1 Autophagy Regulation Mediated by Nanomaterials

Nanomaterials refer to materials with at least one dimension in the nanoscale (i.e., 1–100 nm) or consisting of basic units at the nanoscale (approximately 10–100 tightly aligned atoms). When the three dimensions of materials are all in the nanoscale range, the materials are considered nanoparticles. The fluctuation of electrons and the interaction between atoms will be affected by the size at the nanometer scale; therefore, when the particle size is reduced to a certain size on the nanometer scale, the particle usually shows better physical and chemical properties than conventional materials with the same composition.

The development of nanotechnology and the emergence of new nanomaterials have brought revolutionary achievements to biomedicine. Nanotechnology has been increasingly penetrating the life sciences. An increasing number of nanomaterials are widely used in research on drug delivery, bioimaging, biosensors, medical diagnoses and other biomedical fields. With the increasing application of nanotechnology in biomedicine, increasing research has been performed to explore the cellular processing of nanomaterials and underlying molecular mechanisms. Recently, autophagy induced by nanomaterials has attracted more attention. Many nanomaterials have been shown to induce autophagy both in vivo and in vitro, and some nanomaterials have made some progress in the field of biomedicine.

To date, many nanomaterials have been found to cause autophagy, including gold nanoparticles, graphene, carbon nanotubes, fullerene and its derivatives, silica, α-alumina, iron oxide, dendrimers and cationic liposomes. The characteristics of autophagy regulation by nanomaterials have potential applications in the diagnosis and treatment of autophagy-related diseases, such as cancer and neurodegenerative diseases.

1.1 Activating Autophagy Clearance

Autophagy plays an important role in maintaining cell homeostasis and protecting cells from damage. When cells ingest nanoparticles, they are recognized as foreign bodies and activate the clearance mechanisms of the organism via triggering autophagy. At present, there are three types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy. Nanoparticles usually induce macroautophagy, similar to the autophagic process by which cells remove invasive pathogens. When nanoparticles enter the cell through endocytosis, the nanoparticles in the cytoplasm are isolated and wrap into the membrane structure, forming a closed double-layer membrane structure called the autophagosome. Then, the autophagosomes fuse with lysosomes, forming autolysosomes, and the cargos are degraded by lysosomal enzymes. It is worth noting that the autophagic regulation effect induced by nanomaterials does not necessarily enhance the autophagic scavenging ability of cells. In contrast, when a large number of nanoparticles accumulate in the cells, they will damage the downstream pathway, blocking the autophagic flux and causing cell damage.

1.2 Blocking Autophagic Flux

Autophagic flux induced by nanomaterials can be blocked by disrupting lysosome function. After endocytosis into cells, a large number of nanoparticles accumulate in lysosomes, resulting in enhanced permeability of lysosomal membranes, increased pH and inactivation of lysosomal enzymes. All these cellular and molecular changes prevent the fusion of lysosomes and autophagosomes, which leads to accumulated autophagosomes and blocked autophagic flux. A typical example of this cellular process is autophagy caused by gold nanoparticles. Initially, researchers observed an increase in autophagy in cells after gold nanoparticle treatment. They believe that gold nanoparticles could induce autophagy. However, further studies revealed that the increase in autophagosomes is due to the blockage of autophagic flux rather than the induction of autophagy (Ma et al. 2011).

To better understand the relationship between nanomaterials and autophagy, it is particularly important to dynamically monitor changes in autophagic flux. At present, several methods can be used to detect the formation of autophagic bodies, including transmission electron microscopy, GFP-LC3 dots, LC3 immunofluorescence staining and LC3 western blotting (Klionsky et al. 2016). These methods are also widely used in the detection of autophagy induced by nanomaterials. However, these commonly used methods are relatively static observation methods, and autophagy is a multi-step, dynamic process. The increase of autophagosomes not only increases the ability of cells to degrade autophagic contents but also may be related to the blockage of autophagic flux. Both the increase in autophagic activity and the blockage of autophagic flux can lead to the accumulation of autophagosomes in cells. Therefore, autophagic flux analysis is often used to distinguish the two possibilities, including the use of autophagy-related drugs to detect LC3-I/II turnover, the GFP-LC3 cleavage test, GFP-mCherry-LC3B dual fluorescence analysis and detection of long-lived protein degradation (Klionsky et al. 2016).

