Anti-cancer and neuroprotective effects of conjugated graphene quantum dot in brain tumor-bearing rat model

Glioblastoma has been recognized as a most complex and highly malignant type of primary brain tumor. The rapid progression brain tumor model was developed by direct intracranial administration of ENU at the different focal brain points in the rat brains. The GQD was synthesized by bottom-up technique and functionalized with Trastuzumab and Caspase-8 antibody by Carbodiimide-amidation activation. The in-vitro cytotoxicity MTT assay was performed with all the GQD conjugates in SK-N-SH and N2a cell lines. The acute and chronic toxicity of synthesized GQD was performed in healthy rats and evaluated the hemolytic activity and CRP levels. A synthesized quasi-spherical 2D tiny GQD has a particle size of less than 10 nm and a 12.7% quantum yield. DSL, TEM, AFM, FTIR, and fluorescence spectroscopy characterized the GQD conjugates. In-silico molecular docking was a conformed static interaction between GQD and antibodies. GQD-conjugates showed dose-dependent toxicity in both cell lines and mild acute toxicity in rat blood. The GBM tumor-bearing rats were assessed for the anticancer and neuroprotective activity of the GQD conjugates. Histopathology, immunohistochemistry, metabolic, and inflammatory tumor biomarker estimation showed that the GQD_Caspase-8 conjugate showed better anti-tumor and neuroprotective effects than other conjugates.


DLS
therapies is 21 months, and the survival rate is only 26% [4].These treatments are indistinct and still ineffective to preclude the relapse of cancer [5].Modern therapies have limited clinical compensation due to events of adverse drug reactions and auto-immune disease.In particular, GBM is like a dense tumor, and immunotherapies are unproductive because of the obstacle of immune cell infiltration inside the strangely developed extracellular matrix (ECM) [6].A similar antagonistic effect of chemotherapeutics is caused by the heterogeneity and complexity of the tumor microenvironment (TME), which contains immune suppressants like tumor discharge cytokines [7].As the urge for adjuvant chemotherapy and optional treatment alternatives proceeds, there is extreme attention on producing targeted therapies for GBM.The future of molecular targeting therapies includes the implementation of personalized treatment approaches, primarily with an emphasis on variation in the level of active gene/protein in tumor cells in response to anti-tumor agents.The treatment strategies should also be well planned equally to overcome the hindrance of the blood-brain barrier (BBB) and targeted especially on benign tumor cells.However, these problems are challenging due to the absence or small thickness of BBB ligands and tumor-specific receptors.These challenges can be overcome by utilizing nanoparticulate systems [8].
Since their discovery in 2004, the main themes of graphene quantum dots (GQD) research were applications in cellular optical imaging.Recently, other efforts have been attracted by applying bio-imaging targeting specifically.With these, one could target the specific tissue of live animals (i.e., bone), the particular type of cells ( i.e., cancer cells), and a detailed compartment of cells (i.e., nucleus) using GQD-based systems with high specificity [9].GQD possesses characteristics and advantages like tunable fluorescence, small sizes, prominent biocompatibility, excellent photostability, low cytotoxicity, high drug loading capacity, pH-dependent release, and targeting efficacy, which have shown their promising application in therapeutic and bio-imaging [10].GQD possesses the excellent capability of gene delivery, drug delivery, and anticancer activity, deprived of functionalization.GQD's dual character of anticancer drug carrier and DNA cleavage activity enhancer should be credible in cancer treatment [11][12][13].GQD penetrates the BBB and protects against dopamine neuron loss induced by the α-synuclein protein by inhibiting fibrillation and desegregation in Parkinson's disease [14].Assumed fluorescence of GQD, which easily monitors their real-time movement in the cells without employing external dyes, facilitates the localization of the drug carrier and drug delivery simultaneously [15].
Bovine serum albumin (BSA) is a bio-mimetic template used as the prime tool for drug-protein interaction studies.Interactions of anticancer drugs with BSA may affect toxicity and bioavailability [16].BSA is mainly accumulated by proliferating tumor cells through enhanced permeation and retention (EPR) effect due to poor lymphatic drainage and leakiness of tumor vascularity.The release of drugs from BSA-based nano-theranostic bio-mimetic templates has depended on the nature of TME [17].The targeted therapy by brain tumor-specific antibodies mainly depends on the tumor cells' epidemiology and TME.Here, we have selected two antibodies for active targeting of brain tumors such as Caspase-8 and Trastuzumab.The caspase-8 antibody acts on the intrinsic apoptotic pathway.The caspase-8 mediated cleavage promotes the direct activation of caspase-3, which leads to denaturation of cytoskeletal protein, and DNA fragmentation causes apoptosis [18].Trastuzumab is an anti-HER/2 neu antibody that mainly acts on an extrinsic pathway.It inhibits the juxta-membrane domain of human growth factor receptor 2 (HER2).Tumor treated with trastuzumab undergoes cell cycle arrest in the G 1 phase, reducing cell proliferation.Trastuzumab has been shown to have proteolytic cleavage and antiangiogenic activity [19].
Based on the above premises, the prime inspiration of the present work is to synthesize low-cost, efficient, pristine GQDs and develop their conjugates with brain tumors targeting specific bio-recognized antibodies and proteins.To investigate molecular mechanisms, the anti-tumor and neuroprotective activity of prepared GQD and their conjugates were assessed using various in-vitro and in-vivo models.That would be appropriate for the development of new neuro-on-therapeutics.

Preparation of GQD nanocrystals
The GQD has been produced using a previously developed method [20].In which mainly two bottom-up approaches we employed to synthesize GQD.In the first hydrothermal method, Carbogenic precursors and SPAs were dissolved in deionized water and autoclaved at a high temperature of 150 °C for two h.The solution was centrifuged and filtered after autoclaving to eliminate big agglomerate particles [21].In the second synthesis method, carbogenic precursors were directly subjected to pyrolysis at high temperatures; the liquified mixture changed from dark orange to brown due to carbonization and nucleation of carbohydrates.Then, the mixture was neutralized, centrifuged, and filtered to remove large agglomerate particles [22][23][24].

Fluorescence spectroscopy
Spectra Manager software obtained all 2D fluorescence spectra, emission, and excitation intensity by a Spectrofluorometer FP-6500 (Jasco, Japan).The 3D fluorescence spectra were recorded by the 3D spectrum measurement tool [25].

Measurement of quantum yield (Φ)
The Quantum yield (Q.Y.) of GQD was measured against Quinine sulfate (Q.S.) as a standard has a Q.Y. of about 54.3% in 0.1 N H 2 SO 4 , employing the below equation [26].
Where ΦGQD and ΦQS represent the quantum yield of GQD and Quinine sulfate, I mean the integrated emission intensity; A denotes the optical density, and η is designated the refractive index of the solvent.The Φ of GQD was measured at the 330 nm excitation wavelength.The solution concentration was adjusted for a minimal absorbance of less than 0.1 [27].

Particle size distribution
The nano-size distribution of GQD nanoparticles was performed by dynamic light scattering (DLS) using NanoPartica SZ-100 (Horiba, Japan).The size distribution was recorded in mean diameter, Z-average, and P.I. value, which indicated the mean fraction diameter of standard deviation and poly dispersibility of GQD in the medium [20].

