Biocompatibility and toxicity of graphene quantum dots for potential application in photodynamic therapy

Aim: Achieving reliably high production of reactive oxygen species (ROS) in photodynamic therapy (PDT) remains challenging. Graphene quantum dots (GQD) hold great promise for PDT. However, the photochemical processes leading to GQDderived ROS generation have not yet been fully elucidated. Materials & methods: Physicochemical characteristics of GQDs were comprehensively investigated, including electron paramagnetic resonance analysis of singlet oxygen production. Dark toxicity was assessed in vitro and in vivo. Results: GQDs demonstrated excellent photo-luminescent features, corrosion resistance, high water solubility, high photo/pH-stability, in vitro and in vivo biocompatibility and very efficient singlet oxygen/ROS generation. Conclusion: The enhanced ROS generation, combined with good biocompatibility and minimal toxicity in vitro and in vivo support the potential of GQDs for future PDT application.


Introduction:
Cancer accounts for over 8 million deaths annually.Standard treatment options include surgery, radio/chemotherapy and photodynamic therapy (PDT).The latter can be a particularly effective modality in the treatment of superficial tumors such as basal cell carcinoma, but has limitations due to low yields of reactive oxygen species (ROS) plus unwanted side effects.Therapeutic and diagnostic techniques based on quantum dots (QDs) are emerging as a promising paradigm for the battle against life threatening conditions including cancer, renovascular and cardiovascular disease, intestinal inflammatory bowel disease and neurodegenerative diseases [1-4] ** .The wide-ranging biological applications of QDs are due to their unique particle size, excellent photo-physical properties, luminescent features and biocompatibility [5,6] * .
The photoluminescence features of QDs derived from radiative recombination result in a long-lived electron-hole pair to produce a metastable state similar to the longlived triplet state of photosensitisers (PS) and provide a cytotoxic mechanism for the production of singlet oxygen and other ROS [7,8] .Photo-induced electron transfer reactions of QDs have therefore recently been envisioned as potential PS for PDT.
PDT is a highly selective approach to ablate tumors, without damaging surrounding tissues.Effective PDT requires three key components: a PS; light and oxygen.The combination of these three leads to the production of ROS.These highly reactive species result in a shift of redox balance and trigger the activation of transcription factors implicated in the initiation of cell death signalling via apoptosis and/or necrosis.The efficacy of PDT mainly relies on the yield of singlet oxygen and other ROS production by a PS and this yield depends on the nature of PS involved [9,10] .Despite several leaps forward, a critical challenge still remains in relation to the inadequate oxygen supply in tumors and incapability of selectively localizing diagnostic probes and drugs at tumors sites for the purposes of early diagnosis and effective treatment [11,12] .One emerging strategy is to synthesize nanoplatforms which may achieve higher ROS yield.However the current applications of QDs are often limited by poor water dispersibility, poor photostability, low singlet oxygen and other ROS generation yield, low biocompatibility, and a lack of imaging and therapeutic functions without attaching biomolecules or antibodies [13,14] .
Graphene quantum dots (GQDs) are among the latest frontiers of research on graphene (two dimensional monolayered carbon materials with aromatic substructure) [15] ** .They are typically derived from graphene/graphite or other 3D graphitic materials by various approaches and exist as mono or few-layered structures with a lateral dimension up to 100 nm [16] .Lower production of ROS and the intrinsic toxicity of conventional organic photosensitizers such as liposomes and polymeric nanoparticles limit their clinical applications in PDT [17] .GQDs have many advantages over conventional organic photosensitizers, such as chemical inertness, high water solubility, photo-stability, interplay between optoelectronic features and shape/size, good donors in the fluorescence resonance energy transfer process, high stability in physiological conditions, specific accumulation at the target site and facile surface functionalization.These features therefore make GQDs promising candidates in novel delivery systems for target-specific photosensitization [18 ** -21] due to their photoluminescence (PL) properties [22,23] , quantum confinement and edge effects [24] .
Considerable efforts are being made to understand the interplay of features such as size and shape, in concert with the type and quantity of additional functional groups, for the generation of PL, as well as capacity to act as energy donors for conventional photosensitizers [16,[22][23][24] .The energy transfer between GQDs and cell molecules, such as triplet oxygen, could potentially induce the generation of reactive oxygen species, thus provoking cellular apoptosis.The light-mediated cytotoxicity of GQDs, together with their energy-donor capacity, could therefore open a new area for GQD research in the life sciences -as direct theranostics or as cofactors or components of conventional photosensitizing agents used in PDT.Recent studies have investigated alternative syntheses and PDT-applicability of GQDs with encouraging results [25][26][27] .
Although promising, GQDs will not achieve broad clinical applicability until concerns have been alleviated about toxicity from long term exposure and quantum yield of ROS, as well as surface dependent therapeutic responses and biological corrosion resistance.
In the present study, we report a robust, smart and highly versatile multifunctional GQD which can be synthesized by exfoliation and disintegration from graphite flakes.
We have previously reported a novel effective method to obtain water-soluble GQDs with higher yield (7.1 %), showing a size around 20 nm, and with the majority exhibiting mono-layering [28] .Crucially, we have now demonstrated limited in vivo toxicity after four weeks of high dose administration in rats, and subsequently confirm the enhancement of singlet oxygen and other ROS generation of GQDs.The intensity of the electron paramagnetic resonance (EPR) signal, resulting from singlet oxygen generation, showed an increase which was dependent on the irradiation time.
The ROS production from GQDs could potentially allow precise therapeutic dosing.
In vivo toxicity experiments in rats demonstrate that our GQDs have minimal dark toxicity, which is a key attribute for a photosensitizer for subsequent use in photodynamic therapy.In this study we also have investigated water solubility, corrosion resistance and pH/photo-stability.This study lends further credence to the theranostic potential of GQDs in disease.

