Graphene-Triggered Autophagy: The Savior or Slayer

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GRAPHENE-TRIGGERED AUTOPHAGY: THE SAVIOR OR SLAYER

17

Mamoon Rashid*, Monzurul Amin Roni†, Mehbuba Rahman* Department of Pharmaceutical Sciences, Appalachian College of Pharmacy, Oakwood, VA, United States* Department of Pharmaceutical Sciences, Hampton University, Hampton, VA, United States†

CHAPTER OUTLINE 1 Introduction ................................................................................................................................. 354 2 Overview of Autophagy ................................................................................................................. 354 2.1 The Process of Autophagy ............................................................................................. 354 2.2 Regulation of Autophagy ............................................................................................... 355 2.3 Distinction From Other Cell Deaths ............................................................................... 356 2.4 Effect on Physiological Processes .................................................................................. 356 2.5 Effect on Pathological Processes ................................................................................... 356 3 Chemical Inducers and Inhibitors of Autophagy .............................................................................. 357 4 Is Nanoparticle-Mediated Autophagy Beneficial or Deleterious? ...................................................... 357 4.1 Experimental Intervention of Nanoparticles to Induce Autophagy? .................................... 357 5 Nanomaterials Involving Autophagy Pathways ................................................................................ 358 6 Modulation of Autophagy by Graphene and Related Nanoparticles ................................................... 360 6.1 Autophagy-Mediated Cytoprotection of Graphene ............................................................ 362 6.2 Concentration- and Time-Dependent Induction of Autophagy by GQDs .............................. 363 6.3 Inhibition of Autophagy Prior to Graphene Oxide Treatment ............................................. 364 7 Proposed Mechanism(s) of Nanoparticle-Mediated Induction of Autophagy ...................................... 366 8 Blockade of Autophagic Flux by Nanoparticles ............................................................................... 366 9 Variation in Autophagic Response Induced by Different Graphene Compounds .................................. 367 10 Therapeutic Application and Future Directions ............................................................................... 368 References ........................................................................................................................................ 369

Biomedical Applications of Graphene and 2D Nanomaterials. https://doi.org/10.1016/B978-0-12-815889-0.00017-9 # 2019 Elsevier Inc. All rights reserved.

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1 INTRODUCTION Graphene and its derivatives have gained a great interest in diverse branches of biomedical science including cancer therapy (1–3), diagnostics (4, 5), bioimaging (6–8), and drug delivery (9, 10). Also, a greater concern of safety and toxicity has slowly escalated. This double-edged sword possesses excellent characteristic to be loaded with myriad varieties of therapeutics yet intrinsically bears the stressgenerating cytotoxic properties. Apoptosis, autophagy, and necrosis are the major forms of cell death. At present, the toxicological profiling of graphene and related compounds has not made any distinction among these cell death types. Autophagy may take place simultaneously with apoptosis, and in some cases, the cells may divert into prosurvival autophagy for evading apoptotic fate (11). Graphene and its derivatives are potent inducer of autophagic response, which activates biochemical machineries involved in cytotoxicity or cytoprotection (12). In this chapter, we summarized the biochemical tools and pathways involved in the autophagic process and the relationship of graphene and other nanoparticles to it. Various experimental interventions have been reviewed, and research findings have been condensed to shed light on how graphene may be exploited for the most desired therapeutic outcomes.

2 OVERVIEW OF AUTOPHAGY Autophagy (Greek, self-eating) is a catabolic mechanism that degrades damaged cellular components via lysosomal machinery (13). This process is significantly upregulated by different stimuli such as starvation, cellular stress, infections, and chemicals (14). Upon stimulation, cytoplasmic constituents are sequestered within double-membrane vesicles called autophagosome, which are later joined with lysosomes for degradation. Autophagy is believed to be an adaptive response for cell survival that promotes recycling of nutrient subunits, elimination of unnecessary proteins, and removal of pathogens. However, persistent stress can promote extensive autophagy, resulting in cell death (15, 16). Based on morphological difference, autophagy in mammalian cells has been classified into three types: macroautophagy, microautophagy, and chaperone-mediated autophagy. In general, macroautophagy is the most common type, and it will be referred as “autophagy” in this chapter unless mentioned otherwise.

2.1 THE PROCESS OF AUTOPHAGY Autophagy, a tightly regulated process that involves a complex cascade of proteins, is divided into five steps in mammalian cells: initiation, nucleation, elongation, maturation, and fusion (Fig. 1): 1. Initiation: A semicircular membrane or preautophagosome is formed from endoplasmic reticulum (17) that is activated by Unc-51-like kinase (ULK1). The ULK1 forms a complex with autophagyrelated genes Atg13, FIP200, and Atg 101, which are essential for autophagy (18). 2. Nucleation: In the next step, preautophagosome involves another protein complex containing vacuolar sorting proteins Vps34, Vps15, and Beclin1. ULK1 activates Beclin1 by phosphorylation. The Beclin1 protein complex acts as class III phosphatidylinositol 3-kinase (PI3K) that recruits WD repeat domain phosphoinositide-interacting (WIPI) proteins to the preautophagosomal membrane.

2 OVERVIEW OF AUTOPHAGY

355

Ras

Class I P13K/mTOR inhibitors

Class I P13K

Class III P13K inhibitors ULK1 inhibitors

ATG4 inhibitors Damaged mitochondria

Chloroquine

ATG4 ATG7

Lysosome

LC3/PE

mTOR complex

Class III P13K complex

ULK1 complex

Vsp34 Beclin 1

ERK inhibitors

ERK

Ras

Class I Pl3K/mTOR inhibitors

AMPK

p62 Cargo Isolation membrane

Autophagasome Autophagolysosome Autolysosome ATG5 & 12

Bd-2

ATG7 JNK inhibitors

ATG10

JNK Ras (1) Initiation

(2) Nucleation

(3) Elongation

(4) Vesicle fusion

(5) Degradation

FIG. 1 Schematic overview the five stages of the autophagy pathway. The execution point of where known pharmacological inhibitors act are written in purple. See text for details. Reprinted from Cooper, K. F. Till Death Do Us Part: The Marriage of Autophagy and Apoptosis. Oxidative Med. Cell. Longev. 2018, 2018, 13. https://doi.org/10.1155/2018/4701275.