2 Underlying Mechanisms of Autophagy Modulated by Nanomaterials

As novel autophagy regulators, nanomaterials affect autophagy through various mechanisms. Previous studies have shown that different nanomaterials have distinct autophagy mechanisms due to their different physical and chemical properties. The mechanisms for nanomaterial-modulated autophagy can be broadly categorized into three classes: oxidative stress, direct regulation of autophagic signaling pathways, such as Akt/mTOR, and alteration of the expression level of autophagy-related genes or proteins (Zheng et al. 2016). The possible pathways of nanomaterial-mediated autophagy are shown in Fig. 14.1.

Fig. 14.1
figure 1

The possible pathways of nanomaterial-mediated autophagy

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Oxidative stress is considered to be one of the main causes of cytotoxicity caused by nanoparticles, which plays an important role in the regulation of autophagy (Stern et al. 2012). Reactive oxygen species (ROS) are chemical active molecules containing oxygen. They are natural byproducts of normal oxygen metabolism and play a key role in cell homeostasis. The main sources of ROS include mitochondria, endoplasmic reticulum, peroxisome and NADPH oxidase complexes (Wen et al. 2013). Mitochondria play an important role in the production of ROS in cells. On the one hand, nanomaterials can increase ROS production by interacting with mitochondria. However, metal or organic substances on the surface of nanomaterials can cause redox reactions. In addition, excited electrons on the surface of nanomaterials can also lead to an increase in ROS in cells. For example, gold-coated iron nanoparticles can release Fe2+ ions and participate in ROS generation. ROS are involved in the regulation of the mTOR signaling pathway, which activates or inhibits the activity of mTORC1 in a dose- and time-dependent manner and regulates autophagy.

The degradation of nanoparticles in lysosomes can also directly induce ROS. Lysosomes are considered to be the conventional targets of cytotoxicity and autophagy induced by nanoparticles (Stern et al. 2012). The accumulation of nanoparticles in lysosomes results in lysosome swelling and the release of cathepsin, which are accompanied by an increase in ROS levels and autophagy. In this process, autophagy is upregulated as a protective mechanism to compensate for the inadequacy of lysosomal degradation. At the same time, increasing lines of evidence show that the accumulation of nanoparticles in lysosomes can cause lysosome alkalization and lysosome damage, ultimately leading to the blockage of autophagic flux (Stern et al. 2012).

Nanoparticles can also interact directly with the mTOR signaling pathway. The changes in mTOR activity mediated by nanoparticles are associated with the regulation of the mTORC1 activators AKT and PI3K, as well as the mTORC1 inhibitors AMPK and TSC (Hulea et al. 2016). In the process of endocytosis, nanoparticles may affect the recruitment/activation of PI3K/AKT in the local area of the cell membrane, thus altering the ability of AKT to activate mTORC1 (Hulea et al. 2016). Because AKT can be activated by mTORC2, the change in AKT activity mediated by nanoparticles may be at least secondary to mTORC2 regulation in some cases. In addition, the interaction between nanoparticles and lysosomes may also affect lysosome recruitment and mTORC1 activation. Nanoparticles can also activate TFEB nuclear translocation and overexpression and enhance the transcription of autophagy-related genes (ATGs) and lysosome genes, thereby promoting the occurrence of autophagy (Zhang et al. 2018; Liu et al. 2018).

In conclusion, the mechanisms of autophagy regulation by various nanomaterials may be different due to their diverse physicochemical properties and biological functions; thus, the specific mechanisms need to be further clarified.

3 Biomedical Applications of Autophagy Modulated by Nanomaterials

Autophagy modulation by nanomaterials has potential applications in drug sensitization, abnormal protein aggregate removal and degradation, immunoregulation and cancer treatment. Table 14.1 summarizes the autophagic regulation of some nanomaterials. Next, we will select several nanoparticles that have been extensively studied to illustrate their relationship with autophagy regulation and their application in biomedicine.