Experimental section:
Synthesis and Characterization of GQDs: We prepared GQDs from our previous established route by exfoliating and disintegrating graphite flakes [28] (Also see Supplementary note 2).
Atomic force microscopy (AFM) micrographs were taken by using a VEECO Dimension 3100 Atomic Force Microscope in a tapping mode with a scan rate of 0.5 Hz.Raman spectra were recorded on a Renishaw Raman spectrometer with a 532 nm laser beam.
Ultraviolet-visible (UV/Vis) spectra were recorded by a PerkinElmer Lambda 900 Spectrometer at room temperature.Electrochemical behaviour was examined using a CHI 660C Electrochemical Workstation with a distinctive three-electrode cell: Pt as the auxiliary electrode, Ag/AgCl as the reference electrode and gold as the working electrode (2 mm diameter), in a 0.1 M KCl electrolytic solution.Stability test was performed from 0.2 V to -1.0 V for 2 hours.Impedance measurements were carried out in the frequency range of 100 kHz to 0.1 Hz at OCP with voltage amplitude of 0.005 V. GQDs in water and heavy water solutions were dropped onto the mica substrate followed by drying at 70 °C for 5 h.A digital camera was used to record the images and their contact angles were calculated by PolyPro software package.
Samples were then irradiated for a given time, after which a second spectral acquisition was carried out.Spectral acquisition parameters for trapping of 1 O2 by mM TMP, and irradiated (λmax = 635 nm, 25 J/cm 2 , Aktilite CL-16).Spectra were acquired pre-and post-irradiation and the detection of an increased EPR signal confirmed that our detection system was operating as expected.To measure the time-dependent generation of 1 O2, GQDs were prepared in H2O at a concentration of 1.3 mg/ml with 50 mM TMP. Spectra were acquired following the irradiation of separate samples for 0, 2, 5, 10 and 30 minutes, which equates to light doses of 0, 0.24, 0.6, 1.2 and 3.6 J/cm 2 , respectively.The generation of 1 O2 in D2O was also measured by EPR.GQDs were prepared in H2O or D2O at a concentration of 0.5 mg/ml with 50 mM TMP. Spectra were acquired pre-and post-irradiation (1.8 J/cm 2 ).
Lastly, spectra of GQDs in H2O (0.5 mg/ml) were acquired in the presence of L-Histidine (25 mM), a 1 O2 quencher (1.8 J/cm 2 ).The positive control for spin trapping by DEPMPO consisted of a hypoxanthine (HX) and xanthine oxidase (XO) system [29,30] .We used a solution containing 50 µM HX, 200 µM DTPA, 20 mM DEPMPO and 0.04 U/ml XO in PBS.Following addition of XO, the solution was immediately injected into a quartz flat cell and a spectrum was acquired.GQDs (1.3 mg/ml) were mixed with DEPMPO (20 mM) and a spectrum was acquired pre-and post-irradiation (1.8 J/cm 2 ).An additional mixture, containing GQDs, DEPMPO and DTPA (200 µM) was also made and treated in the same manner.