3. Elongation: The preautophagosome elongates and continues to add more proteins including ubiquitin cargo-binding proteins p62 and NBR1, which acts as partial selective receptors for some cellular substrates (19). Activation of this process requires involvement of certain protein molecules including Atg5, Atg12, and Atg16 and microtubule-associated light-chain protein 3 (LC3-1). The cargo is recognized by phagophores and sequestrated into double-membrane vesicles called autophagosomes. 4. Maturation: The phagophore elongates into a mature autophagosome that wraps around the cargo. During this process, LC3-1 is activated by Atg7 and bound to Atg3. 5. Fusion: Autophagosomes are fused with lysosome via soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins and Rab7, leading to the formation of autolysosomes. Autophagosomes release the cargo inside lysosome, and they are degraded by the lysosomal hydrolases.

2.2 REGULATION OF AUTOPHAGY Autophagy is induced by external or internal stressors including nutrient starvation, hypoxia, the lack of growth factors, and nanomaterials. The initiation step is downregulated by many factors such as growth factors and mammalian target of rapamycin (mTOR). The three major kinases that regulate autophagy are cAMP-dependent protein kinase A (PKA), AMP-activated protein kinase (AMPK), and mammalian target of rapamycin complex 1 (mTORC1) (19). These kinases, in addition to other regulatory proteins, respond to a variety of intracellular and extracellular signals to regulate autophagy. TOR and PKA pathways sense nutrient status and negatively regulate autophagy. AMPK is the major

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Table 1 Comparison Among Autophagy, Apoptosis, and Necrotic Cell Death

Cell death Autophagosome Lysosomal fusion Caspase-dependent ATG gene-dependent

Autophagy

Apoptosis

Necroptosis

 + +

+

+

+ +

energy-sensing kinase in the cell and responds to intracellular AMP/ATP levels to regulate autophagy. When activated by low energy levels, AMPK can phosphorylate and inhibit mTORC1. Under basal condition, mTORC1 localizes onto lysosomal membrane and phosphorylates transcription factor EB (TFEB) to prevent its translocation to the nucleus (20). Under stress, mTORC1 is released from the lysosomal membrane, resulting in translocation of TFEB into the nucleus and activation of coordinated lysosomal expression and regulation (CLEAR) gene network that regulate the formation of autophagosome and subsequent fusion with lysosome.

2.3 DISTINCTION FROM OTHER CELL DEATHS Autophagy is a unique form of programmed cell death. Apoptosis and programmed necrosis (necroptosis) are other kinds of programmed cell deaths that are morphologically and mechanistically different from autophagy. For example, autophagy utilizes autophagosome and autolysosome for cellular degradation, which are not common in other types of cell deaths. In addition, autophagy is either prodeath or prosurvival, whereas apoptosis and necroptosis are always prodeath (11). The differences between autophagy and other types of cell deaths are shown in Table 1.

2.4 EFFECT ON PHYSIOLOGICAL PROCESSES At basal level, autophagy occurs at a relatively low rate. Autophagy is stimulated under stress including nutrient starvation, to degrade cytoplasmic components into metabolites for cellular survival. Moreover, autophagy degrades damaged or excess organelles. Intracellular toxic or nanomaterials are also cleared from the cell by autophagy. Similarly, autophagy is important for degradation of intracellular pathogens.

2.5 EFFECT ON PATHOLOGICAL PROCESSES Excessive self-degradation by dysfunctional autophagy is harmful for the body. Autophagic dysfunction is associated with pathological conditions such as cancer; metabolic disorder; and liver, heart, and lung disorders (21). Abnormal autophagy is associated with excess accumulation of protein and lipid aggregates that are linked to pathogenesis of lysosomal storage disease and neurodegenerative disorders including Parkinson’s disease (22).

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Table 2 Chemical Inducer and Inhibitors of Autophagy Inducers of Autophagy

Inhibitors of Autophagy

Chemotherapy (25) Simvastatin (26) Rapamycin (28) Nanomaterials (e.g., graphene) (29) NF 449 (31) Tunicamycin (32) Thapsigargin (32) Brefeldin A (32)

Chloroquine (24) Wortmannin (27) LY294002 (27) 3-Methyladenine (30)

3 CHEMICAL INDUCERS AND INHIBITORS OF AUTOPHAGY In recent years, dysfunctional autophagy has been targeted for the development of new therapies. For example, autophagy is induced by a variety of chemotherapeutic drugs. Tumor cells utilize autophagy to develop tolerance against the deficiency of nutrition, hypoxia, or acidic environment, which ultimately reduce the therapeutic effect of anticancer drugs (23). To enhance the effects of chemotherapy, different autophagy inhibitors such as chloroquine have been used as adjunct therapy (24). A number of chemical agents are now available that can inhibit or induce autophagy. A list of agents is shown in Table 2.

4 IS NANOPARTICLE-MEDIATED AUTOPHAGY BENEFICIAL OR DELETERIOUS? Healthy cells maneuver autophagic mechanism of scavenging or downsizing their resources in order to primarily survive the environment deficient of nutrient or growth factor. Also, the ER stress and invasion of foreign particles including microorganisms may force the cell to follow autophagy. However, apart from these prosurvival schemes, an uncontrolled or excessive autophagy may lead to cell death (33–35). Cancerous and tumor cells targeted with cytotoxic therapeutics will gain synergistic benefit of graphene or related nanoparticle in a prodeath arrangement. In the contrary, healthy cells exposed to these nanomaterials will experience untoward oxidative stress, imbalanced reactive oxygen species (ROS), autophagy, or even cytotoxicity. In the latter case, an inhibitory mechanism for the autophagy will be beneficial.