Table 14.1 Autophagy modulation by nanomaterials
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3.1 Carbon Nanomaterials

Carbon nanomaterials are one of the earliest studied nanomaterials. Carbon nanomaterials, including carbon nanotubes, graphene, fullerene C60 and nanodiamond, are new materials that are widely studied and applied. Among them, the first three types of carbon nanomaterials are the most mature. These new carbon materials possess many excellent physical and chemical properties and are widely used in biosensors, gene and drug delivery and stem cell tracking. Studies have shown that carbon nanomaterials can induce autophagy in different cell types.

3.1.1 Fullerene and Derivatives

Fullerenes (C60) are spherical or ellipsoidal conjugates composed of closed, hollow five- and six-membered rings. Unlike other nanomaterials, fullerenes and their derivatives have an antioxidant effect by scavenging free radicals and protecting cells from ROS damage. Fullerenes and their derivatives can also play an important role in autophagy regulation. For example, C60 fullerene-pentoxifylline nanoparticles were found to induce autophagy as a protective mechanism against β-amyloid peptide-induced cytotoxicity in neuro-2A cells (Lee et al. 2011). Therefore, this fullerene derivative has potential application value in drug design for amyloid-related diseases, such as Alzheimer’s disease (AD). In addition, fullerene C60 and its derivatives have an autophagy-dependent chemosensitization effect on cancer cells. Studies have shown that fullerene C60 and its derivative C60 (Nd) nanoparticles can induce autophagy at very low concentrations in cells, and the autophagy effect of C60 (Nd) is more potent (Wei et al. 2010). By modulating autophagy at the nontoxic level, cancer cells are more sensitive to doxorubicin (DOX), and the killing effect of DOX is enhanced (Zhang et al. 2009). Notably, the method is also effective for drug-resistant cell lines. In summary, fullerene combined with traditional anticancer drugs (such as DOX) shows a synergetic effect on cancer cells by regulating autophagy, thereby improving the curative effect and reducing the potential side effects.

3.1.2 Graphene Oxide

Graphene has attracted increasing attention because of its excellent physicochemical properties and low cost since its first discovery in 2004. Graphene, especially graphene oxide, is widely used in biomedical fields such as bioimaging, targeted drug delivery, cancer therapy and biological detection. As a precursor of graphene, graphene oxide has the advantages of simple synthesis, good biocompatibility and good water dispersion. It has been found that graphene oxide can induce autophagy in RAW264.7 macrophages in a concentration-dependent manner. Graphene oxide can activate autophagy in CT26 colon cancer cells, trigger autophagy and TLR-4/TLR-9 signaling cascades, which have anti-tumor effects (Chen et al. 2014). In addition, the combination of graphene oxide and cisplatin (CDDP) can improve the chemosensitivity of cancer cells to anti-cancer drugs (Chen et al. 2015). In addition to initiating autophagy, graphene oxide may induce the opposite autophagy effect due to the different sizes of materials, synthesis methods, surface functional groups, and so on. Graphene oxide quantum dots (average diameter 3.28 + 1.16 nm) can inhibit lysosomal degradation by reducing the activity of cathepsin B in GC-2 and TM4 cells, thus blocking autophagic flux (Ji et al. 2016). Our latest research also shows that graphene oxide nanocolloids can inhibit the fusion of autophagosomes and lysosomes by increasing the pH of lysosomes and the permeability of lysosome membranes in mouse embryonic stem cells, ultimately blocking the autophagic flux (Wei et al. 2019). These different conclusions also illustrate the complexity of the modulation of autophagy by nanomaterials. We need to conduct a concrete analysis of each specific question when using nanomaterials to regulate autophagy.