Preparation and Basic Characterization of GQDs
GQDs were prepared by following our previously established technique of exfoliating and disintegrating graphite flakes (GFs) [28] .The fundamental mechanism of the synthesis is illustrated in Supplementary Figure 1.The GQDs used herein have an atomic force microscopy (AFM) size around 20-40 nm (Figure 1a).AFM of the dispersed GQDs gave an average thickness of ~ 1 nm (Figure 1a inset), similar to the majority of other QDs due to equivalent size-dependent luminescent features of these QDs, in combination with intrinsic properties of mono-layered graphene [28,31,32] .
GQD crystallization was confirmed by Raman spectroscopy (Figure 1b); two characteristic D (defect) and G modes of graphene were determined at around 1354 and 1592 cm -1 , respectively.The G peak of the GQDs was slightly blue-shifted from that of pure graphite and chemically-reduced graphene oxide (~1588 cm -1 ) [33,34] , since GQDs prepared via this route have oxidized edges, making them water soluble [28] .The FTIR spectrum had two peaks: an absorption peak centered at 1637 cm -1 , and a broad peak at 3402 cm -1 revealing O-H bonding (Supplementary Figure 2a).
The absorptions at 1255 cm -1 and 1078 cm -1 also revealed the existence of C-H and C-O, respectively.The functional groups (O-H, C-H, and C=C) located at the surface of the GQDs act as a passivation layer.This self-passivated surface layer facilitates the water solubility of GQDs [35,36] .The maximum luminescence peak was centered at around 460 nm (from 370 -700 nm) at an excitation wavelength of 360 nm as previously reported by us [28] with quantum yield of 7.12% (Supplementary Table 1 and Supplementary Note 1).The time resolved PL (TRPL, time-correlated single photon counting technique) spectra of the GQDs recorded at 460 nm with 360 nm excitation laser source is shown in Figure 1c.The lifetime of the luminescence can be fitted well with a tri-exponential function: 0.32 (14.2%, we cannot exclude that this fast decay may arise from the instrument response), 2.09 (39.3%) and 5.53 ns (46.5%) respectively.The largely nanosecond decay of luminescence suggests that the GQDs are promising candidate materials for optoelectronic and biologic applications.This synthesis and basic characterization demonstrates the reproducibility of our previously reported novel fabrication route.

Photo-stability, water dispersibility and pH stability:
The propensity of GQDs for anti-biofouling depends on the physicochemical properties of the QDs surface; key properties include the hydrophilicity of the surface and photo-stability.Incorporation of heavy atoms into the QDs increases the probability of intersystem crossing and also increases spin orbit coupling of an electron (known as the internal heavy-atom effect).This effect consequently increases the levels of reactive oxygen species generation (see below).Water dispersibility and photo-stability were also measured in heavy water.The water contact angle (CA) was used to evaluate the wettability, hydrophilicity of the GQD surface and water dispersibility.The natural wettability of the surface is reflected by the CA parameter measured immediately through water droplets on the material surface.As shown in Figure 2 a-b, the contact angle for water soluble GQDs was 11°, indicating the high hydrophilicity due to the existence of hydrophilic carboxylic groups [37] .The contact angle of 16° for heavy water soluble GQDs was comparable to water soluble GQDs as well as 21.8° on mica substrates.The hydrophilic behaviour of surface amphiphilicity suggested that water and heavy water soluble GQDs both are expected to be an attractive candidate for administration in living systems.The GQDs also showed good water solubility (Supplementary Figure 3).
To further investigate the effectiveness of GQDs as an optical bio-imaging agent, the stability under various pH conditions was also verified.As shown in Figure 2 c, no obvious luminescence quenching of the corresponding PL peak (460 nm) was observed in various buffered solutions, demonstrating that GQDs exhibit no attenuation in PL intensity and that PL is largely independent of pH, which is favourable for bio-imaging applications.Photo-stability was also investigated over a period of >200 hours (Figure 2 d), showing that GQDs are highly photo-stable, which is also beneficial for biomedical applications.GQDs exhibited superior photo-stability compared to conventional QDs [38,39] .Overall, the good water dispersibility and solubility, effective pH stability and extraordinary photo-stability render GQDs an attractive alternative probe for efficient imaging in biological and biomedical applications.

