that after the conjugation of functionalized graphene nanosheets with folic acid, liver, spleen and lungs uptakes %ID/g intensively decreased with impressive increase of tumor uptake %ID/g. Additionally, the folate-receptors tumor targeted radiotracer, IFGO, demonstrated maximum tumor accumulation (~ 14%ID/g) representing a superior prominence over the previously published tumor targeted, vascular endothelial growth factor receptor (VEGFR) and follicle stimulating hormone receptor (FSHR), GO radiotracers with potential tumor uptake ~ 8 and 11.5%ID/g, respectively (Shi, et al. 2015; D. Yang, et al. 2016).

Fig. 11
figure 11

Uptake %ID/g organ (liver, spleen, lungs and tumor) of radioiodinated functionalized graphene nanosheets (IFGO) and radioiodinated conjugated graphene nanosheets (ICGO) in tumor-bearing mice mode

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Target/nontarget ratio represents the uptake %ID/g organ of tumor muscle/normal muscle that confirms the selectivity of the injected agent for differentiating between cancer and normal cells from the same tissue origin (Mahmoud et al. 2022). As presented in Fig. 12, it is clear that T/NT of ICGO (7.27 and 7.19 at 60 min and 90 min, respectively) showing in average 4–5 times folds of T/NT of IFGO (1.9 and 1.56 at 60 min and 90 min, respectively). Furthermore, our synthesized radiotracer (IFGO) demonstrated higher tumor accumulation with lower RES-organs’ uptakes when compared to 131I-labeled PEG-functionalized reduced nano-GO (131I-RGO-PEG) (L. Chen, et al. 2015). IFGO showed a significant reduction in accumulation within the spleen, liver, and lung when compared to amino-functionalized GO labelled with radioactive gold nanoparticles (198,199Au-labeled amino-functionalized GO) as a SPECT imaging and therapeutic agent (Fazaeli et al. 2014).

Fig. 12
figure 12

Target/nontarget (T/NT) of IFGO and ICGO in tumor-bearing mice model

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Methods

Chemicals and equipment

The natural graphite powder (99.9%), potassium permanganate (KMnO4, 95%), concentrated sulfuric acid (H2SO4, 98%), hydrochloric acid (HCl, 37%), hydrogen peroxide (H2O2, 37%), sodium hydroxide (NaOH, > 98%), chloroacetic acid (ClCH2COOH, 99%), N-(3-dimethylaminopropyl-N’-ethylcarbodiimide) hydrochloride, EDC (C8H17N3·xHCl), N-Hydroxysuccinimide, NHS (C4H5NO3, 98%), Dimethyl sulfoxide, DMSO ((CH3)2SO, > 99%), folic acid (C19H19N7O6, > 97%), Dulbecco’s minimum essential medium (DMEM), fetal bovine serum (FBS) as well as MF-Millipore® Membrane Filter (0.22 µm pore size) were purchased from Sigma-Aldrich company (Darmstadt, Germany). All solutions preparation as well as sanitation processes were performed utilizing ultrapure water (a Milli-Q purification system (Merck, Massachusetts, USA). Shimadzu UV-1700 spectrophotometer (Thermo Fisher Scientific, USA) geared up with 1 cm quartz optical length cuvettes was applied to confirm the proper formation of the nanosheets. Ultrasonic bath P30H (Elma Schmidbauer GmbH, Germany) operating at 200 W was applied for dispersion purposes. Photo correlation spectrometer (PCS) was used using Brookhaven 90 plus (Zetasizer Nano™, Beckman Coulter, Miami, FL, USA). UV–visible Spectroscopy was performed using a Cyber lab UV-100 double beam spectrophotometer (Thermo Fisher Scientific, USA). Fourier transform infrared (FT-IR) spectra were obtained on Perkin Elmer Spectrum 100 Spectrometer (Mattson Instruments, Inc., New Mexico, USA). No-carrier-added [131I]NaI was received as a gift from RPF (Radioisotopes-Production-Facility), Egyptian Atomic Energy Authority (EAEA). A NaI (Tl) scintillation counter (Scaler Ratemeter SR7 model, the United Kingdom) was used for γ-ray radioactivity measurement. Thin layer chromatography (TLC) was carried out on the plates of silica gel (Merck Kiesel gel 60F254, BDH) to monitor the progress of the radiosynthesis process.

Synthesis and functionalization of graphene nanosheets (FGO)

Graphene oxide nanosheets (GO) were synthesized according to an improved Hummers method (J. Chen, et al. 2013; K. Yang, et al. 2013). Briefly, the natural graphite powder (1 g) was dissolved in a concentrated solution of sulfuric acid (23 ml) under vigorous magnetic stirring in an ice-bath. This is followed by slow addition of potassium permanganate (3 g) in which the reaction temperature should not exceed 20 °C. The suspension flask was moved into a water bath at 40 °C and vigorously stirred for 30 min. Then, the reaction temperature was raised to 70 °C in which the suspension color changed to brown. Subsequently, a definite volume of water (50 ml) was slowly added and the reaction proceeded at 95 °C for 15 min followed by another addition of 150 ml of water. Next, hydrogen peroxide (10 ml) was added turning the dispersion color to yellow, which indicates the formation of GO nanosheets. Finally, the purification process was performed through centrifuging the dispersion at 12,000 rpm for 20 min followed by washing the resultant precipitate with 10 ml hydrochloric acid (10% v/v) to remove the metal ions and then water washing was utilized until neutral pH was achieved. The GO aqueous dispersion was exfoliated by 60 min bath-sonication process to obtain small nano-sized flaks. The exfoliated dispersion was centrifuged for 15 min at 12000 rpm in order to remove the un-exfoliated flaks. The carboxylic acid functionalization of GO was performed according to previous literatures (Song & Xu 2013; K. Yang, et al. 2011). Sodium hydroxide (0.12 g/ml) and chloroacetic acid (0.1 g/ml) were fully dissolved in an aqueous dispersion of GO (2 mg/ml, 10 ml). This mixture was forced for 3 h bath-sonication. The resulting mixture was neutralized with dilute hydrochloric acid and purified by repeated rinsing and filtration until well dispersion (carboxyl-functionalized graphene oxide (FGO) was obtained.