4.1 EXPERIMENTAL INTERVENTION OF NANOPARTICLES TO INDUCE AUTOPHAGY? Graphene oxide (GO) has a relatively large surface area and unique π-π interaction in the solid-water interface, enabling its physicochemical sorption of various chemicals including other nanoparticles (36). However, the effect of the surface interaction of graphene oxide with other chemicals in the determination of their fate in the biochemical pathways including cytotoxicity is still unclear (37).

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Nanoparticles-induced autophagy

PO4

TFEB

mTORC1 Lysosome

TFEB

Nan

tio n

PO

mTORC1

Activation of CLEAR gene network

Tra n

sloc ation

als r tic opa

d nt trie Nu

ef ici en cy /st ar va

4

Transcriptional activation of autophagy

TFEB

Lysosome

FIG. 2 Mechanism of nanoparticle-induced autophagy.

5 NANOMATERIALS INVOLVING AUTOPHAGY PATHWAYS Lysosomes securely chamber pH-dependent hydrolytic enzymes that degrade digested proteins and detoxify foreign particles. Degradation via autophagy requires the fusion of autophagosomes with lysosomes, which is controlled by the CLEAR gene network. Translocation of dephosphorylated TFEB, a master gene for lysosomal biogenesis (38), into the nucleus is a prerequisite of the activation of CLEAR gene network (20, 22). The mammalian target of rapamycin protein kinase complex 1 (mTORC1) serves to be a sensor of nutrient, energy, growth signals, or redox condition of the cell and controller of various protein syntheses including the players of autophagic scavenging (39, 40). During normal cellular homeostasis, TFEB remains phosphorylated by mTORC1 and thereby incapable of translocation to the nucleus (39). Under stressed condition, mTORC1 loses contact of lysosomal membrane and thereby dephosphorylates and capacitates the TFEB to translocate into the nucleus. As a result, the transcriptional activation of the CLEAR gene network and autophagy machineries commences (40). As Fig. 2 shows, along with conditions such as diminished nutrients and oxidative stress, the activation of TFEB and CLEAR gene network also gets activated by aberrant accumulation of intracellular materials (22). Also, cellular uptake of 2-hydroxypropyl-beta-cyclodextrin (41), quantum dots (42), and polystyrene nanoparticles (43) demonstrated translocation of TFEB and subsequent initiation of the autophagy process. Nanomaterials are able to both accumulate inside the autophagosomes and independently initiate the autophagy process. Quantum dots (44) are able to induce autophagy, and various other nanomaterials including silica (45), gold (46), alpha alumina (47), rare earth oxides (48), and fullerenes (49) have been found to accumulate within the autophagosome. Graphene quantum dots (GQDs) have promising application in various branches of diagnostic and therapeutic approaches with potentially unresolved issue of toxicity. Externalization of

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phosphatidylserine, activation of the caspase pathways, and DNA fragmentations are common indicators of apoptosis, while the formation of autophagic vesicles, conversion of LC3-I/LC3-II, and degradation of autophagic target p62 are manifested in apoptosis. Electrochemically generated GQD that irradiates a blue light with 470 nm is capable of producing ROS and kills U251 human glioma cells through the oxidative stress, which follow both apoptotic and autophagic cell death routs. Also, incorporation of short hairpin RNA (shRNA) to inactivate the autophagy-inherent protein LC3B nullified the photodynamic cytotoxicity of GQD (29). Although a large number of studies have clearly documented the integral role of lysosome in the degradative completion of autophagy process in response to nanoparticles, the exact nature of the autophagic response upon nanomaterials’ uptake remains unclear. The phenotypic variance in the biochemical response greatly relies on the physicochemical property including the surface chemistry of the nanoparticle. In its simplest form, the autophagy commences as a cytoprotective maneuver to evade the foreign particles including nanomaterials. In other words, anything that the cell perceives as toxic may induce, to some extent, autophagic response (50). Alternative models that have been hypothesized include nanomaterial-induced toxicity through lysosomal dysfunction (51), oxidative stress (46, 52), mitochondrial damage (53) and selective degradation of mitochondria by autophagy aka mitophagy (54), and direct effects on regulatory genes (55). Upon internalization of the nanoparticles, if the lysosome-autophagy system is transcriptionally activated, then it may lead to an enhanced autophagic purgatory outcome or blockade of the autophagic flux due to impaired downstream execution of the autophagic orchestra. Ceria (cerium oxide) nanoparticles are gaining importance owing to their application in heterogeneous catalysis and becoming more prone to human exposure (56) and have found to enhance autophagic clearance (57). Nanoceria removes the free radicals from the cell and thereby protects it from oxidative or radiative injury (58). Nanosilvers are gaining popularity in both the personal care products and antimicrobial preparations (59) and activate autophagy along with induction of oxidative stress and extensive cytotoxicity (60). However, whether the autophagy in the case of nanosilver stems from the response to the foreign material or to ROS-induced damage is debatable (61). Since the elevated amount of autophagy initiation is not balanced with higher number of lysosomic fusion, it is assumed that autophagic flux by nanosilver gets block downstream. As discussed above, TFEB regulates the translational upregulation of the biochemical machineries required for the fusion of lysosome and progression of the autophagic process (20). Therefore, modulation of TFEB appears to be a potential instrument to enhance the clearance of intracellular nanoparticles and endogenous undesired aggregates (62). An in vitro model of aberrant cellular accumulation of aggregated distinct biological materials was developed by Kilpatrick and colleagues in which autophagy was induced by 2-hydroxypropyl-beta-cyclodextrin (63). This cyclodextrin analogue is FDA approved and widely used for ameliorating dissolution in the formulations. It was found that this cyclodextrin-analogue-mediated activation of TFEB is independent on its ability of changing the cellular level of cholesterol (63). Properties of the nanomaterial surface also decide their route of autophagic response. N-Acetylglucosamine, PEG, and polyvinylpyrrolidone were engineered to modify the surface of ceria nanomaterials and hereby stabilize inside the cell, which resulted in TFEBmediated expulsion from the cell. Autophagic response may be amplified or reduced by altering the chemistry of nanoparticles. Lanthanide nanocrystals lose their ability to induce autophagic activity when high-affinity surface-coating peptides were used. As opposed to this, the combination of a high-binding-affinity moiety with the arginine-glycine-aspartic acid (RGD) cell adhesion domain with the help of a bifunctional peptide significantly activated the autophagy (64).