3.1.3 Carbon Nanotubes

Carbon nanotubes can be regarded as curled graphene sheets. According to the number of graphene sheets, carbon nanotubes can be divided into single-walled carbon nanotubes and multi-walled carbon nanotubes. In biomedical research, increasing attention has been paid to the biological function and toxicity of carbon nanotubes. It has been found that the effects of nanomaterials on autophagy vary depending on the surface properties of the nanomaterial. For example, carboxyl-modified (COOH–) single-walled carbon nanotubes induce autophagic death in A549 cells through the AKT-TSC2-mTOR pathway, while polyaminobenzene sulfonic acid (PABS) and polyethylene glycol (PEG) modified carbon nanotubes cannot induce autophagosome formation (Liu et al. 2011). By changing the surface groups of carbon nanotubes, autophagy can be regulated to a controllable level or even completely avoided. To better understand the role of the surface chemistry of carbon nanotubes in autophagy regulation, Yan et al. systematically studied the autophagy effect of 81 multi-walled carbon nanotubes modified by different combinations of chemically modified surface ligands (Wu et al. 2014). They found that multi-walled carbon nanotubes with different chemical compositions induced autophagy to different degrees by activating different signal pathways, which further demonstrated the flexibility and specificity of nanoparticles in regulating autophagy. Although nanoparticle-induced autophagy can cause nanotoxicity, proper regulation of autophagy may have therapeutic potential. In addition, functionalized single-walled carbon nanotubes can reverse the abnormal activation of mTOR signaling and lysosomal protein hydrolysis defects, thus helping to eliminate autophagic substrates (Xue et al. 2014). Lysosomal dysfunction has been recognized as a major cause of Alzheimer’s disease, Parkinson’s disease and other neurodegenerative diseases. These findings suggest that carbon nanotubes might serve as potential neuroprotective therapeutics for neurodegenerative diseases.

3.2 Metal and Metal Oxide Nanomaterials

Metal and metal oxide nanomaterials, including gold nanoparticles, silver nanoparticles, titanium dioxide, iron oxide, alumina, zinc oxide and a variety of rare earth metal oxides, have been reported to regulate autophagy.

3.2.1 Gold Nanoparticles

Gold nanoparticles, also known as colloidal gold, are biocompatible nanomaterials widely used in the fields of drug delivery, cancer treatment and cell imaging. Gold nanoparticles can induce oxidative stress and autophagy regulation in cells, and the levels of autophagy-related proteins LC3-II and ATG7 are upregulated. Further studies show that the accumulation of autophagosomes and the increased LC3B-II expression induced by gold nanoparticles are due to the blockade of autophagic flux (Ma et al. 2011). In fact, gold nanoparticles cause the accumulation of autophagosomes and autophagic substrate p62 by blocking the fusion of autophagosomes and lysosomes. The uptake of gold nanoparticles is particle-size-dependent. Compared with smaller (10 and 25 nm) particles, larger (50 nm) gold nanoparticles are more likely to be internalized into cells and lead to more autophagosome accumulation (Ma et al. 2011). The engulfed gold nanoparticles in lysosomes result in lysosome alkalization, which weakens the lysosome degradation ability and ultimately blocks autophagic flux. Nanoparticles can either induce autophagy or block autophagy, both leading to the upregulation of LC3-II and the accumulation of autophagosomes. Therefore, it is necessary to detect the autophagy process dynamically and comprehensively when nanomaterials are used as autophagy regulators.

3.2.2 Metal Oxide Nanoparticles

Metal oxide is a binary compound composed of oxygen and another metal element, such as iron oxide, zinc oxide, alumina, titanium oxide or manganese oxide. These metal oxide nanoparticles have autophagy regulation characteristics to varying degrees. It was found that iron oxide nanoparticles could kill cancerous cells by inducing excessive autophagy in A549 lung cancer cells but had no obvious cytotoxicity to normal human lung fibroblasts (IMR-90) (Khan et al. 2012). Another study on the relationship between autophagy and photothermal therapy mediated by iron oxide nanoparticles in cancer showed that the photothermal effect of iron oxide nanoparticles can upregulate the autophagy level in cancerous MCF-7 cells, and inhibiting autophagy can enhance the killing effect of photothermal therapy by increasing cell apoptosis (Ren et al. 2018).