Transmission electron microscopy (TEM)
The TEM images of GQD were recorded by Tecnai-20 S-TWIN (Philips, Netherlands), a system operating at 200 kV of accelerating voltage.TEM samples were prepared by putting a drop of GQD solution on a copper grid and then placing it onto the sample holder, adjusting the objective aperture, and selecting the optimum area to enhance the contrast by blocking electron diffraction at a high angle [28].

Atomic force microscopy (AFM)
The 2D topography of GQD was recorded by Multimode-8 AFM (Bruker Scientific, USA).GQD sample was prepared on a glass piece of 1 cm 2 area and placed on the microscope stage.The cantilever, laser, and detector were maneuvered to avoid integral and noise with the fine-tuning before scanning for better tracking and particle size by plane-fitting and flattening [28].
2.3.7. 13C-Nuclear magnetic resonance (NMR) analysis 13 C-NMR was conducted to identify the carbon isotope interaction of functional groups within the organic and organometallic compounds.The experimental process was performed using Avance-III (Bruker Scientific, USA), 400 MHz liquid state FT-NMR spectrometer using tetramethyl-silane (TMS) as a reference standard [29].

Development of GQD-biomarkers conjugates
Two different methods performed the conjugation preparation between GQD and tumor biomarkers:

PEGylation method
Surface adsorption of polymeric material on GQDs is done by solution-phase incubation and sonication.The physical interaction primarily depends upon the surface morphology and hydrophobicity of GQDs and the geometry and electron density of other molecules.The hydrogen bond situated on the edge of GQD and the opposite molecule supports the surface adhesion through non-covalent interaction, for which the PEGylation reaction was performed under two different temperature conditions as GQD required a higher temperature for the annealing of surface functional groups.The required quantity of PEG was dissolved in the GQD solution by bath sonication for 30 min at room temperature.The solution concentration of 4 mg ml −1 PEG in GQD was then taken, sealed in a glass container, and autoclaved at 150 °C for two hours.
Similarly, the second reaction was carried out at 100 °C for two hours under constant stirring [30,31].Then, all reaction solutions were allowed to cool to room temperature.Finally, 1 ml of 10% w/v BSA solution, 1 μl of 1 mg ml −1 Caspase-8, and one μl of 1 mg ml −1 Trastuzumab were added into 10 ml GQD-PEGylated solution and incubated for 30 min at room temperature.The unreacted PEG and antibody/protein residues were removed by dialyzed through a dialysis membrane (MWCO, 10-12 kDa).

EDC/NHS Carbodiimide activated amidation coupling reaction
Carbodiimide oxidation was carried out through our previously developed method.Briefly, 8 mg of EDC and 12 mg of NHS have been added into 10 ml GQDs solution.The solution was taken and sealed in a glass container autoclaved at 150 °C for two hours, and the second reaction was carried out at 100 °C for two hours under constant stirring.Then, all reaction solutions were allowed to cool to room temperature.Finally, 1 ml of 10% w/v BSA solution, 1 μl of 1 mg ml −1 Caspase-8 antibody, and one μl of 1 mg ml −1 Trastuzumab antibody were added to the GQD-diimide solution, and the resulting mixture was incubated for 30 min at 37 °C.The unreacted EDC/NHS and antibody/protein residues were removed by dialyzed through a dialysis membrane (MWCO, 10-12 kDa) [20,32,33].

Fourier transform infrared (FT-IR) spectroscopy
The FTIR spectra of GQD and its antibody/protein conjugations were carried out by spectrophotometer FT/IR-6100 (Jasco, Japan) using JASCO Spectra manager software with a resolution of 4 cm −1 [25].
2.6.In-silico molecular docking simulation and virtual screening Molecular docking software such as AutoDock tool v1.5.6,PyRx v8.0, Discovery Studio Visualizer v19.1.0.18287, and PyMOL v2.3.1 have been used for virtual screening and identification of structural-based molecular interactions.The 3D co-crystal structure of antibody/protein with ligand was gained from the RCSB-PDB website (http://rcsb.org/).The list of active site residues (amino acid) of mentioned antibodies and protein are mentioned in table 1.The obtained structure comprises ligand and water molecules, which must be removed using Discovery Studio Visualizer before docking.The 2D design of GQD used in the docking was prepared by ChemDraw ultra v 12.0.2.1076 software and then converted into 3D structure in SDF format and PDB using Open Babel software [34,35].The ligand was docked with the protein's active binding site of the PDB file using AutoDock vina of PyRx software.The ligand binding affinity was measured in kcal/mol, a unit of docking score.It ranked and provided all possible conformation/orientation of the GQD complex [36].Interactions and molecular simulation of docked GQD with the different antibodies/proteins were characterized by H-bond interactions, hydrophobic interactions, binding patterns with residual sites, and bond length and types through Discovery Studio Visualizer [20].

2.7.
In-vitro % cell viability (MTT) assay Sowed 1 × 10 4 cells of SK-N-SH and N2a per well of a 96-microtiter plate in DMEM culture media, including two controls, to adhere cells incubated for 24 h at 37 °C in 5% CO 2 .Added 100 μl of freshly prepared GQD and its antibody/protein conjugations in different concentrations of 5, 10, 20, 50, 100, 200, and 400 μg ml −1 and incubated for 24 h.All the serial dilution was made in DMSO for better dissolution and results.Then, ten μl of MTT solution (5 mg ml −1 in PBS) was added into each reactant well and incubated under 5% CO 2 for 3 to 4 h until the purple precipitate of formazan was visible.After incubation, 100 μl of SDS-HCl detergent solution was added to each well and mixed gently by shaking.After four hr of incubation at room temperature in dark conditions, the absorbance was at 570 nm using an ELISA microplate reader (Robonik, Mumbai) at every six hr interval.Calculate the average reading of triplicates and substrate from the average value of the control.The results were represented in terms of % of cell proliferation/viability against test concentrations [37][38][39][40].

In-vitro wound healing (Scratch test) assay
The SK-N-SH cells were seeded in 6 well microtiter plates, including control, and incubated for 24 h at 37 °C in 5% CO 2 .Once confluency of about 80%-90%, the wound in the cell monolayer was made by scratching the micropipette tip in a straight line.Then, cells were treated with serial dilution of GQD solution concentrations of 10, 20, and 50 μg ml −1 and incubated for 48 h.Every six h interval during the incubation period, images of a cell monolayer were taken with a digital microscope, and observed the gap width and surface area of the wound.The results were represented in % wound closure against time intervals [40,41].
2.9.In-vitro hemolytic toxicity assay of GQD in rat blood A fresh blood sample of 0.2 ml was collected from healthy rats from a retro-orbital vein and centrifuge.Prepared the stock solution of RBCs by diluting 0.2 ml with 3 ml of PBS.Added 0.2 ml of stock RBCs solution in serial dilution of GQD concentration of 10, 25, 50, 100, 200, and 500 μg ml −1 and incubated at 37 °C for 3 h.After the incubation, the supernatant was collected via centrifugation at 10,000x g for 3 min.Measured the U.V. absorbance of hemoglobin at 541 nm and 650 nm as a reference wavelength.For Negative and Positive control, 0.2 ml of stock RBC solution was added to 0.8 ml of PBS and water, respectively.The % hemolysis of RBCs was calculated using the following equation [42,43].
( ) Hemolysis sample absorbance negative control absorbance positive control absorbance negative control absorbance 100 2

Acute in-vivo toxicity studies of GQD in rat model
The Latex turbidimetry method estimated the CRP levels using a commercially available kit (Accucare, Labcare diagnostic kit).Ten μl serum sample was added to 1 ml of working reagent, and absorbance was taken immediately after 4 min of incubation at 540 nm [44].