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6 MODULATION OF AUTOPHAGY BY GRAPHENE AND RELATED NANOPARTICLES Table 3 has summarized the experimental intervention of various cell lines with graphene and related nanoparticles along with the results. Several studies have confirmed the autophagy-inducing and cytotoxic properties of nanoparticles (46, 55, 74–76). Also, depending on the intensity and physicochemical nature of the stress or signaling cross talk, an autophagic event may lead to both apoptotic and nonapoptotic death fates (77–79). Fullerene C60 nanocrystal-treated HeLa cells demonstrated the formation of microtubule-associated light-

Table 3 Experimental Intervention of Various Cell Lines With Graphene and Related Nanoparticles and Observed Outcome Cell Line

Treatment

Observation

References

CT26 colon cancer cells

Graphene oxide

(65)

Cultured primary murine peritoneal macrophages Epithelial (BEAS-2B)

Graphene oxide

Autophagic flux was diverted, nuclear import enhanced, increased necrotic cell death, and enhanced antitumor therapeutic effect Induced the accumulation of autophagosome bodies and impairment of lysosomes Cationic nanoparticles proved highly toxic Cationic nanoparticles found to be resistant Selective cytotoxicity on cancer cells only but not on healthy lung fibroblasts The toxicity of PCB 52 was attenuated only when GO is pretreated

Hepatoma (HEPA-1) Human cancer cells

Human-hamster hybrid AL cells

Human microvascular endothelial cell (HMEC) Human neuroblastoma cell line (SK-N-SH) Macrophage (RAW 264.7) Michigan cancer foundation-7 (MCF-7) breast cancer cells

60 nm NH2-labeled polystyrene (PS) nanospheres 60 nm NH2-labeled polystyrene (PS) nanospheres Iron oxide nanoparticles

Graphene oxide was treated (a) concurrently, (b) before, and (c) after the administration of cytotoxic chemical polychlorinated biphenyl (PCB) 52 60 nm NH2-labeled polystyrene (PS) nanospheres Synthetic prion protein (PrP) (106–126) 60 nm NH2-labeled polystyrene (PS) nanospheres Docetaxel was delivered with poly(lactide-co-glycolide)-based nanoparticle

(15)

(66) (66) (67)

(36)

Cationic nanoparticles found to be resistant

(66)

GO-induced autophagic flux is protective against PrP (106– 126)-mediated neurotoxicity Cationic nanoparticles proved highly toxic Autophagy decreased drug efficacy, which was restored by inhibitors 3-MA or chloroquine

(68)

(66) (69)

6 AUTOPHAGY BY GRAPHENE AND RELATED NANOPARTICLES

361

Table 3 Experimental Intervention of Various Cell Lines With Graphene and Related Nanoparticles and Observed Outcome—cont’d Cell Line

Treatment

Observation

References

Oral and colorectal cancer cells Pheochromocytoma (PC-12) Porcine kidney cells

Iron core-gold shell nanoparticles

Enhanced autophagy and lowered viability Cationic nanoparticles found to be resistant Abundant autophagy

(67)

(29)

Graphene oxide

Cell death happens through oxidative stress, showing signs of apoptosis and autophagy Inflammation, autophagy, and apoptosis are activated Autophagy is increased

Graphene oxide

Autophagy is decreased

(72)

Micrometer-sized graphene oxide (MGO, 1089.9  135.3 nm), submicrometer-sized GO (SGO, 390.2  51.4 nm), nanometersized GO (NGO, 65.5  16.3 nm), and graphene quantum dots

Autophagy is induced by SGO and NGO, but not by MGO and quantum dots

(73)

U251 human glioma cells Macrophage (THP-1 monocyte) Adenocarcinomic human alveolar basal epithelial (A549) cells Macrophage (RAW 264.7) cells Vascular endothelial cells (Human Umbilical Vein Endothelial Cells (HUVECs))

60 nm NH2-labeled polystyrene (PS) nanospheres Quantum dots containing (a) cadmium selenide and (b) indium gallium phosphide Graphene quantum dots

Graphene quantum dots

(66) (70)

(71) (72)

chain protein 3 (LC3) dots in a dose-dependent manner, indicating execution of autophagy. Authentic autophagy in this approach was further fortified by treating green fluorescent protein (GFP)-tagged LC3 protein with nano-C60 in the presence of 3-methyladenine (3-MA) and rapamycin, the wellknown inhibitor and inducer of autophagy, respectively (80). Porcine kidney cells were found to undergo abundant autophagy upon treating with quantum dots containing (a) cadmium selenide and (b) indium gallium phosphide, which manifest that nanomaterials in general may trigger autophagic action (70). The cytotoxic effect of various metal oxide nanoparticles was evaluated on three respiratory cell lines such as human adenocarcinoma A549 cells, human nonsmall cell lung cancer H1650 cells, and human nasopharyngeal carcinoma CNE-2Z cells. While TiO2 (10 nm), TiO2 (32 nm), Fe2O3, and Fe3O4 demonstrated no significant difference in the cell viability from the control, SiO2 posed mild toxicity, and CuO was found to be the most toxic in this group. The CuO-induced cell death was not mediated through apoptosis, as validated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and analysis of caspase-3 protein, the two reliable methods of apoptosis detection. However, experiments with enhanced green fluorescent protein (EGFP)-tagged LC3 and knockdown autophagy gene ATG5 confirmed that CuO nanoparticles drive the cells toward autophagy only (81). Other studies have confirmed that the nanoparticles of metal oxide are responsible for genotoxicity, mitochondrial dysfunction, and enhanced cytotoxicity in vitro (82–84).