3 Biological corrosion resistance
Corrosion resistance is known to play an important role in the biocompatibility assessment of bionanomaterials [40,41] .The possible release of metallic ions and particles through electrochemical corrosion phenomena induces inflammatory and toxic effects which in turn reduces the biosafety and biocompatibility of nanomaterials to solve real-world clinical problems [40,42] .Corrosion resistance depends on morphology and number of layers of quantum dots, exposure environment and electrochemical fluids.It is unclear whether GQDs are resistant to biological corrosion when employed in the context of bio-applications.Biological corrosion occurs in a complex aqueous environment through electrochemical processes, [43] during which GQDs are oxidized to form ions which then migrate away from the GQD surface as free ions [43] .To understand these electrochemical processes, we initially measured their cyclic voltammetry (CV) curves.As shown in Figure 3a, CV curves of GQDs had high currents and good rectangular shapes.Pairs of distinctive redox peaks occurred around -8.85 (at 0.098 V, 50 mV.s - ), 11.4 (at 0.151 V, 150 mV.s -1 ), and -9.57μA (at -0.171 V, 100 mV.s -1 ), accompanied with a small redox peak that indicated a multistep glassy carbon ion intercalation/deintercalation [44] .These high redox currents suggested a good corrosion resistance of the GQD passivation layer.
They also suggested that the passivation layer on the surface of GQDs could block chemical reactions between the GQDs and body tissues during bio-imaging and therapy treatment, preventing corrosion, and allowing for a potentially longer implantation time.In the CV curve, there was also a small redox peak at ~0.1 V (vs Ag/AgCl), most likely arising from the multiple valence states of GQDs. Figure 3b shows the stability curve of the GQDs recorded in an electrolytic solution.The stability curve shows a negligible decrease in current density, again indicating excellent stability, even after 2 h of continuous operation.These characteristics of GQDs with active sites can be attributed to the high-temperature-induced strong coordination between the (QDs) surface and electrolyte, and also demonstrates their potential as a sensitive material for electrochemical sensing and bio-imaging.We further used electrochemical impedance spectroscopy (EIS) to monitor the interfacial properties of the self-passivated electrode.The Nyquist plot is shown in Figure 3c, revealing a high frequency of electron transfer to the GQD surface, which can be ascribed to the charge-transfer resistance (Rct1= 31.70Ω) and the double layer capacity (CPE1= 0.05, n=0.542) occurring at the interface between the electrode and the electrolyte [45] .These results confirmed that the network of GQDs provides remarkable electronic conductivity.In this Nyquist plot, the curve at higher frequencies again indicates good corrosion resistance for biological applications [46,47] .
The potentiodynamic polarization curve of GQDs was also determined (Figure 3d); the initial potential was 0.2 V with a corresponding current value of -0.919 μA.The polarization curve obtained in this study is consistent with literature [40,48] revealing that polarization plot could be divided in three potential domains: (1) preliminary with the onset of polarization as soon as the GQDs comes in contact with the saline environment; (2) the region having the maximum value of current was labelled as partially active.There was a shift in the corrosion resistance behaviour with a positive potential and current value at -0.999 V and 2.6X10 -6 A respectively.Furthermore, it was again shifted towards corrosion inhibition at -0.78 V and (3) the formation of the passive layer started with a rise in potential and fall in the current density.This behaviour demonstrated that GQDs act as a corrosion-resistant barrier because a passive layer develops over a short period of time.In addition, these results suggested that the immobilized and passivated GQDs on the electrode could retain their native bioactivity [44]. .