Development of ENU-induced brain tumor animal model
Twelve healthy three-month-old adult S.D. rats weighing 200-250 gm were randomly picked into four groups, each containing three male rats.They were procured from a registered, licensed breeder and kept under wellcontrolled conditions of temperature (22 ± 2 °C), humidity (55 ± 5%), and 12 h/12 h light-dark cycle keeping three males in a cage Conventional laboratory diet and water were provided ad libitum.The protocol of the experiment was approved by the Institutional Animal Ethics Committee (IAEC) as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (IP/PCEU/PhD/27/2020/44).No animals are subjected to harm during the experimentation; investigators employ euthanasia techniques for the ethical termination of the animals.Tumor induction was carried out by multiple-dose administration of ENU for two weeks [45,46].In the first week, 1st dose of 4 mg kg −1 of ENU was administered to animals via different routes: (1) Normal control (Placebo), (2) Intraperitoneal (I.P.) injection, (3) Indirect focal bregma injection, (4) Indirect focal lambda injection.In the second week, 2nd dose of 4 mg kg −1 of ENU was injected into each group, and an additional 10 μl of the TNF-α gene was administered in bregma and lambda groups.Then, animals were kept under neurobehavioral and physical observation for a further two weeks (a total of 28 days) for confirmation of the tumor.Each animal group's body weight, food, and water intake were measured during induction.
2.12.Pharmacological evaluation of ENU-induced brain tumor animal model 2.12.1.Neurological assessment by gait analysis The rats from tumor induction groups were subjected to visual investigation for their neuromuscular deviation and movement coordination after two weeks of tumor induction.Functional gait disturbance (FGD) was examined in the postural stability during walking.Walker et al standardized the scheme for evaluating FGD stability.They created an assessment consisting of 10 tasks: surface level of gait, speed alteration, horizontal and vertical turns, pivot turns, ambulating backward movement, stepping over obstacles, narrow base support, and eye-closed gait movement [47].The scoring of FGD for each task was in the range of 0 for severe impairment to 3 for average performance; the highest scoring is not possible over 30.

Nociception alteration
Nociception threshold was performed by tail-flick and hind paw allodynia in an analgesiometer after four weeks of tumor induction.The temperature of the hot plate of the analgesiometer was adjusted to 50 °C before experiments.Rats were handled on apparatus, and their tail and paws were subjected to a heat stimulus.Tailflick time was measured seconds from the initial heat source activation until the tail-flick.Hind paw withdrawal was measured in seconds from the initial sensitivity of pain.The overall latency was measured for 30 s, twice for each rat, after 30 min of interval.The average of two readings was considered, and the result was represented in terms of the nociception threshold in seconds [48].

Locomotion alteration
A photo-actometer carried out locomotor activity after four weeks of tumor induction.The apparatus is made by the 2D square (450 × 450 mm 2 ) dark box with a 30 cm height bottom frame support.The frame comprises a 16 × 16 infrared laser beam for optimal detection of locomotor movements in both horizontal and vertical directions.Rats were handled on an apparatus and subjected to free action under dark conditions.Locomotion activity is measured by breakdowns in a movement-sensitive laser beam, converted into activity counts.The locomotion was measured in total distance traveled (cm) for 60 s [49].

Estimation of metabolic tumor biomarkers 2.13.1. Serum glucose estimation
The 10 μl of serum samples were poured on GLU-PIII slides provided in the Dry-chem, Fujifilm, diagnostic kit.Following deposition, the sample was spread uniformly on the spreading layer and allowed to diffuse into the underlying layer.Swipe the Q.C. card obtained from the GLU-PIII slide package to initiate the test acquisition.
Slides were incubated at 37 °C for 2 min and set on a DRI-CHEM NX500i analyzer.The absorbance of each serum sample was recorded at 505 nm by reflective spectrophotometry.Glucose concentration was calculated from the absorbance of red dye using a preinstalled calibration curve.

Serum LDH estimation
The 10 μl of serum samples were poured on LDH-PIII slides provided in the Dry-chem, Fujifilm, diagnostic kit.Following deposition, the sample was spread uniformly on the spreading layer and allowed to diffuse into the underlying layer.Swipe the Q.C. card obtained from the LDH-PIII slide package to initiate the test acquisition.
Slides were incubated at 37 °C for 2 min and set on a DRI CHEM NX500i analyzer.The absorbance of each serum was recorded at 540 nm by reflective spectrophotometry.LDH concentration was calculated from the absorbance of formazan dye using a preinstalled calibration curve.

Development of anti-tumor and neuroprotective GQD treatment model in ENU-induced brain tumor rats
Thirty-six healthy three-month-old adult S.D. rats weighing 200-250 gm were picked into six groups, each containing six male rats.They were procured from a registered, licensed breeder and kept under well-controlled conditions of temperature (22 ± 2 °C), humidity (55 ± 5%), and 12 h/12 h light-dark cycle keeping three males in a cage.Conventional laboratory diet and water were provided ad libitum.The protocol of the experiment was approved by Institutional Animal Ethics Committee (IAEC) as per the guidance of the Committee for Control and Supervision of Experiments on Animals (CPCSEA), Government of India (IP/PCEU/PhD/27/2020/45).
No animals are subjected to harm during the experimentation; investigators employ euthanasia techniques for the ethical termination of the animals.After successfully evaluating the induced brain tumor model, treatment induction was carried out for four weeks.In the first week, 1st dose of 4 mg kg −1 of ENU was injected in the groups mentioned above via the previously investigated route: (1) Normal control (Placebo), (2) Disease control (Lambda brain tumor rats), (3) GQD treatment group (50 μg ml −1 kg −1 of body weight), (4) GQD-BSA conjugation treatment group (50 μg ml −1 kg −1 of body weight), (5) GQD-Caspase eight conjugation treatment group (50 μg ml −1 kg −1 of body weight), (6) GQD-Trastuzumab conjugation treatment group (50 μg ml −1 kg −1 of bodyweight).In the second week, the subsequent 2nd dose of 4 mg kg −1 of ENU was injected, followed by the first week.After two weeks (14 days) of tumor induction, treatment induction was initiated by administering GQD 50 μg ml −1 kg −1 of body weight and their conjugates containing equivalent one μg of antibodies/protein for another 14 days at one-day intervals.Each animal group's body weight, food, and water intake were measured during induction.
2.15.Estimation of brain tumor diagnostic biomarkers 2.15.1.Serum TNF-α estimation The Sandwich ELISA method was used to estimate the serum TNF-α level per the product assay kit.Added 100 μl of prepared standard concentrations and serum samples into the wells and incubated for two h at 37 °C.
Working biotin-conjugated antibody was prepared freshly, added 100 μl into each well, and incubated for one hour at 37 °C.Working streptavidin-HRP solution was prepared freshly, added 100 μl into each well, covered the wells, and incubated for 30 min at 37 °C.100 μl of Tetramethylbenzidine (TMB) substrate was added into each well and incubated for 20 min at 37 °C, under dark light.Then 50 μl of stop solution was added into wells, and measured the absorbance at 450 nm wavelength.Calculate the result using a preinstalled standard regression curve and process the data using the four-parameter logistic (4-PL) curve-fit method.