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The antibacterial effect of UVA was significantly amplified when it was applied after pretreatment with silver nanoparticles, but the irradiation with other exposure methods created no significant difference from the control (85). Another study pretreated C60(Nd), which is C60 spherical cage enclosing neodymium (Nd) atom, for 24 h and observed significantly augmented cytotoxicity of the chemotherapeutic agents and reduced drug resistance obtained by means of prodeath autophagy (86). Autophagy offers a protective mechanism for the cell against damaged and malfunctioning organelles, xenobiotics, and long-lived proteins by means of scavenging them and funneling a fresh supply of amino acids to the biosynthetic pool. In the light of that, inducers of autophagy may potentially serve as cell protectants. GO has proved to be protecting AL cells from cytotoxic and mutagenic effects of PCB 52 (36). Silver nanoparticles, likewise, may induce the cytoprotective autophagy (87), inhibition of which may render the cancer therapeutics more effective. Prosurvival autophagy has been displayed by paramontroseite VO2 nanocrystals too, through upregulating the expression of heme oxygenase-1 (88). The cytoprotective autophagy may provide an escape route for the cancer cells and thereby diminish the therapeutic efficacy of antineoplastic drugs. For instance, the activated autophagy in the cancer cells prevents synergistic antitumor effect of curcumin and temozolomide, and the inhibition of autophagy enabled the drug combination to kill the tumor cells (88).

6.1 AUTOPHAGY-MEDIATED CYTOPROTECTION OF GRAPHENE Polychlorinated biphenyls (PCBs) contain chlorine attached to their aromatic ring and are cytotoxic, mutagenic, and harmful for the environment (89). GO is heavily loaded with oxygen moieties on their large specific surface area and edges, which provides its ability of adsorbing heavy metals and PCBs (90, 91) and therefore augments cell survival. Liu and colleagues (36) conducted a study to elucidate the detoxifying nature of graphene oxide against PCB 52 on the human-hamster hybrid AL cells. Treating the cells with graphene oxide over a concentration range of 0–100 μg/mL proved to be inducing no toxic effect, per se, and that is why two lower concentrations (2 and 20 μg/mL) were used over the PCB 52-treated cells. In order to further divulge the up- and downstream molecular involvement within this cell death induction and prevention process, the cells were exposed to toxic PCB 52 and graphene oxide in three different fashions. The first pattern involved treating the AL cells with 2 or 20 μg/mL concentration of graphene oxide for 4 h and then adding PCB and incubating the cells for 24 h before they were assayed for survival. The second fashion swapped the reagents, that is, addition of PCB 52 took place first, and then, GO was added. The third fashion involved addition of PCB 52 and GO simultaneously and incubating the cells for 24 h before the cell survival assay. The second or third fashion of order of administration did not alter the cell survival significantly from those of the cells treated with PCB 52 alone, the control. Interestingly, the first approach where the cells were treated with GO prior to the exposure of PCB 52 had a significant increase in the cell viability. Two microgram per milliliter concentration of GO increased the cell survival by about 10% points (from the control 67.7% to treated 77.8%), and the 20 μg/mL concentration enhanced it by 20% points (from the control 67.7% to treated 86.3%). This observation was confirmed by cell survival assay and fortified by cell morphology and counting assays. PCBs execute their genotoxicity through increasing the DNA breakage (92). The abovementioned group also employed CD59 gene mutation in the AL cells, which serves to be the representative end point for genotoxicity (93). It was found that the AL cells pretreated with GO and then exposed to PCB 52 displayed a significant reduction in the mutation yield. As opposed to this, cells receiving the PCB 52 exposure before the GO treatment or simultaneous treatment of both did not show