After exposure to human umbilical vein endothelial cells (HUVECs), copper oxide deposits in lysosomes destroyed lysosome function, resulting in impaired autophagic flux and the accumulation of undegraded autophagosomes (Zhang et al. 2018). However, blocking the deposition of copper oxide in lysosomes can significantly reduce the death of HUVECs. Further studies show that inhibiting copper oxide nanoparticle deposition in lysosomes reduces the release of copper ions, which is considered to be a key factor involved in the toxicity of copper oxide (Zhang et al. 2018).

When titanium dioxide nanoparticles are exposed to human immortalized keratinocyte HaCaT at a noncytotoxic level, autophagy is induced at a low dose (0.16 μg/mL) but blocked at a higher dose (25 μg/mL), indicating that the concentration of nanoparticles has a great influence on autophagy regulation (Lopes et al. 2016). Therefore, special attention should be paid to the dosage of nanoparticles when using them to regulate autophagy for biological applications.

3.2.3 Rare Earth Metal Oxide Nanoparticles

Rare earth metal oxides are one of the most important types of metal oxides. They have excellent application prospects in nano-labeling, diagnosis and treatment. Since neodymium oxide (Nd2O3) was found to induce autophagy in NCI-H460 non-small-cell lung cancer in 2005, several rare earth metal oxides, including samarium oxide (Sm2O3), europium oxide (Eu2O3), gadolinium oxide (Gd2O3) and terbium oxide (Tb2O3), have been found to induce autophagy (Yu et al. 2009). In addition, the nano-sized rare earth metal oxides, yttrium oxide (Y2O3), ytterbium oxide (Yb2O3) and lanthanum oxide (La2O3), were found to induce autophagy in HeLa cells (Zhang et al. 2010). Researchers note that the autophagy-inducing effect may be a universal biological effect of rare earth metal oxide nanoparticles.

3.3 Liposomes and Polymer Nanomaterials

3.3.1 Cationic Liposomes

Liposomes are spherically structured lipid bilayers that form when phospholipids are dispersed in water. Cationic liposomes, such as commercialized lipofectamine, are positively charged on the surface. They are widely used as gene/drug carriers due to their ability to transport DNA, RNA and macromolecule substances. Cationic liposomes can induce autophagy through a non-mTOR-dependent mechanism, and the transfection efficiency of cationic liposomes is also affected by the level of cell autophagy (Man et al. 2010). The autophagy regulation effect of cationic liposomes is helpful for improving the efficiency of gene and drug delivery. However, it is noteworthy that the “proton sponge effect” of cationic nanoparticles may lead to lysosome dysfunction, which may lead to the failure of lysosome recruitment and the activation of mTORC1, thus exerting a certain influence on autophagy (Xu et al. 2015).

3.3.2 Poly(D,L-Lactide-Co-Glycolide)

Poly (lactic-co-glycolic acid) (PLGA) is a degradable organic compound that is randomly polymerized by lactic acid and glycolic acid. PLGA has been certified by the Food and Drug Administration of the United States (FDA) and has been formally included in the United States Pharmacopoeia as a pharmaceutical excipient. Studies show that PLGA nanoparticles can induce autophagy and are regulated by the class III PI3K complex. The combination of PLGA nanoparticles loaded with docetaxel (DTX) and the autophagy inhibitors 3-methyladenine (3-MA) or chloroquine can significantly enhance the anti-tumor effect of DTX, both in vivo and in vitro (Zhang et al. 2014). This is because PLGA nanoparticles can induce autophagy, degrading the loaded drug and thus reducing its efficacy. When combined with autophagy inhibitors, nanocarrier-induced autophagy was inhibited, and the pharmacodynamics were significantly improved. It is suggested that the relationship between nanocarriers and autophagy should be taken into account when designing nanodrugs to fully demonstrate the advantages of nanodrugs.