Synthesis of folate conjugated graphene oxide nanosheets (CGO)

Folic acid (FA) was conjugated to carboxylated graphene oxide through covalent bond formation between the carboxyl groups of FGO and the amine group of FA (Eivazzadeh-Keihan, et al. 2022; Qin, et al. 2013). Briefly, a definite concentration of EDC and NHS (5 mg/ml and 1 mg/ml, respectively) were added to the dispersion of FGO in DMSO (1 mg/ml) and then the mixture was sonicated for 30 min. Another portion of EDC was further added while the sonication continues for another 30 min. Then, FA (2 mg/ml) was added and the mixture was preceded under stirring overnight at room temperature. Finally, the mixture was centrifuged and washed twice with water and ethanol to get rid of excess DMSO as well as any unconjugated FA. The mixture was filtered using 0.22 µm membrane filter and then the product (folate conjugated graphene oxide; CGO) was re-dispersed in water for washing and then filtered again, left for air-drying then collected.

Characterization of FGO and CGO

The morphological appearances, sizes and surface charges of both of functionalized graphene nanosheets and conjugated graphene nanosheets were evaluated using SEM and PCS instruments. FT-IR analysis was performed for free folic acid, functionalized graphene nanosheets (FGO) and conjugated graphene nanosheets (CGO) to evaluate the surface function groups and confirm the conjugation between folic acid and functionalized graphene nanosheets.

In vitro cytotoxicity assay

Normal human lung fibroblast cell line (WI-38 cells) that deposited overnight on 96-well plates (4000/well) were refined using Dulbecco's modified Eagle's medium (DMEM) loaded with 10% fetal bovine serum, 1% streptomycin–penicillin antibiotics. The cells were then cultivated at 37 °C for 48 h in a 5% CO2 saturated humid incubator in the presence of different concentrations of FGO as well as CGO. Control cell line was assessed without incorporating any of the tested samples. After the incubation period was complete, the cell growth was spectrophotometrically analyzed to identify the absorbance of the plates with the help of crystal violet solution (1%) at tested wavelength 490 nm.

Radiosynthesis of radioiodinated functionalized graphene nanosheets (IFGO) and radioiodinated folate conjugated graphene oxide nanosheets (ICGO)

Radioiodination procedure was carried out by studying different amounts for both the synthesized FGO and CGO (100–700 µL) solutions, then (50–500 µg) of freshly prepared chloramine-T (CAT) solution were added. After that, 10 µL of [131I]NaI (4 MBq) was added to the mixture of the reactions, and the pH values were adjusted from 5 to 10. The reactions were kept at room temperature up to 24 h. After a definite time, the reactions were quenched using sodium metabisulphite solution. All these parameters were carefully studied in order to obtain the highest radiochemical purity (RCP). Formation of [131I]I-FGO and [131I]I-CGO were monitored by using TLC using methanol (70%). 2 µL from each reaction mixture was spotted at a point of 2 cm from lower edge of TLC paper strip (13 cm length and 1 cm width), then the strip was developed ascendingly in a closed glass tube containing a freshly prepared methanol (70%) as a mobile phase. free iodide was migrate with the solvent at Rf = 0.7–1; while [131I]I-FGO and [131I]I-CGO remained at origin (Rf = 0–0.1), then the RCP was calculated. After reaction completion both [131I]I-FGO and [131I]I-CGO were purified from free iodine using TLC technique.

Biological evaluation of radioiodinated functionalized graphene nanosheets (IFGO) in normal mice model

Radioiodinated functionalized graphene nanosheets (IFGO) were evaluated in vivo in normal mice model to determine its biodistribution behavior with different time intervals (30, 60, 90, 120 and 150 min). The different percentage injected dose per gram (%ID/g) values for each organ and fluid were calculated ex vivo after extracting all mice parts at the assigned time points (El-Ghareb, et al. 2020; Sakr, et al. 2018; M. M. Swidan, et al. 2019). Three mice were examined at each time point. Statistical evaluation for the results was applied. All the approvals for ethics regulations were obtained from Egyptian Atomic Energy Authority.

In vivo comparative study between radioiodinated functionalized graphene nanosheets (IFGO) and radioiodinated conjugated graphene nanosheets (ICGO) in tumor-bearing mice model

IFGO and ICGO were evaluated in vivo in tumor-bearing mice model to determine their accumulation in tumor tissues besides the relevant organs such as lung, liver, spleen and normal muscle. Tumor was induced in right thigh muscle of mice using Ehrlich ascites carcinoma obtained from the National Cancer institute, Egypt. Mice selected organs were collected and evaluated for %ID/g to compare between the targeting ability of both agents in vivo and to evaluate their retaining ability in the other relevant organs (Aljuhr et al. 2021; Essa et al. 2020; Sakr et al. 2020).

Conclusion

The presented work succeeded to develop two different potential non-toxic nano-radiopharmaceutical agents based on graphene oxide nanosheets that were able to target cancer tissues selectively either passively or actively. It was also proven that the decoration of these graphene oxide nanosheets with selective tumor targeting moiety intensively enhances its tumor targeting ability and accordingly uptake and retention. This preliminary study highlights the usefulness of using such a multifunctional agent to image and treat tumor tissues.