Serum IL-6 estimation
The Sandwich ELISA method was used to estimate the serum IL-6 level per the product assay kit.Added 100 μl of prepared standard concentrations and serum samples into the wells, covered the wells and incubated for two h at 37 °C.Working biotin-conjugated antibody was prepared freshly, added 100 μl into each well, covered the wells, and incubated for one hour at 37 °C.Working streptavidin-HRP solution was prepared freshly, added 100 μl into each well, covered the wells, and incubated for 30 min at 37 °C.100 μl of TMB substrate was added into each well and incubated for 20 min at 37 °C, under dark light.Then 50 μl of stop solution was added into wells, and measured the absorbance at 450 nm wavelength.Calculate the result using a preinstalled standard regression curve and process the data using the 4-PL method.

Serum CNTF estimation
Sandwich ELISA was used to estimate the serum CNTF level.Added 50 μl of the prepared standard into standard wells.Added 40 μl serum samples into the sample's wells, then added 10 μl anti-CNTF antibody into the sample's wells and 50 μl of streptavidin-HRP to sample and standard wells, cover the wells and incubated for one hour at 37 °C.Added 50 μl of substrate solution A and substrate solution B into each well, incubated for 10 min at 37 °C, under dark light.Then 50 μl of stop solution was added into wells, and measured the absorbance at 450 nm wavelength.Calculate the result using a preinstalled standard regression curve and process the data using the 4-PL method.

Histopathology of brain tissue
After 28 days of tumor progression and tumor treatment induction, the rats were deeply anesthetized with ether and decapitated; the brains were removed quickly and immersed in cold saline for 10 min.The brains were fixed in 10% formaldehyde, then dehydrated and embedded in melted paraffin wax, followed by alcohol washing.Paraffin block was mounted on the microtome and taken 50 μm vertical sections of brain centers.The tissue sections were fixed on the glass slide and finally subjected to hematoxylin and eosin (H & E) staining.The tissue sections were mounted on an inverted light microscope (Olympus CKX41, Japan), and images were taken using a photomicroscope attached to it (100x) [50].
2.17.Immunohistochemistry (IHC) by glial fibrillary acidic protein (GFAP) staining of brain tissue Brain tissues were rinsed, dehydrated, and fixed in melted paraffin, followed by alcohol washing.Paraffin block was mounted on the microtome and took 50 μm vertical sections of brain centers.The sections were fixed on the glass slide and finally subjected to GFAP staining through the following steps: deparaffination, antigen retrieval, blocking endogenous enzyme and background, incubation with primary and secondary antibodies, chromogen substrate binding, counterstaining, and clearing.The sections were mounted on an inverted light microscope (Olympus CKX41, Japan), and images were taken using a photomicroscope attached to it (100x) [51].

Biostatistical analysis
The data were expressed as Mean ± SEM per animal protocol and groups.Comparisons among the group were performed by one-way ANOVA test.The significances were established at * p < 0.05, ** p < 0.01 and *** p < 0.001.All the statistics were done by Prism software v8.0.3 (GraphPad, USA).

Synthesis of GQD
Here, two approaches for synthesizing Pristine-GQD from carbohydrate precursors, citric acid monohydrates and cane sugar, are used through hydrothermal/solvothermal treatment and direct pyrolysis.The first hydrothermal/solvothermal method treated the carbohydrates at high temperatures (150 °C for two h) in aqueous and organic solvent with or without SPAs.The second direct pyrolysis method melted carbohydrates until a liquified solution was obtained and then neutralized in an alkaline medium.The quantum size, functionalities, and biological activities of GQD are dependent on the approaches elected for synthesis [52].
When the carbohydrate core was heated above its melting point temperature, hydronium ions decomposed and catalyzed the subsequent reaction.A hydrophilic polymer was formed after polymerization of the carbogenic core, followed by aldol condensation.Further condensation and cycloaddition-endorsed aromatization of hydrophilic polymer formed aromatic clusters.Finally, the burst nucleation occurs when the concentration of aromatic clusters is reached above the critical supersaturation phase, and GQD is produced [24].The developed GQD has excitation-dependent fluorescence emission under U.V. dark blue light [13,20].

Spectro-fluorescence measurement of GQD
The fluorescence overlay spectrum has indicated that GQD possessed excitation-dependent fluorescence emission under exposure to UV radiation.Figure S1 shows that GQDs prepared by different approaches exhibited discrete bright fluorescence when exposed to 360 nm U.V. dark blue light.Figure S1(A) showed that GQD prepared by C.A. pyrolysis produced bright green fluorescence emission at 500 nm wavelength when excited at 330 nm U.V. exposure.Like figure S1(B), GQD designed from C.S. alkaline hydrothermal passivation produced light blue fluorescence at 410 nm when exposed to the 310 nm U.V. dark blue light.Figure S1(C), GQD prepared by peroxide hydrothermal passivation of C.S. produced yellow fluorescence at 540 nm when exposed to the 480 nm U.V. dark blue light.Figure S1(D), red fluorescence GQD prepared from acidic hydrothermal passivation of C.S. emitted a volatile fluorescence shift to light green due to quantum quenching of lone pair of electrons in the last p-orbits of a carbon atom [22,53].Hence, due to the high tunable and bright fluorescence of GQD synthesized by the pyrolysis method from citric acid, it was elected for further investigations.

Measurement of quantum yield (Φ)
The fluorescence emission of the GQD was dependent upon the size, shape, solubility, surface functional groups, edge defect, and a fraction of the sp 2 -hybridized domain inside the sp 3 -matrix.The spectro-fluorescence overlay of Q.S. and GQD prepared from C.A. and C.S. was illustrated below in figures 1(A)-(D).The mathematical equation for quantum yield measurement is mentioned in section 2.3.3.The summary of values obtained for both GQD is in supplement table S1.The 12.7% Q.Y. was found for GQD prepared from pyrolyzed C.A. In contrast, a miserable % Q.Y. was reported for CS-based GQD because it showed very low optical density and emission intensity compared to quinine sulfate standard in a similar concentration [20,26].

Particle size distribution of GQD by DLS
The DLS was employed to measure particle size in three different phases of formulation: raw synthesized GQD, purified and dialyzed GQD, and Diluted GQD.Figures 2A(a), (b) Without surface passivation, the purified GQD was about 36 nm, significantly reduced to 5.2 nm after proper dilution in deionized water.Similarly, after purification of surface passivated GQD through PEG-6000, the particle size was increased near to 84 nm, which decreased to 56 nm after proper dilution, as shown in figures 2B(a), (b).The raw synthesized GQDs have shown larger diameters and narrow distributions at a 130°scattering angle in a system adjusted to a polydisperse medium.While the medium and scattering angle were corrected manually to monodisperse at 90°, the size of GQD was significantly decreased to 27 nm.So, based on the above illustrated, we investigated that particle size distribution of GQD was affected by the synthetic approach, carbogenic precursor, purification, and dilution process.By manifesting the above factors, we control and manipulate the particle size of GQD [54].