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any protection from the genotoxicity. Therefore, it may be concluded that GO pretreating offers a defense against the cytotoxic and genotoxic effect of PCB 52. Now, in light of the above observation, a curious investigation would be determining whether autophagy is playing any role here. A hallmark of autophagy is the conversion of the microtubuleassociated light-chain protein LC3 into its lapidated form LC3-II, which may be verified with the Western blot. GO significantly increased the formation of LC3-II in a dose-dependent manner, confirming activation of autophagic pathway. The GO nanosheets were labeled with fluorescein isothiocyanatelabeled bovine serum albumin (FITC-BSA) (94) and examined under confocal laser scanning microscope revealing that only a small portion was taken up into the cytosol and the majority was present on the cytoskeletal membrane. The mTOR signal downstream pathway also remained undisturbed, and GO nanosheets failed to inhibit the phosphorylation property of mTOR and its substrate p70 S6 kinase. This confirms that the induction of autophagy was mediated through mTOR-independent pathway. The autophagy inhibitor bafilomycin A1 prevents the fusion of lysosome with autophagosome (95). An interesting observation was AL cells pretreated with GO showed higher level of LC3-II than those obtained from treating GO or bafilomycin A1 alone, indicating probable de novo formation of autophagosome was induced by GO. The execution of autophagy is validated by the degradation of sequestosome-1 (SQSTM1)/p62. Pretreating the AL cells with GO for 4 h dramatically dropped the level of p62 protein, which proves that a genuine autophagy took place upon the administration of GO. Another curious question would be testing if the elevated level of ROS plays any role in the protective nature of GO in this particular phenomenon. The total and mitochondrial oxidative stress were quantified, and it revealed that there was no significant difference in the ROS level of the cells treated with GO alone and that of the cells treated with both. This suggests that the combined toxicity is probably not due to the global ROS generation. Surface-passivated carbon dots are capable of accessing the intracellular space, but their localization and distribution are not fully understood (96). The precedence of internalization and distribution/accumulation of graphene oxide by AL cells is different from what Markovic and colleagues observed in the case of the GQDs (29). U251 human glioma cells examined under confocal microscope after incubating 4 h with photoexcited GQD displayed a green fluorescence, which indicates a significant accumulation of cell-associated GQD. More precise intracellular localization was confirmed with TEM, which revealed that a noticeable vacuolization took place after 12 h. Since most of the GQDs are contained within cytoplasmic vesicles, endocytosis is perhaps the means of entry for GQD to these cells. At the same time, the presence of engulfed cellular components including the mitochondria suggests that the autophagic degradation is also in progress. In this type of situation, autophagy is induced by photodynamic therapy as a means of scavenging the oxidatively damaged organelles and injured proteins (97); however, classical quantum dots themselves are capable of inducing autophagic cellular responses (70, 74).

6.2 CONCENTRATION- AND TIME-DEPENDENT INDUCTION OF AUTOPHAGY BY GQDS GQDs exert autophagic action on macrophage in both concentration- and time-dependent manner. Macrophage (THP-1 monocyte) cells were treated with GQD at the concentrations of 10, 50, 100, and 200 μg/mL for 24 h, and the green fluorescence of the autolysosomes was found to be increased by 1.3-, 1.6-, 5.7-, and 6.8-fold, respectively (71). When analyzed with flow cytometry, these treatments showed 2.0-, 4.2-, 4.6-, and 5.1-fold increase of the autophagy, respectively. Also, the cells exposed to 100 μg/mL concentration showed 2.7-, 4.3, 5.1-, 5.8-, and 6.4-fold increase of the autophagy for the treatment periods of 8, 1, 2, 24, and 48 h, respectively. Also, the autophagy-associated marker

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proteins were found to be upregulated. Beclin1 increased by 1.5-, 1.8-, 2.3-, and 2.4-fold for the GQD treatment at the concentrations of 10, 50, 100, and 200 μg/mL, respectively. Also, GQD caused the overexpression of LC3-I and LC3-II, clearly indicating autophagy. At low doses, GQD significantly induced autophagy but not apoptosis—which suggests that macrophage adopts the GQD-induced autophagy as a means of cellular defense.

6.3 INHIBITION OF AUTOPHAGY PRIOR TO GRAPHENE OXIDE TREATMENT 3-MA and Wortmannin (Wmn) are common inhibitors of autophagy and are commonly used to elucidate the intracellular trafficking via autophagic machinery. In the absence of any autophagy inhibitor, graphene oxide-treated cells show an augmented level of the lapidated form of the microtubuleassociated light-chain protein (LC3-II), a classic hallmark of the autophagy progression. However, the cells treated with either 3-MA or Wmn and then exposed to graphene oxide displayed a reduced expression of LC3-II (Table 2, rows 1 and 2). The AL cells exposed to cytotoxic PCB 52 after receiving pretreatment with graphene oxide only had a cell viability of about 65%, which reduced to about 43% and 38% if the autophagy was blocked in the first place with 3-MA and Wmn, respectively. This indicates that graphene oxide drives the cells toward autophagy-mediated prosurvival pathways. However, the inhibitors, when applied alone on the cell line, exerted no effect on the cell viability (Table 2, rows 3–7). As discussed earlier, graphene oxide reduced the mutation yield; however, the mutation yield is amplified upon impeding the autophagy with 3-MA or Wmn, suggesting that the protective effect of graphene oxide against genotoxicity is truly by means of autophagy (Table 2, rows 8 and 9). ATG5 is one of the pivotal proteins involved in autophagy, which may be translationally downregulated by small interfering ribonucleic acid (siRNA). Incorporating the cells with ATG5 siRNA prior to graphene oxide treatment and then exposing to cytotoxic PCB 52 increase cytotoxicity (Table 2, rows 10 and 11). This also indicates that the cytoprotective nature of graphene oxide is mediated through autophagy. However, this should be noted with caution that the cytotoxic effects commonly seen with photodynamic and nanoparticle treatments involve both apoptotic and nonapoptotic cell death pathways (44, 53, 97, 98). The cause-and-effect relationship between autophagy and a cell death has not been completely established (99); rather, an ongoing autophagy may be diverted from a conservative prosurvival scheme to suicidal prodeath direction if the autophagic activity is uncontrolled or excessive (100). Various nanoparticles including polymeric nanomaterials (101) and iron oxide nanoparticles (53) may activate cell death mechanism by means of enhanced autophagosomes. The most critical step of autophagic degradation is the fusion of lysosome with autophagosome and subsequent proteolytic digestion of the contents. Depending on the desired destiny of the cell, the upregulation of the autophagosome may be either beneficial or detrimental for the intervention. A malfunctioning autophagosome incapable of fusing with lysosome will lead to lysosomal overload (102) and eventually block the autophagic flux. This type of blockage has been documented in the case of several types of nanomaterials including fullerenol (103), silver (60), rare earth oxide (104), and gold (51) nanoparticles. Blocked autophagic flux, autophagosome accumulation, and cytotoxicity have been reported from cellular uptake of carboxylated multiwalled carbon nanotubes. Application of low-dose bafilomycin A1 served to be partly beneficial at this condition, since it promoted escape of the nanomaterials from cytosolic sacs to the extracellular environment (105). Most nanoparticles remain unharmed by lysosomal proteolytic enzymes, for which exocytosis is the most supported means of escape for them (22) (Table 4).