In vitro and In vivo cytotoxicity:
The toxicity and biosafety of GQDs has not yet been comprehensively examined [49] and uncertainties remain about how such nanomaterials interact with biological substrates, since this is also dependent on the particular physicochemical characteristics of different formulations.In vivo toxicity will further depend on To examine the in vivo cytotoxicity of GQDs, we performed a complete blood count (CBC), serum biochemistry and histological study of vital organs of control and treated rats in a dose-dependent manner (8 doses spread over a 30 day period of either 5, 10 or 15 mg/kg GQD).No clear toxic effect of GQDs on CBC was observed (Supplementary Figure 4 a-l) although there was a slight 6% reduction in platelet numbers in the 10 mg/kg group at 1 week, which normalised by weeks 3 and 4; monocyte and eosinophil fractions similarly underwent a modest dose-dependent reduction, although the proportion of lymphocytes remained stable and total white cell count was unaffected.When serum biochemical parameters were compared between the groups, the results similarly did not indicate any acute toxicity.An increase in total protein was observed at 4 weeks (for all three doses), although the levels remained within the normal range (Supplementary Information, Figure 5 a-e).
A comprehensive post mortem histological study was then performed to assess any tissue interactions with GQDs.Sections of heart, kidney, liver and lung were examined for histopathological changes thirty days after GQD administration (at doses of 5, 10 and 15 mg/kg GQD).No gross abnormalities were observed, although some minor changes were particularly noted in the liver and lungs at the 10 and 15 mg/kg doses of GQDs (Figure 3b and supplementary table 2 and 3 corresponding to their histological findings).No significant cardiac histology changes were evident.In the liver, some apoptotic bodies, binuclear hepatocytes, and activated Kupffer cells were identified, indicating minor alterations to liver histology, but no infiltration of polymorphic inflammatory cells; hepatocyte swelling due to hepatic sinusoidal obstruction syndrome suggests some pathological effects related to GQDs.A small degree of pigmentation was observed in the kidneys of rats treated with GQDs, possibly indicating lipofuscin deposition, but again no infiltration of inflammatory cells.
Within the lungs, occasional giant histiocytes were observed, associated with an inflammatory cell infiltrate, potentially indicative of a mild foreign body reaction to the GQDs.Taken together, these data indicate that GQD treatment did not result in overt acute toxicity, although the minor histopathological changes, particularly in the liver and lungs, warrant long-term observation in future studies.