Morphological particle size by TEM
The TEM images (figure 3) were showed that prepared GQD exhibited a quasi-spherical shape and well distributed in an aqueous medium.The hydrophilic nature of GQD from C.A. produced a lateral size of about 6.36 nm, and after the PEGylation, the length increased by approximately 24.10 nm, as shown in figures 3A(a), (b).However, the GQD from C.S. showed a much larger particle size above 136.75nm, and after PEGylation, particle size increased above 200 nm).So, based on the above results, it was observed that CS-derived GQD have a broader and higher size distribution range, which is unobeyed the rationale of the quantum dots size less than 10 nm [26].

Topographical size distribution by AFM
AFM was employed to measure the topographical appearance of any nanomaterials found in the orange subfraction of the 2D and 3D diagrams.Figures 3(C) shows that CA-based GQD were well dispersed in solution and persevered as an individual particle; the size distribution histography showed that the 2D GQD have lateral sizes around 6.85 nm diameter and thickness distributed within 1.0-3.5 nm. Figure 3(D) illustrated that after PEGylation of GQD, the diameter has increased by about 27.5 nm [28].

Characterization of GQD-antibody/protein conjugates 3.3.1. 2D electrophoretic assay in agarose gel
Several trials were performed with prepared conjugations using different loading dyes.But non-obvious band migration was observed in all after half run without any fluorescence; it is supposed to be due to the highly hydrophilic nature of the GQD.The conjugations were successfully loaded directly onto an agarose gel using CoralLoad dye.It contains three marker dyes, orange, pink, and yellow, that discrete the migration distance of protein and optimization of gel run time [20,31].From figure S3(A), the post-electrophoretic run showed that two separated bands of conjugation and total gel run time were observed in EDC/NHS amidation conjugation.
In contrast, in PEGylation, no spare bands of protein were observed [55].So, it was concluded that EDC/ NHS amidation successfully conjugated GQD with BSA rather than PEGylation.As shown in figure S3(B) of the electrophoretic run of GQD-antibody/protein conjugates, it was observed that trypan blue dye easily differentiated antibody migration in the gel matrix.Caspase-8 and Trastuzumab antibodies were seen as a distinct and sparse band.

FT-IR characterization of GQD-antibody/protein conjugations
The prepared conjugation of GQD-antibodies/proteins was characterized by FT-IR spectroscopy.The characteristics of functional groups present in the GQD-antibody/protein conjugations were summarized in table S2.As shown in figure S4(A   additional weak band at 1738.10 cm −1 was observed in EDC/NHS complex due to -N-H bending of amide bond present between GQD and active site residual amino acids of BSA protein [24,30,56].Figures S4(B) GQD-Caspase 8 and (C) GQD-Trastuzumab antibodies have illustrated that two additional weak peaks at [6] 1212.24 cm −1 and [7] 1049.71cm −1 were observed in EDC/NHS conjugation due to an oxidative state of the amino group toward blue shift and aromatic/aliphatic stretching of -C-NH functional group respectively formed between GQD and active site residual amino acids of antibodies [57].So, from the obtained results, it was concluded that EDC/NHS amidation conjugation was stable and rapid conjugation as compared to PEGylation; hence, we recruited carbodiimide conjugations of GQD with antibodies/protein for further investigation of the anti-tumor and neuroprotective activities.

3.4.
In-silico molecular docking simulation and virtual screening AutoDock vina in PyRx tool performed superior and standard physiological interactions of GQD with antibodies/proteins.Moreover, it provided the score of the 09-best conformation of the GQD.The least negative binding energy of the ligand in contrast to the target protein is considered 'binding affinity,' which makes it possible to understand how the ligand interacts with the receptor.
Hence, the interactions mainly depend on the ligand's physio-chemical properties [58].The GQD.mol ligand interacted with the target.pdbfile, the total fitness energy values in kcal/mol, comprises mainly Vander Waals interaction.The length of H-bond polar interaction in the GQD interactions was found in a range of 1.9°A to 3.9°A [59].Figure 4 shows the best-docked conformation/orientation of GQD interactions with target macromolecule visualization done by the post-docking analysis features in the Discovery studio visualizer.They mainly concerted on hydrogen bonding and hydrophobic interactions to measure GQD conjugations [60].A summary of binding energies and developed conformations interaction is shown in table 2 below.The GQD-BSA conjugation, as shown in figure 4(A), whose receptor binding affinity is −10.2 kcal mol −1 , GQD showed the closed covalent hydrophilic interactions with Ala258 and Lys286 amino acid residues.The Ala258 has formed singular H-bond polar interaction with -CONH 2 (amide) of GQD, a length of about 2.18°A, while Lys286 created dual -C=O bond polar interactions with GQD about 2.23°A and 2.55°A.GQD has shown hydrophobic interactions with the BSA protein's Lys262, Pro282, and Leu283 residues.
The GQD-Caspase 8 conjugation has a binding affinity of −9.3 kcal mol −1 ; GQD showed closed covalent hydrophilic interactions with Gln32 and Leu205 amino acid residues.The Gln32 formed singular H-bond polar interaction with -CONH 2 (amide) of GQD is length about 2.35°A, while Lys286 created dual -CONH 2 (amide) bond polar interactions with GQD are length about 2.57°A and 2.81°A.Aside from traditional interactions, GQD has shown hydrophobic interactions with the Ala20 and Phe24 amino acid residues of the Caspase 8 antibody, as shown in figure 4(B).The GQD-Trastuzumab conjugation has a binding affinity of −10.3 kcal mol −1 ; GQD shows the closed covalent hydrophilic interactions with Phe118, Pro119, Ser121, and Ser131 amino acid residues as illustrated in figure 4(C).The Phe118, Pro119, and Ser131 formed singular H-bond polar interaction with -CONH 2 (amide) of GQD in length about 3.06°A, 2.76°A, and 2.71°A respectively, while Ser121 created -C=O bond polar interactions with GQD is length about 1.92°A.GQD has shown hydrophobic interactions with Val133, Leu135, and Ser176 amino acid residues of the Trastuzumab antibody.

3.5.
In-vitro % cell viability (MTT) assay 3.5.1.Effect of concentration of GQD and its conjugates on in-vitro % cell viability of SK-N-SH cell line The % cell viability of SK-N-SH cells in the culture after exposure to GQD and its conjugates was determined by the intensities of purple formazan color through colorimetric analysis at different post-exposure time intervals such as 6, 12, and 24 h [61].As shown in figure 5(A), after 6 h of incubation, GQD and their conjugations exhibited dose-dependent toxicity.The assay results showed significant 50% cell death after six h of incubation at 20 μg ml −1 of GQD concentration and their conjugations compared to standard control.The 50 μg ml −1 concentration of plain GQD showed lethal cell death, while the same attention to conjugations showed reduced cell death after six h incubation [62].As the incubation time increased to 12 h, significant cell death was observed in the GQD, and its conjugations are shown in figure 5(B).Still, the concentration of 50 μg ml −1 of GQD conjugates exhibited lower cell death than plain GQD in the same attention.Similar results were obtained after 24 h of incubation, as shown in figure 5(C).The lethal cell viability was observed at 50 μg ml −1 of plain GQD concentration after 24 h of incubation, but the same concentrations of GQD-conjugates showed a reduction in cell death.