3.4 Quantum Dots

Quantum dots (QDs) are a new type of nanomaterial with excellent fluorescence properties. They are usually between 1 and 10 nm in size with excellent optical properties, such as strong photostability, high fluorescence quantum yield and narrow excitation spectrum. QDs can be used to locate and trace intracellular target molecules and have been widely used in bioimaging and other related fields. However, the cytotoxicity of QDs has led to a bottleneck, restricting their clinical application, and has attracted extensive attention. Eleverstov et al. observed for the first time that QDs could induce autophagy in cells. They found that smaller QD525nm QDs could effectively induce significant autophagy in bone marrow mesenchymal stem cells, while QD605nm (twice the size of QD525nm) could not induce autophagy in cells, indicating that the size of QDs played an important role in autophagy induction (Zabirnyk et al. 2007). In addition, the toxicity of CdSe (average particle size 5.1 nm) QDs is several times that of InGaP (average particle size 3.7 nm) QDs with similar sizes. Different element compositions may be the main reason for the difference in intracellular toxicity of QDs, and this toxicity is positively correlated with autophagy. Most QDs are composed of heavy metal ions (such as Cd2+), which may lead to potential toxicity that hinders their practical application. Cadmium telluride (CdTe) QDs can enhance the sensitivity of cells to cadmium ions (Cd2+) through autophagy (Li et al. 2014). Therefore, although CdTe QDs dissociate fewer cadmium ions (approximately a quarter) into cells than CdCl2 solution treatment at the corresponding concentration, CdTe quantum dots are far more toxic to cells than CdCl2. In addition, CdTe/CdS/ZnS quantum dots can protect differentiated PC12 cells from cell damage induced by 1-methyl-4-phenylpyridine ion (MPP+) and hinder the accumulation of α-synuclein induced by MPP+ (Li et al. 2014). The autophagy-inducing activities of CdTe/CdS/ZnS QDs have a good protective effect on the cell model of PD in vitro.

4 Prospect

Diverse materials induce different autophagy phenomena. Even the underlying mechanisms of autophagy modulation by nanoparticles with the same nanomaterial but with different sizes, shapes and surface modifications are different, which provides various models for studying autophagy. Autophagy initiated by nanomaterials has two aspects: on the one hand, it can enhance the ability of cells to remove foreign bodies; on the other hand, excessive autophagy can also cause type II programmed cell death. Suppression of abnormal mTOR signaling by nanoparticles can be used in anticancer and neuroprotective therapies. At the same time, the potential toxicity of nanoparticles to normal cells should also be considered. In addition, due to the nondegradability of most nanomaterials, the accumulation of a large number of nanomaterials in cells or the toxicity of the materials themselves will cause damage to the corresponding organelles and eventually lead to the blockage of autophagic flux.

Although some progress has been made in the regulation of autophagy by nanomaterials, many challenges remain unsolved. The autophagy regulation effect of nanomaterials is influenced by many factors, such as material composition, particle size, shape, surface modification, dosage, treatment time, synthesis method and cell lines. Therefore, the results of autophagy regulation mediated by nanoparticles are variable under different situations. The degree and mechanism of autophagy induced by various nanomaterials still lack a relatively uniform standard. Furthermore, most of the studies on the regulation of autophagy by nanomaterials are carried out in vitro. It is difficult to truly reflect the effect of nanomaterials on autophagy modulation in organisms. At the same time, how to reduce the toxicity of nanomaterials in the process of autophagy and how to use nanomaterials to control the level of autophagy and apply it to clinical diagnosis and treatment of diseases are still large challenges. Although there are still many problems to be further elucidated, with the further study of the autophagy mechanism of nanomaterials, it is believed that autophagy modulation by nanomaterials will provide more possibilities for the development of biomedicine.

5 Summary

The autophagy regulation effect of nanomaterials not only has potential applications in the treatment of cancer and neurodegenerative diseases but also provides new choices for drug discovery and development. At the same time, nanomaterials also provide excellent models for autophagy research. In addition, different molecular mechanisms of autophagy are induced by different nanomaterials, and related research is still in its infancy. Further research is needed to effectively regulate autophagy induced by nanoparticles and transform this autophagy regulation effect into a new method for disease treatment.