Background

The International Organization for Standardization define nanoparticles (NPs) as structures whose sizes in one, two, or three dimensions are within the range from 1 to 100 nm. Apart from size, NPs may be classified in terms of their physical parameters, e.g., electrical charge; chemical characteristics, such as the composition of the NP core or shell; shape (tubes, films, rods, etc.); and origin: natural NPs (NPs contained in volcanic dust, viral particles, etc.) and artificial NPs, which are the focus of this review.

Nanoparticles have become widely used in electronics, agriculture, textile production, medicine, and many other industries and sciences. NP toxicity for living organisms, however, is the main factor limiting their use in treatment and diagnosis of diseases. At present, researchers often face the problem of balance between the positive therapeutic effect of NPs and side effects related to their toxicity. In this respect, the choice of an adequate experimental model for estimating toxicity between in vitro (cell lines) and in vivo (experimental animals) ones is of paramount importance. The NP toxic effects on individual cell components and individual tissues are easier to analyze in in vitro models, whereas in vivo experiments make it possible to estimate the NP toxicity for individual organs or the body as a whole. In addition, the possible toxic effect of NPs depends on their concentration, duration of their interaction with living matter, their stability in biological fluids, and the capacity for accumulation in tissues and organs. Development of safe, biocompatible NPs that can be used for diagnosis and treatment of human diseases can only be based on complete understanding of the interactions between all factors and mechanisms underlying NP toxicity.

Medical Applications of Nanoparticles

In medicine, NPs can be used for diagnostic or therapeutic purposes. In diagnosis, they can serve as fluorescent labels for detection of biomolecules and pathogens and as contrast agents in magnetic resonance and other studies. In addition, NPs can be used for targeted delivery of drugs, including protein and polynucleotide substances; in photodynamic therapy and thermal destruction of tumors, and in prosthetic repair [1,2,3,4,5,6]. Some types of NPs are already successfully used in clinic for drug delivery and tumor cell imaging [7,8,9].

Examples of the use of gold NPs have been accumulating recently. They have proved to be efficient carriers of chemotherapeutics and other drugs. Gold NPs are highly biocompatible; however, although gold as a substance is inert towards biological objects, it cannot be argued that the same is true for gold NPs, since there are no conclusive data yet on the absence of delayed toxic effects [10]. In addition to gold NPs, those based on micelles, liposomes [11], and polymers with attached “capture molecules” [12] are already used as drug carriers. Single- and multiwalled nanotubes are good examples of NPs used for drug delivery. They are suitable for attaching various functional groups and molecules for targeted delivery, and their unique shape allows them to selectively penetrate through biological barriers [13]. The use of NPs as vehicles for drugs enhances the specificity of delivery and decreases the minimum amount of NPs necessary for attaining and maintaining the therapeutic effect, thereby reducing the eventual toxicity. This is especially important in the case of highly toxic and short-lived chemo- and radiotherapeutic agents [14].

Quantum dots (QDs) constitute another group of NPs with a high potential for clinical use. QDs are semiconductor nanocrystals from 2 to 10 nm in size. Their capacity for fluorescence in different spectral regions, including the infrared one [15], makes them suitable for labeling and imaging cells, cell structures, or pathogenic biological agents, as well as various processes in cells, tissues, and body as a whole [16,17,18], which has important diagnostic implications [19, 20]. NPs based on superparamagnetic iron oxide are efficiently used as contrast agents in magnetic resonance tomography (MRT) for imaging liver, bone marrow, and lymph node tissues [21]. There is also an example where radioactively labeled single-walled carbon nanotubes functionalized with phospholipids were used for labeling integrin-containing tumors and their subsequent detection by means of positron emission tomography in experiments on mice [22].

Nanoparticles have also been used in designing biosensors, including those based on carbon nanotubes for measuring the glucose level [23], detecting specific DNA fragments and regions [24], and identifying bacterial cells [25].

Silver (or silver-containing) NPs exert antimicrobial and cytostatic effects; for this reason, they are widely used in medicine, e.g., for treating bandages, surgical instruments, prostheses, and contraceptives [13, 22]. Silver NPs have been reported to serve as effective and safe preservation agents in the cosmetic industry [26].

However, NPs may still be highly toxic, even if the safety of using many of their chemical constituents in medicine has been proved. The toxic effect may be caused by their unique physical and chemical properties, which underlie specific mechanisms of interaction with living systems. In general, this determines the importance of studying the causes and mechanisms of the potential toxic effect of NPs.

Mechanisms of Nanoparticle Toxicity

The toxicity of NPs is largely determined by their physical and chemical characteristics, such as their size, shape, specific surface area, surface charge, catalytic activity, and the presence or absence of a shell and active groups on the surface.