Effect of concentration of GQD and its conjugates on in-vitro % cell viability of N2a cell line
The N2a cell line used in this study displayed a typical growth curve characterized by a short lag phase (slow cell growth), followed by a long log phase (exponential proliferation of cells and consumption of nutrients of the culture medium) until a maximum density of ≈ 100,000 cells/well is reached, and a late stationary/senescence phase (reduced cell proliferation).So, the cell viability evaluation in early exposure was inappropriate and unjustified the assay results [63].As shown in figure 5(D), after 24 h of incubation, GQD and their conjugations exhibited insignificant dose toxicity.The assay results showed significant 50% cell death after 24 h at 20 μg ml −1 of plain GQD concentration compared to standard control.The 50 μg ml −1 concentration of plain GQD showed lethal cell death.
In contrast, the same attention to BSA, Caspase 8, and Trastuzumab conjugations showed reduced cell death after 24 h incubation [64].The time of incubations increased up to 48 h, significant cell death was observed in the GQD, and its conjugations are shown in figure 5(E).The 50 μg ml −1 concentration of BSA and Trastuzumab conjugates exhibited lower cell death than the same concentration of plain GQD and Caspase 8 conjugate.The above results and interpretations concluded that 50 μg ml −1 of GQD showed a protective effect on the brain tumor cells compared to higher concentrations.Therefore, to unveil the neuroprotective effect of GQD, hypothetical doses of 10, 20, and 50 μg ml −1 were chosen for further investigations on brain tumor neuronal cell lines.

In-vitro wound healing (Scratch test) assay
At a chosen hypothetical concentration of plain GQD, a wound healing assay was conducted to investigate neuroprotective efficacy.As illustrated in figures S5A (c), 50 μg ml −1 of GQD concentration showed a higher SK-N-SH cell migration to the wound surface after 12 h of exposure compared to lower concentrations.The microscopic images (figure S5(B)) of wound closure at different time intervals indicated that 50 μg ml −1 concentration treated cells exhibited faster cell migration within 12 h and reduced wound gap width and surface area [40,64].Henceforth, it was assumed that GQD possessed neuroprotective activity, which would be further investigated through in-vivo animal experiments in brain-tumor-bearing animal models.

In-vitro hemolytic toxicity assay of GQD in rat blood
The RBCs were exposed to the GQD, and the color of the supernatant became darker with an increase in concentration, indicating that more hemoglobin was released from RBCs [43].The % of hemolysis of RBCs was measured by U.V. absorbance of hemoglobin, and hemolytic activity was calculated using the equation (2). Figure 6(A) showed that significant hemolysis of RBCs was not observed up to 100 μg ml −1 concentration of GQD and then increased linearly.An attention greater than 100 μg ml −1 causes cell lysis and rupture of the cell  membrane, resulting in more hemoglobin being released by the RBCs.Therefore, the lethal toxic dose in animal model hypotheses must be less than 100 μg/cubic cell/ml [42].
3.7.Acute in-vivo toxicity studies of GQD in rat model Figure 6(B) showed that the GQD concentration was reciprocal to the increased CRP levels in both serum and plasma.The level of CRP in serum and plasma increased significantly after administration of 500 mg kg −1 GQD by body weight in rats.Concentrations greater than 500 mg kg −1 showed toxic effects and inflammation; however, no mortality was reported after 14 days of exposure to GQD in rats [65].

Pharmacological evaluation of anti-tumor and neuroprotective GQD treatment model in ENU-induced brain tumor rats
The newer brain tumor model developed by I.P., focal brain bregma, and lambda administration of ENU 4 mg kg −1 of body weight in respective animal groups.The neurobehavioral assessment and serum metabolic biomarker estimation of the brain tumor-induced group showed rapid tumor progression in the lambda lambda-induced group.There was a slight progression of the tumor in the I.P. and bregma induction groups.As a result, we recruited the lambda tumor induction model for further investigation of the anti-tumoral and neuroprotective activity of GQD and their conjugates.The preliminary dose of GQD was 50 μg ml −1 kg −1 , and its conjugations were 50 μg ml −1 kg −1 containing equivalent one μg antibodies/protein administered in respective animal groups.

Neurological assessment by gait disturbance
CNS tumors could result in cranial neuropathy, insensitivity, and depression.FGD has assessed the general motor impairment of the brain tumor model.Gliomagenesis often leads to motor deficits, cognitive impairment, and gait disturbance.An individual animal was taken in a dark chamber and subjected to visual investigation for their neurobehavioral deviations after two weeks of tumor induction [49].The detailed summary of the FGD score in each animal group is described in table 3. It was shown that the lambda focal administration of ENU in rat brains exhibited a lower FGD score than normal control and other tumor-induced groups due to severe impairment in gait disturbance, cognition, and neuromotor deficits that might be associated with tumor progression.

Effect of ENU on nociception alteration in brain tumor-induced rats
The result of the nociception threshold showed a significant reduction in tail-flick and hind paw withdrawal latency in intraperitoneal, bregma, and lambda-induced animal groups compared to normal control.Figures 7A(a), it was observed that the bregma-induced group showed the most significant decrease (p < 0.001) and altered nociception.While I.P. and lambda-induced groups showed significantly decreased (p < 0.05) nociception compared to a normal control group [66].The effect of GQD and their conjugates on the nociception threshold showed a significant increase in tail-flick and hind-paw withdrawal latency in all treatment groups, as illustrated in figures 7A(b).The GQD-Caspase 8 and GQD-Trastuzumab antibodies treated group showed more significance (p < 0.01) increased nociception, while the plain GQD and GQD-BSA protein treated group showed significance (p < 0.05) increased nociception alteration compared to the disease control group.All the treatment groups restored the perception and sensitivity of neurons in brain tumor-induced rats.Only the plain GQD-treated group showed a slightly significant (p < 0.05) decrease in serum glucose levels compared to other treated groups.Hence, it showed that brain tumor cells exhibited stiff and robust oncemetabolic activity through Warburg effect/aerobic glycolysis [67].

Estimation of serum LDH level in ENU-induced brain tumor animal model
Figures 7D (a) shows that I.P. and bregma-induced groups showed a significant (p<0.01)elevation in serum LDH level.While the lambda-induced group showed a higher significance (p < 0.001) rise in serum LDH level compared to normal control and other tumor-induced groups [68].From figures 7D (b), it was observed that plain GQD, GQD-BSA, and GQD-Caspase 8 conjugations treated groups showed a significant (p < 0.01) reduction in serum LDH level.While GQD-Trastuzumab treated group showed a higher significance (p < 0.05) rise in serum LDH level compared to other induced groups.So, it showed that GQD and its BSA and Caspase 8 conjugates downregulate lactate secretion and enhance immune regulation in the tumor cells.Healthy, normochromic, well-aligned cells with no intracellular space were present in the cortex and hippocampus regions of the normal control group.In I.P., the bregma-induced group showed altered and damaged cells changed descriptively, with more extracellular spaces, scar formation, and hippocampal gliosis.The lambda-induced group exhibited more hippocampal gliosis, scar formation, and intracellular space observed active inflammation and tissue damage [72].