Singlet oxygen generation and reactive oxygen species:
To further investigate the potential of GQDs for PDT, the singlet oxygen generation capacity of GQDs was verified and their PL in H2O and D2O was also measured (Supplementary Figure 6 shows the absorbance and PL of GQDs in H2O and D2O.).
The lifetime of singlet oxygen in heavy water is 10 times longer than in water [50] ; solutions were therefore prepared by adding 0.1 mg of GQD powder into 5 mL of H2O or 5 mL of D2O, respectively.It was observed that the blue shift in D2O was only a little quicker than in H2O because the singlet oxygen is formed on the GQDs surface by energy transfer from the GQDs.The O2 molecules involved in oxidation are likely to intercalate onto the surface [50] .Hence, singlet oxygen was responsible for the PL shift of the GQDs. Figure 5 a shows a schematic of the mechanism for the singlet oxygen generation from a PDT photosensitizing agent.To further monitor the singlet oxygen generation, EPR spectra were recorded under different irradiation conditions and it was observed that the intensity of the signal, resulting from singlet oxygen generation, showed an increase which was dependent on the irradiation time (Figure 5 b).EPR signals arising from the spin trapping of superoxide were also observed (Figure 5 c).These results confirm that energy transfer from GQDs to oxygen is responsible for the excitation of ground state oxygen [51,52] .By utilising the nitroxide spin trap, TMP, in conjunction with EPR spectrometry, we were able to demonstrate that, upon irradiation, GQDs generate 1 O2 and that this occurs in a light dosedependent manner.TMP traps 1 O2, producing the stable radical TEMPOL, which has hyperfine splitting of a N = 16.3.The small TMP signal observed prior to irradiation (0 J/cm 2 ) was attributable to low-level contamination from the manufacturing process of TMP, as this signal was also observed in solutions of TMP alone (data not shown).As the irradiation time (and thus the light dose) was increased, the concentration of 1 O2 generated increased in a linear manner (R 2 = 0.98, data not shown), as indicated by an increasing signal.To further demonstrate that the observed signal(s) were arising from the trapping of 1 O2 by TMP, experiments were also carried out in D2O and in the presence of the 1 O2 quencher, Lhistidine.When generated in D2O, 1 O2 has a significantly longer lifetime, which was demonstrated here by a 44% increase in the concentration of the TMP reaction product detected post-irradiation.In the presence of L-histidine, no appreciable EPR signal was observed, indicating complete quenching of 1 O2 [53,54] .The complete lack of signal, including that arising from the previously described contamination, is likely to be due to the generation of other reactive oxygen species (as described below), which are known to oxidise the product, arising from the reaction of 1 O2 with TMP, to the "EPR silent" hydroxylamine form [55] .Thus, in the absence of any 1 O2 generation, the signal is completely eliminated.In addition, L-histidine only chemically and physically quenches/captures singlet oxygen and does not do the same with electrons.Thus the formation of the TEMPOL adduct must be caused by singlet oxygen reacting with TMP and not by electron transfer between photoexcited GQDs and TMP.If a proportion of the adduct formation was caused by electron transfer, then we would expect to see a decreased signal, not the complete absence of a signal that we observed with L-histidine.Evidence supports the view that L-histidine does not affect electron transfer [54,55] .We have also established that GQDs generate respectively.Addition of the metal chelator DTPA inhibited the generation of hydroxyl radicals [56] , leaving the DEPMPO-OOH signal (a N = 13.4,a P = 52.5, a H = 11.9;PDT has been regarded as a minimally invasive treatment modality and widely applied in clinics for various types of cancer treatment [57][58] .Compared to conventional therapies, PDT exhibits certain distinct advantages, since the cytotoxic photosensitizing agent can be selectively activated by manipulating the location of light exposure.However, there are tremendous hurdles to overcome with standard PDT technology because of limited ROS production, lack of deep tissue penetration by the excitation wavelengths utilised, lack of selectivity towards tumor cells and rapid removal from the body.Assembly of luminescent graphene-related materials properties ideal for in vivo optical imaging and PDT is therefore highly potential prospect [60,61] .Recent advances in the GQD field have provided unique optical properties, plus remotely controlled release of therapeutic agents in vitro and in vivo [25,26,62] .The use of any precursor as carbon source and their chemical processing to isolate GQDs, their size tunability and fluorescence quenching can also have a significant effect on ROS generation ability.For example GQDs prepared from graphene oxide are actually graphene oxide QDs and they have different functional groups (such as carbonyl, carboxylic, or hydroxyl groups) as compared to pristine GQDs and which in turn affect the ROS generation and toxicity of GQDs in living systems.Surface functional groups are an especially important parameter in controlling the biological activities of these QDs [9,28,63,64] .In general, several synthesis techniques are available and have been widely used for the preparation of GQDs, such as lithography [65,66] , hydrothermal cutting [65,66] and electrochemical preparation [67] .The drawbacks of these methods are the low production yield and the problems associated with the separation and purification of the condensed amorphous carbon phase from low crystalline carbon blacks and fibres [28,49,67] .The resultant product of this partial separation and purification is an oxidized graphite framework and graphene oxide QDs rather than GQDs [65] .Efficient large scale and cost effective production of GQDs remains an important challenge.The features of the different synthetic methods used for the preparation of GQDs have been compared in Supplementary Table 4.Our study demonstrates a novel and facile approach to GQD fabrication through the exfoliation and disintegration of graphite flakes, resulting in GQDs with an excellent repertoire of physicochemical properties, including biocompatibility, water dispersibility, plus high photo and pH stability with higher quantum yield of 7.12 %, showing a size around 20 nm, and the majority of them are single layered.Further investigation shows that these GQDs exhibit strong luminescence with high quantum yield, which is highly desirable for the real world application of GQDs.Because of the carbene structures in the zigzag planes, the obtained GQDs exhibit exciting luminescence with high quantum yield.On the basis of these fascinating features, GQDs may be optimized for cytocompatibility and surface bioactivity, leading to unique bio-functionality not achieved through existing synthesis approaches.Crucially, they have ideal luminescent features and can generate high yields of singlet oxygen (and other oxygen radicals), above and beyond those currently achievable with state-of-the-art PDT agents.The quantum yield of our GQD preparation compares favourably with that shown in a recent study by Ge et al [25] in which the authors use an alternative strategy for GQD preparation, based on a hydrothermal method using polythiophene derivatives.These two contrasting approaches to GQD fabrication result in nanoparticles with comparable physicochemical properties, indicating that GQDs can be robustly and easily produced with manufacturing processes which could be upscaled for future therapeutic applications while conforming to GMP guidelines.These complementary studies demonstrate that GQDs are therefore highly efficacious for PDT targeting of both cervical carcinoma xenografts in rats and breast cancer xenografts in mice [25] , indicating the potentially wide applicability of this technology in a range of tumour types.Building on previous studies, we also investigated the inherent toxicity of GQDs, which is a pivotal consideration for future therapeutic application.
Encouragingly, our data indicate that GQDs have minimal toxicity in fibroblasts or epithelial cells cultured in vitro.GQDs were then injected at a range of doses into healthy rats, which were monitored over a four week period.The rats had no overt signs of discomfort or ill health.Post mortem analysis revealed only limited histological changes in the liver and lungs, suggestive of a mild inflammatory reaction, but no notable changes were observed in the heart or kidneys.These findings support the biocompatibility of GQDs and alleviate concerns about the toxic sideeffects of this treatment approach, although future studies should address long-term toxicology.This investigation also demonstrated that the intensity of the electron paramagnetic resonance signal, resulting from singlet oxygen generation, showed an increase which was dependent on the irradiation time.Singlet oxygen generation was also carried out in D2O and in the presence of the 1 O2 quencher, L-Histidine.When generated in D2O, 1 O2 has a significantly longer lifetime, which was demonstrated here by a 44% increase in the concentration of TEMPOL detected post-irradiation and the addition of the transition metal chelator DTPA inhibited the production of • OH, leaving a O2 •--specific electron paramagnetic resonance spectrum.
In summary, we have demonstrated a straightforward and facile approach for GQD manufacture, generating GQDs with high singlet oxygen yield, high biocompatibility and corrosion resistance, plus the high photo/pH-stability, indicating their potential as a flexible, multifunctional nanoplatform for cancer therapy.The applications of GQDs are wide-ranging and could be easily extended into other unique functional materials and open new doors for precision nanomedicine in future clinical applications.