Table 4 Schematic Representation of the Effect of Various Autophagy Inhibitors and Gene Silencer on Human-Hamster Hybrid AL Cells in the Presence and Absence of Graphene Oxide and PCB Compound, PCB 52

Summarized from the research findings of Liu et al. (36).

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7 PROPOSED MECHANISM(S) OF NANOPARTICLE-MEDIATED INDUCTION OF AUTOPHAGY The exact mechanism of how graphene oxide induces autophagy is yet to establish. The formation of LC3 dots, a common indicator of autophagy reported by various groups, is due to both induction of the autophagic process and blockade of the downstream flux (36). Therefore, understanding the integrity of the GO-elicited autophagic flux is warranted. While some nanoparticles possess the autophagyinducing properties, it is not a general hallmark of all nanoparticles, since nanoparticles with great similarity but varying sizes may fail to produce autophagic flux, as in the example of larger quantum dot’s inability to induce autophagy in human mesenchymal stem cells (44). Surface chemistry of the nanoparticles may play decisive role too, since A549 cells exhibited higher amount of autophagy when treated with cationic polyamidoamine (PAMAM) dendrimer but not the anionic PAMAM one (55). Single-walled carbon nanotubes failed to induce autophagy if they are functionalized with polyaminobenzene sulfonic acid and PEG. Collectively, the size, shape, and characteristics of the surface functional groups determine whether the cell will direct toward autophagy or not (98). Growth factors and ROS, among others, induce autophagy (18, 106). Treatment of graphene oxide to human-hamster hybrid AL cells induced autophagy that was mediated by not only pure graphene oxide but also the imbalanced ROS moieties (36). It is possible, however, that graphene and its derivatives may have dissimilar or diverted autophagic pathways yielding contrasting observations, depending on the cell type being treated or the nature of the exposure (36). Right on the membrane of the nascent autophagosome sac, the LC3 protein undergoes a covalent linking with the lipid phosphatidylethanolamine (107), which contributes to the closure of the autophagosome (108) and enables the docking of adapter protein p62. Sequestosome-1 or p62 is also known as the ubiquitin-binding protein p62, which was surprisingly found to sharply decrease during the first 4 h of pretreating the human-hamster hybrid AL cells with graphene oxide (36). It has also been found that the expression level of p62 may not always inversely correlate with the autophagic activity, and events like prolonged deprivation of the nutrients may restore the protein to its basal level (109).

8 BLOCKADE OF AUTOPHAGIC FLUX BY NANOPARTICLES The mechanical strength and ability of the cytoskeleton to retain integrity during the autophagic episodes are essential for the unobstructed progression of autophagosome (110). The cell first contacts the nanoparticles at the site of cytoskeleton and confines them within the vicinity of the cytoskeleton. Alteration of the cell mechanics and subsequent impediment of the autophagic flux may result primarily from the interaction between the nanoparticle and the cytoskeleton of the housing cell (111). Induction of oxidative stress, which is an indirect effect of the nanomaterial uptake, is able to halt autophagic flux too (112). Completion of the autophagic degradation relies on the successful fusion of lysosomes with autophagosomes. The most widely accepted hypothesis of nanoparticle-induced blockade of the autophagic flux is lysosomal dysfunction (22). Gold particles and carbon nanotubes have been detected inside the lysosomes, which are known to induce autophagy-directed cell death (51, 98). The mechanical fusion of the two intact bodies may be hindered by faulty lysosomal integrity. Even upon fusion, catalytically

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inactive enzyme system of lysosome may fail to achieve autophagic goal. Either of these may lead aberrant accumulation of autophagosomes within the cytosol (113, 114). The mechanism of how lysosomal dysfunction may be induced from nanoparticles is not fully understood, but (a) permeabilization or osmotic swelling of lysosomes (66), (b) induction of oxidative stress (60), and (c) alkalinization of the lysosome (53) have been hypothesized. Five different cell lines (RAW 264.7, BEAS-2B, HMEC, HEPA-1, and PC-12) were exposed to 60-nm amino-labeled polystyrene nanospheres by Xia and colleagues (66) who found that the nanosphere toxicity depends on the endocytic and mitochondrial injury pathways that are quite specific to the type of the cell being investigated. They coined a “proton sponge” effect that is founded on the high-proton-buffering capacity of the polystyrene nanoparticles that interact with the low pH content of the lysosome, which leads to impairing the proton pump activity followed by permeabilization of the lysosomal membrane. When a proton pump inhibitor was used, the endosomal acidification was prevented, and the nanosphere-induced toxicity was lowered.