Histopathological evaluation of GQD-treated brain tumor-induced rats
H & E staining of GQD and its conjugates treated tumor-induced rat brain sections (50 μm) illustrated in figure 9(A).The normal control group showed normochromic, healthy neurons with intact cells and intracellular spaces.The lambda tumor-induced group, plain GQD treated, GQD-BSA conjugate treated, and GQD-Trastuzumab conjugate treated groups showed disturbed and altered cell bodies with extracellular space, scar formation, and hippocampal gliosis.While the GQD-Caspase 8 conjugate-treated group showed recuperate changes in damaged cell structures, reduced inflammation, and neurodegeneration [71,72].
3.9.3.Immunohistochemical evaluation of GQD-treated brain tumor-induced rats GFAP staining in GQD and its conjugates treated tumor-induced rat brain sections (50 μm) illustrated in figure 9(B).The GFAP stain showed more accumulation of activating tangled astrocytes in the cortex and hippocampus of the lambda tumor-induced group, GQD-BSA conjugate, and GQD-Trastuzumab conjugate treated rat brain sections due to neuron damage and gliosis.The more tangled astrocytes staining, the more information there is about inflammation and neurodegeneration.Plain GQD and GQD-Caspase 8 conjugate treated group showed very little or no tangled astrocyte accumulation compared to tumor-induced and other treatment groups.Hence, it showed that GQD-Caspase 8 conjugate treatment restored the astrocytic damage and reduced neurodegeneration.So, it was concluded that Caspase 8 conjugated GQD exhibited anti-tumor as well as simultaneous neuroprotective effects in brain tumor-induced rats [70,73,74].

Discussion
The present work is to develop GQD by simple, efficient, and low-cost fabrication by hydrothermal treatment and pyrolysis of citric acid at a relatively low temperature through a less time-consuming process.We explain the correlation between the route of the tunable fluorescence emission with the crystal formation's shape and the quantum particles' size [24].As the quantum size of the particles increases, the construction of sp 2 -hybridized atoms inside the sp 3 -hybrid matrix due to delocalization and transformation of π-bond electrons results in fluorescence [54].Therefore, the minor GQD emission shifted to red when absorbing longer-wavelength photons and increasing bandgap energy (Eg) [13].The maximum size limit of that fluorescence quantum particle is typically ∼10 nm [75,76].Spectro-fluorimetry analysis showed that GQD possesses excitationdependent fluorescence emission when exposed to UV radiation.The larger hydrodynamic diameter seen in DLS can be due to the hydration effects of the crystalline structure inside the aqueous medium, which may also be influenced by surface charges and the ion cloud surrounding the particles [77].Based on the TEM image, GQDs were observed to have a quasi-spherical shape and a narrow distribution in an aqueous medium.AFM's 2D and 3D diagrams indicated that GQD remains well dispersed and individual particles in the solution.Henceforth, based on the above discussion, it was justified that GQD prepared from citric acid has a small and uniform size distribution because it undergoes incomplete carbonization at lower temperatures to produce sp 2 -hybridized carbon clusters [52]. 13C-NMR spectrum indicated that GQD has a strong carbonic core containing a sp-and sp 2 -hybridized cluster inside the large sp 3 -domain.GQD also have electronegative oxygen-connected carboxylic and ester functional groups situated on the surface, allowing the conjugation as therapeutic targeting ligands or components for biomedical applications such as bio-imaging, bio-sensors, gene, and drug delivery [57].
Here, we reported two approaches for conjugating GQD with brain tumor-targeted antibody/protein: [1] PEGylation and [2] carbodiimide-activated amidation.The surface annealing of GQD was carried out in two different temperature conditions.Surface adsorption with solution-phase incubation formed non-covalent interactions that enabled GQD to be PEGylated and conjugated to an antibody or protein [78].In carbodiimideactivated amidation, the EDC/NHS cross-linker molecule first binds to the GQD surface by non-covalent physical adsorption through π-π stacking and hydrophobic interactions then covalently attached to the biomolecules [33].The EDC was reacted first with a carbonyl group on the surface of GQD and formed an unstable, reactive O-acylisourea intermediate.The NHS was added to convert a semi-stable amine into an active NHS ester.The amine group of antibody/protein then covalently conjugated with the NHS-ester modified GQD to produce a stable complex [32].
The 2D electrophoresis result reveals the credibility and stability of the GQD-antibody/protein complex.Based on spectroscopic interpretation, we observed that the well-controlled micropattern of EDC/NHS crosslinker fabricates on the graphene surface through π-π stacking.The reactive succinimide ester groups of -NH 2 have interacted exactly with the -COOH functional group of antibody/protein.The FT-IR data reveals the formation of an amide bridge between the -COOH and -NH 2 functional groups of GQD and BSA vice-versa.
The molecular docking initiated by the ligand depends on its affinity for the binding with the targeted amino acid residues of the receptor protein.The higher negative value of the docking score results from the better intermolecular force produced between ligand and receptor and vice-versa.The GQD has exhibited dosedependent toxicity in both cell lines.The concentration of GQD conjugates at 50 μg/ml showed lower cell death than plain GQD after 24 h and 48 h incubation in SK-N-SH and N2a cell lines, respectively.It was assumed that 50 μg ml −1 concentration of GQD conjugations showed a protective effect on brain tumor cells compared to higher concentrations.Thus, a hypothetical concentration of 50 μg ml −1 of GQD was taken for further in-vitro investigation to unveil the neuroprotective effect in brain tumor cell lines.The scratch assay results showed that a 50 μg ml −1 concentration of GQD showed a higher rate of cell migration in the SK-N-SH cell line after 12 h of wound exposure compared to lower concentrations [62].The effect of GQD concentration on the hemolysis of RBCs might be correlated with in-vivo nanomaterial-induced toxicity and therapeutic activity.The % hemolysis of RBCs linearly increased with the concentration of GQD.The cell membrane ruptured, and cell lysis was shown in a higher concentration above 100 μg ml −1 after exposure [42].The elevated CRP level in blood is associated with exogenous materials-induced toxicity and tissue damage.The dose administration above 500 mg kg −1 day −1 body weight in rats showed inflammation and toxic effects.Nonetheless, there was no mortality.
The results of ten combined FGD tasks showed that lambda focal administration of ENU in rats exhibited severe cognition and neuromotor deficits compared to other induction groups and normal control, which might be associated with tumor progression.The results of nociception alteration showed a reduction in tail-flick and hind paw allodynia withdrawal latency in a bregma-induced group compared to normal control and other induced groups.The impairment of the cortex neuron functioning and neuro-gliosis often cause delusions and false perceptions [48].The locomotion alteration showed that the lambda induction group significantly declined neuromotor activity compared to other induced groups [49].
Upregulation in glucose influx was associated with tumor growth and onco-metabolism.The increase in glucose influx has been associated with vasculogenesis and angiogenesis [67].Glutaminolysis-induced lactate secretion was strongly associated with angiogenesis, tumor migration, and growth [79].The highest elevated serum LDH level was observed in the lambda-induced group compared to normal control and other induced groups [68].Histopathological analysis of tumor-induced rat brain cortex and hippocampal regions showed tissue damage.The lambda-induced group showed maximum hippocampal gliosis, damaged cells, more intra and extracellular spaces, and scar formations compared to other induced groups, which is associated with tissue damage and CNS inflammation [50].The neurobehavioral, serum metabolic biomarker and histopathological evaluation of the brain tumor-induced group showed rapid tumor progression in the lambda-induced group.Hence, we recruited the lambda tumor induction model for further investigation of the anti-tumor and neuroprotective activity of GQD and their conjugates.The hypothetical dose of GQD 50 μg ml −1 kg −1 and its conjugations 50 μg ml −1 kg −1 containing equivalent to one μg of antibodies/protein were administered in respective animal groups at one-day intervals for 14 days.The effect of GQD and their conjugates on the nociception alteration showed a significant increase in withdrawal latency in every treatment group.GQDantibodies conjugates showed a more substantial increase in nociception than plain GQD and its protein conjugate.Hence, the antibody treatment restores pain perception and neuron sensitivity in brain tumorinduced rats.While GQD and their conjugates treatment unaffected cognitive impairment and locomotor activities.The biochemical analysis of metabolic serum biomarkers showed insignificantly elevated serum glucose levels because high on-metabolic activity promotes hyperglycemia.Plain GQD and its BSA and Caspase 8 conjugates significantly downregulated the serum LDH, reduced lactate secretion, and enhanced immune regulation in treatment groups.
Activated astrocytes, followed by tumor migration, release inflammatory cytokines from the macrophages, further promoting neuroinflammation and tumor progression in the CNS.Secretion of neurotrophic factors followed by releasing inflammatory cytokines and growth factors triggered the neuroprotection against CNS injury and tissue damage.Elevated serum TNF-α level is associated with tumor growth, invasion, and gliosis.The immunoassay results indicated that GQD and their conjugates insignificantly reduced serum TNF-α compared to the tumor-induced group [80].Elevated serum IL-6 level is associated with cell proliferation and treatment resistance [70].The immunoassay results revealed that all the treatment groups of GQD had significantly reduced serum IL-6 levels compared to the tumor-induced group.The GQD-Caspase 8 conjugate has the most concentrated serum IL-6 considerably compared to other treatment groups.Decreased serum CNTF level is associated with anti-tumor and neuroprotective activity of treatment [82].The immunoassay results showed that plain GQD and its Caspase 8 conjugates significantly reduced serum CNTF levels.
Based on the histopathological investigations of treatment-induced rat brain cortex and hippocampus region, it was observed that GQD and its BSA and Trastuzumab conjugates treated group showed maximum tissue damage, scar formation, brain injury, and gliosis like the tumor-induced group.At the same time, GQD-Caspase 8 conjugate has reduced tissue injury, inflammation, and neurodegeneration in tumor-induced rat brains.Immunohistochemistry of tumor-induced and treatment-induced rat brain cortex and hippocampus section showed positive GFAP-stained activated tangle astrocytes because of neurodegeneration, tissue injury, and damage.The maximum tangled astrocytic growth was seen in lambda tumor-induced, GQD-BSA, and GQD-Trastuzumab conjugate-treated group rat brain sections due to gliosis and neuronal damage.But plain GQD and GQD-Caspase 8 conjugate treated group showed little or no tangled astrocyte accumulation.Thus, the above results have indicated that GQD-Caspase 8 conjugate treatment reduces neurodegeneration and reinstates astrocytic damage in tumor-induced rat brains.The developed GQD-Caspase 8 antibody conjugate by carbodiimide method has shown anti-tumor and simultaneous neuroprotective activity on newly developed brain tumor-bearing animal models.