Conclusion
We herein report the enhanced yield of singlet oxygen and associated ROS from a newly-synthesized GQD with potential as a smart and promising PDT agent.
These graphite flake-derived GQDs demonstrated excellent photo-luminescent features, particle diameter, excellent corrosion resistance, high water solubility, high photo/pH-stability, good in vitro and in vivo biocompatibility and very efficient singlet oxygen/ROS generation.This investigation also demonstrated that the intensity of the electron paramagnetic resonance signal, resulting from singlet oxygen generation, showed an increase which was dependent on the irradiation time.

References:
[1] Choi, H.S., et al.Renal clearance of quantum dots.Nat.biotechnol.25, 1165-1170 (2007)   ** -This is an important study on intravenous administration of quantum dots in rodents and their ultimate renal clearance, filtration and urinary excretion of such nanodots.
[2] Yaghini, E., Seifalian, A. M., & MacRobert, A. J.Quantum dots and their potential biomedical applications in photosensitization for photodynamic therapy.Nanomedicine, 353-363 (2009).** -This review presents the potential usefulness of quantum dots as photosensitizer in photodynamic therapy compared with traditional agents such as organic dyes which have low yield of generation of cytotoxic reactive oxygen species.[7] Yaghini, E., Pirker, K. F., Kay, C. W., Seifalian, A. M., & MacRobert, A. J. Quantification of reactive oxygen species generation by photoexcitation of pegylated quantum dots.Small, 10, 5106-5115 (2014) photosensitising agent, were prepared at a concentration of 10 µM, mixed with 50 Statistical analysis was performed between two treatment groups by unpaired Student's t-test, and between multiple treatment groups by oneway analysis of variance (ANOVA) with Tukey post-hoc testing or two-way ANOVA with Bonferroni post-hoc test, using Graphpad Prism 5 software.Results are presented as mean ± s.d, unless otherwise indicated.The value of p<0.05 was considered significant.