9 VARIATION IN AUTOPHAGIC RESPONSE INDUCED BY DIFFERENT GRAPHENE COMPOUNDS Owing to the characteristic physicochemical properties including the large surface area and ability of both accepting and donating free electrons, graphene, and other derivative nanoparticles may show different intensities of autophagic response based on cell type and nature of the nanoparticle. For instance, the treatment of graphene oxide on adenocarcinomic human alveolar basal epithelial (A549) cells showed an increase of autophagy while on macrophage (RAW 264.7) cells was found to have a downregulation of autophagy, indicated by the alteration of the LC3-I to LC3-II conversion (72). Also, its toxicity and biological responses greatly depend on various intrinsic factors including lateral size, stiffness, hydrophobicity, surface functionalization, dose administered, and purity (115–117). Smaller particles of GQDs are quickly eliminated through renal excretion, but larger ones (40 nm) are retained in the hepatic cells, which has been proved to be hepatoprotective (12). Lectin concanavalin A is a potent inducer of acute inflammatory liver injury, which is attenuated by large GQDs. In this case, both the apoptosis and autophagy mechanisms are downregulated resulting in higher cell survival and hepatoprotection. This result is interesting and contrasting to other observations that GQDs induce apoptosis (11, 29, 44, 71, 118). GO-mediated cytotoxicity has been reported to be dependent on the size of the particle being used. Mi-Hee Lim and colleagues constructed four types of particles, namely, micrometer-sized GO (MGO, 1089.9  135.3 nm), submicrometer-sized GO (SGO, 390.2  51.4 nm), nanometer-sized GO (NGO, 65.5  16.3 nm), and GQDs, and evaluated their effect on cell viability and apoptosis (73). The submicrometer- and nanometer-sized species markedly induced apoptotic cell death via the activation of autophagy in the vascular endothelial cells. This is to be noted that the autophagy and apoptosis may function cooperatively, synergistically, or even antagonistically based on the cell type (119–121). The SGO and NGO particles showed the autophagic response in a dose-dependent manner. However, larger graphene oxides (1089.9  135.3 nm) or GQDs did not induce any autophagy. GO nanosheets induce autophagy in a concentration-dependent manner. In murine macrophagelike cells (RAW 264.7), GO induced the formation of autophagosome-like vacuoles and cell death from low to high concentrations (5–100 μg/mL) (122). The TUNEL assays confirmed that the cell deaths were not primarily from apoptosis. Transmission electron microscopy revealed that the autophagic

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vacuoles surrounded GO nanosheets. Both small and large GO nanosheets with lateral dimension from 2 to 350 nm induced autophagy in RAW 264.7 cell line, indicating both are internalized by macrophages with equal efficiencies (122). Furthermore, GO nanosheets also induced autophagy in colon cancer cells (CT26) and produced antitumor effects (123). GO alone or in combination with cisplatin increased cytosolic colocalization of LC3/p62 and LC3/lysosome-associated membrane protein 2 (Lamp-2) in various cell lines including CT26, ovarian cancer (Skov-3), and prostate cancer (A549) (123). GO in osteosarcoma cancer cells (MG-63) induced autophagy by characteristic autophagosome formation, autophagy flux, and increased expression of autophagy-related proteins (LC3I to LC3II conversion, ATG5, and ATG7) (124). The intensity of autophagy-related protein increased significantly in a concentration- and time-dependent manner. Interesting, GO did not induce marked autophagic vesicles in K7M2, another osteosarcoma cell line, in the same study (124). Apart from cancer cell lines, GO can induce autophagy in neuronal cell lines. For example, GO activated autophagic flux in SK-N-SH neuronal cells (68). The GO nanoparticles dose-dependently increased LC3 and decreased p62 protein levels and prevented prion-mediated mitochondrial neurotoxicity by inducing autophagy. Notably, GO-induced autophagy had neuroprotective effects (68). Additionally, graphene nanoplatelets (GNP) induced autophagy in human bronchial epithelial cell line (BEAS-2B) (125). The nanoplatelets were located within autophagosome-like vacuoles with increased autophagy-related proteins. Conversion from LC3I to LC3II, a late stage-related protein in autophagy, increased after GNP exposure in a dose-dependent manner. The level of p62 protein was also increased dose-dependently, but early stage-related proteins including ATG5 and Beclin1 did not increase (125). In addition to GO nanomaterials, effects of PEGylated GO on autophagy have recently been studied. GO-PEG nanoparticles induced the expression of autophagy modulators ATG3 and DNA damageregulated autophagy modulator 2 (dram2) in vaginal epithelial cells (126). However, PEG-GO was not found to be associated with autophagy in human hepatoma HepG2 cell lines (127).

10 THERAPEUTIC APPLICATION AND FUTURE DIRECTIONS Our current knowledge on the induction, progression, or impairment of autophagic flux sensed by nanoparticles is too inadequate to custom-tailor a nanomaterial capable of executing a specific autophagy-associated event within the cell. Needless to mention, the safety concern and the activation of the autophagy to enhance the cargo delivery largely depend on the rational designing of the nanomaterials to regulate their intracellular interactions. Since nanomaterials are proved inducer of autophagy and, based on their surface chemistry and physicochemical nature, capable of shunting the cells both to prosurvival and prodeath destinations, pathological manifestation originating from aberrant accumulation of protein or other xenobiotics may be encountered with nanoparticles. One such disease condition may be late infantile neuronal ceroid lipofuscinosis (63), fibroblasts obtained from which show enhanced cellular clearance of ceroid lipopigment (128). The therapeutic efficacy of nanomaterial-based drug delivery may be untowardly affected due to commencement of the nanomaterial-sensed autophagy. Zhang and colleagues delivered poly(lactide-co-glycolide) polymer-based anticancer drug docetaxel into MCF-7 breast cells, but the

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efficacy of the drug was greatly increased when autophagy inhibitor 3-MA or chloroquine was used (69). The laboratory of Wu and others investigated multiwalled carbon nanotubes of 81 different structural diversities and screened their ability and mode of inducing autophagy. It was found that the carbon nanotubes functionalized with surface ligands were able to activate autophagy but the ligands alone failed to do so (129, 130). This indicates that the autophagy activation is the outcome of the nanomaterial uptake. Therefore, if the autophagy is desired as part of the therapeutics, then a drug may be designed to be loaded on nanomaterials as the ligand. Autophagic flux may be blocked to render cell death projected therapeutic outcome. Iron oxide nanoparticles elicited cytotoxicity through enhanced autophagosome formation when applied on human cancer cells, but not on healthy lung fibroblasts (53, 67). Also, enhanced autophagy and reduced cell viability of colorectal and oral cancer cells have been documented upon treatment with iron coregold shell nanoparticles (67).

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