Conclusion
In conclusion, we developed a simple one-pot synthesis of water-soluble 2D fluorescence GQD nanocrystals done by bottom-up techniques.GQD has a diameter of less than 10 nm and a narrow size distribution range confirmed by the DLS, TEM, and AFM.Spectro-fluorescence reports revealed that GQD has excitation dependence fluorescence emission.The synthesized GQD comprised robust sp-and sp 2 -hybridized clusters formed inside the large sp 3 -domain, and functional groups such as carbonyl carbon, carboxylic acid, and ester are located at the edge of the GQD.Spectro-fluorimetry analysis, gel electrophoresis, FT-IR, and DLS reports suggested that carbodiimide-activated amidation conjugation produced a stable complex between GQD and antibodies/protein compared to the PEGylation process.In-silico molecular docking and virtual screening hypothesized that possible regular interactions are formed during the conjugation between GQD and targeted antibodies/proteins.In-vitro cell viability result was concluded that GQD and its conjugates have dosedependent toxicity.In-vitro hemolytic results indicated that GQD has lethal toxicity, but in-vivo elevated CRP levels do not induce inflammation in rats.ENU was mildly successful in induced brain tumors assessed by neurological disturbance, depression, cognitive decline, cranial neuropathy, metabolic biomarkers, and histopathology investigations.The neurobehavioral assessment, estimation of metabolic serum biomarkers and brain tumor-specific biomarkers, histopathology, and immunohistochemistry of rat brain tissue sections exhibited that GQD-Caspase 8 conjugate has significant anti-tumor and neuroprotective activity in brain tumor-induced animal models.Based on the above hypothesis, further investigation has been necessary to evaluate the molecular and cellular pathways.

3. 2
.6.13 C-NMR of GQD FigureS2illustrates the13 C-NMR of Synthesized GQD.The intense signals of sp 3 -hybridized carbon atoms have appeared at 39 ppm.The chemical shift of sp-hybridized C-C bond and carbon atom connected to electronegative oxygen and nitrogen atoms was obtained at 72 ppm.The sp 2 -hybridized chemical shift of the C=C bond was obtained at 130 ppm.Lower intense signals at 140 ppm indicated that aromatic carbon atoms connected to a hydrogen atom.The fierce signal between 160-185 ppm indicated that carbonyl carbon atoms are present in GQD[29].

Figure 6 .
Figure 6.Toxicity studies for primary dose screening in animal model: (A) In-vitro % hemolytic toxicity of GQD on RBC (B) Acute effect of GQD concentration on (a) serum and (b) plasma CRP levels in rat blood.
(a)) showed a decrease in locomotor activity after 14 days of brain tumor induction in all animal groups.The lambda induction group significantly decreased (p < 0.05) locomotion activity, while I.P. and bregma induction groups were statistically less reduced (p < 0.01) in locomotion activity compared to normal control.Figures7(B(b)) showed that GQD and their all-conjugation treatment groups showed insignificantly increased locomotor activity in brain tumor-induced rats.Hence, it was observed that all treatment groups were inadequate to rehabilitate motor neuron deficit and cognitive impairment in brain tumor-induced rats[49].3.8.4.Estimation of metabolic tumor biomarkers in ENU-induced brain tumor animal model3.8.4.1.Estimation of serum glucose level in ENU-induced brain tumor animal modelFrom the biochemical analysis, it was observed that ENU-induced brain tumor rats exhibited hyperglycemic conditions.All the induced animal groups showed a significant increase (p < 0.001) in serum glucose level compared to normal control as shown in figures 7(C (a)).The GQD-treated groups demonstrated an insignificant reduction in the hypoglycemic effect induced by tumor metabolism, as shown in figures 7(C (b)).

3. 8 . 5 .
Estimation of brain tumor diagnostic biomarkers in GQD-treated brain tumor-induced rats 3.8.5.1.Serum TNF-α estimation The results of the ELISA assay in figure8(A) showed treatment with GQD and its conjugates did not significantly change serum TNF-α levels compared to disease control.However, there was observed a slight decrease and statistically significant (p < 0.05) reduction in TNF-α levels in GQD-Caspase 8 and GQD-Trastuzumab treated groups[69].

Table 1 .
Active binding site residual amino acid of antibody/ protein.

Table 2 .
Summary of binding affinities and interactions of the GQD with active site residual amino acids of antibody/protein generated by discovery studio visualizer.

Table 3 .
Summary of FGD scoring of an individual gait task in brain tumor-induced rats.