Figure 1 .
Figure 1.Basic characterization of GQDs.(A) Representative Atomic Force Microscopy image of GQDs deposited on mica substrate indicating size around 20 -40 nm, with a particle diameter spectrum showing the complete size distribution of the GQDs (overlaid); (B) Raman spectrum demonstrating peaks for D (defect) and G (graphene) at around 1354 and 1592 cm -1 respectively; (C) Time resolved photoluminescence spectrum of the prepared GQDs, demonstrating the emission lifetime (460 nm) following GQD excitation (360 nm) at room temperature.

Figure 2 .
Figure 2. Water dispersibility, pH stability and Photostability of GQDs.(A, B) Representative images and quantification of wettability in H2O and D2O, as measured by water contact angle, and in comparison with mica substrate; "mica substrate" is the control without GQD.A digital camera was used to record the images and their contact angles were calculated by PolyPro software package.(C) pH-stability of GQDs in H2O and D2O indicating that no obvious luminescence quenching of the corresponding photoluminescence peak (460 nm) was observed in various buffered solutions (shown as mean ± SD from four different experiments) and (D) photostability of GQDs in H2O and D2O over a period of 200 hrs.
pharmacokinetic and pharmacodynamic parameters as influenced by the route of administration, dose and time period of exposure.In vitro cell viability testing using GQDs at concentrations ranging from 1.2 -100 µg/ml demonstrated no obvious toxicity following a 48 hr incubation with either a human bronchial epithelial cell line, 16HBE14o -, or murine NIH3T3 fibroblasts (Figure 4 a and b); both cell types continued to proliferate in the presence of GQDs.

Figure 4 .
Figure 4.In vitro and in vivo toxicity of GQDs.(A) Effect of GQDs on human bronchial epithelial cell (16HBE14o-) viability after 48 hours incubation, relative to untreated control cells; (B) Effect of GQDs on murine fibroblast (NIH3T3) viability after 48 hours incubation, relative to untreated control cells.Data were analysed by one-way ANOVA with a Tukey multiple comparisons test; no significant differences were observed; (C) Histological evaluation of the vital organs of rats at 4 weeks after intravenous injection of GQDs.Tissues of low, medium and high dose of GQDtreated rats were similar to those of the control group.In the control livers, hepatocytes were arranged in cord like pattern (arrowhead).In this section, the O2 •-and • OH and during irradiation.Irradiation of GQDs in the presence of DEPMPO resulted in an EPR signal comprised of the DEPMPO-OOH and DEPMPO-OH spin adducts (a N = 13.4,a P = 52.5, a H = 11.9 and a N = 14, a P = 47, a H = 13; Figure 5 d, upper spectrum), indicating the generation of superoxide and hydroxyl radicals,

Figure 5 .
Figure 5. Singlet oxygen and reactive oxygen species generation by GQDs.(A) Schematic diagram of singlet oxygen generation through energy transfer with an excited photosensitizer; this figure has been adopted with permission from reference 9. (B) Time-dependent electron paramagnetic resonance signals of GQD solutions mixed with TMP.Signal intensity increased with increasing irradiation time (λmax = 365 nm).(C) Electron paramagnetic resonance spectra following irradiation of GQDs (in H2O and D2O) in the presence of TMP.Addition of the 1 O2 quencher, L-histidine (L-his) completely attenuated the signal.(D) Electron paramagnetic resonance spectra of GQDs in DEPMPO.No signals were observed in the dark, but clear signals were observed following irradiation (λmax

[ 3 ]
photosensitizers for use in photodynamic therapy.[5] Cai, W. et al.Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects.Nano let.6, 669-676 (2006) * -This paper reports the preparation of arginine-glycine-aspartic acid peptidelabeled quantum dots for use in vivo imaging of tumor vasculature in athymic nude mice following subcutaneous U87MG human glioblastoma tumors and intravenous administration of quantum dots.[6] He, X. P., & Tian, H. Photoluminescence Architectures for Disease Diagnosis: From Graphene to Thin-Layer Transition Metal Dichalcogenides and Oxides.Small, 12, 144-160 (2016) * -This review provides key insights into the recent developments in the synthesis and use of graphene and transition metal dichalcogenides based single-layer materials for single-target and multiplexed detection of a variety of biomarkers and theranostics owing to their low off-target toxicity and therapeutic responses.