The small size of NPs allows them to penetrate through epithelial and endothelial barriers into the lymph and blood to be carried by the bloodstream and lymph stream to different organs and tissues, including the brain, heart, liver, kidneys, spleen, bone marrow, and nervous system [27, 28], and either be transported into cells by transcytosis mechanisms or simply diffuse into them through the cell membrane. Nanomaterials can also increase access to the blood stream through ingestion [29, 30]. Some nanomaterials can penetrate the skin [31, 32] and even greater microparticles can penetrate skin when it is flexed [33]. Nanoparticles, because of their small size, can extravasate through the endothelium in inflammatory sites, epithelium (e.g., intestinal tract and liver), tumors or penetrate microcapillaries [34]. Experiments modeling the toxic effects of NPs on the body have shown that NPs cause thrombosis by enhancing platelet aggregation [35], inflammation of the upper and lower respiratory tracts, neurodegenerative disorders, stroke, myocardial infarction, and other disorders [36,37,38]. Note that NPs may enter not only organs, tissues, and cells, but also cell organelles, e.g., mitochondria and nuclei; this may drastically alter cell metabolism and cause DNA lesions, mutations, and cell death [39].

The toxicity of QDs has been shown to be directly related to the leakage of free ions of metals contained in their cores, such as cadmium, lead, and arsenic, upon oxidation by environmental agents. QDs may be absorbed by mitochondria and cause morphological changes and dysfunction of the organelles [40]. Entry of cadmium-based QDs into cells and formation of free Cd2+ ions causes oxidative stress [41, 42].

Recent studies have shown that contact of lung tissue with NPs about 50 nm in size leads to perforation of the membranes of type I alveolar cells and the resultant entry of the NPs into the cells. This, in turn, causes cell necrosis, as evidenced by the release of lactate dehydrogenase [43]. There is evidence that QD penetration increases the cell membrane fluidity [44]. On the other hand, the formation of reactive oxygen species (ROS) induced by peroxidation of membrane lipids may lead to the loss of membrane flexibility, which, as well as an abnormally high fluidity, inevitably results in cell death.

Interaction of NPs with the cytoskeleton may also damage it. For example, TiO2 NPs induce conformational changes in tubulin and inhibit its polymerization [45], which disturbs intracellular transport, cell division, and cell migration. In human umbilical vein endothelial cells (HUVECs), damage of the cytoskeleton hinders the maturation of coordination adhesive complexes which link the cytoskeleton to the extracellular matrix, thereby disturbing the formation of the vascular network [46].

In addition, the NP cytotoxicity may interfere with cell differentiation and protein synthesis, as well as activate proinflammatory genes and synthesis of inflammatory mediators. It should be specially noted that normal protective mechanisms do not affect NPs; macrophage uptake of large PEGylated nanoparticles is more efficient than uptake of small ones, which leads to accumulation of NPs in the body [47]. Superparamagnetic iron oxide NPs have been demonstrated to disturb or entirely suppress osteogenic differentiation of stem cells and activate the synthesis of signal molecules, tumor antigens, etc. [48, 49]. In addition, interaction of NPs with the cell enhances the expression of the genes responsible for the formation of lysosomes [50], disturbs their functioning [51], and inhibits protein synthesis [52, 53]. A study on the toxic effects of NPs of different compositions on lung epithelial cells and human tumor cell lines has shown that NPs stimulate the synthesis of inflammation mediators, e.g., interleukin 8 [54]. According to Park, who studied the expression of proinflammatory cytokines in vitro and in vivo, the expressions of interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) are enhanced in response to silicon NPs [55].

Oxidation, as well as action of various enzymes on the shell and surface of NPs, results in their degradation and release of free radicals. In addition to the toxic effect of free radicals expressed as oxidation and inactivation of enzymes, mutagenesis, and disturbance of chemical reactions leading to cell death, degradation of NPs leads to alteration or loss of their own functionality (e.g., the loss of the magnetic moment and the changes in the fluorescence spectrum and transport or other functions) [56, 57].

In summary, the most common mechanisms of NP cytotoxicity are the following:

  1. 1.

    NPs may cause oxidation via formation of ROS and other free radicals;

  2. 2.

    NPs may damage cell membranes by perforating them;

  3. 3.

    NPs damage components of the cytoskeleton, disturbing intracellular transport and cell division;

  4. 4.

    NPs disturb transcription and damage DNA, thus accelerating mutagenesis;

  5. 5.

    NPs damage mitochondria and disturb their metabolism, which leads to cell energy imbalance;

  6. 6.

    NPs interfere with the formation of lysosomes, thereby hampering autophagy and degradation of macromolecules and triggering the apoptosis;

  7. 7.

    NPs cause structural changes in membrane proteins and disturb the transport of substances into and out of cells, including intercellular transport;

  8. 8.

    NPs activate the synthesis of inflammatory mediators by disturbing the normal mechanisms of cell metabolism, as well as tissue and organ metabolism (Fig. 1).

Fig. 1
figure 1

Mechanisms of cell damage by nanoparticles. (1) Physical damage of membranes [43, 67, 75]. (2) Structural changes in cytoskeleton components [45, 46]. (3) Disturbance of transcription and oxidative damage of DNA [61, 62]. (4) Damage of mitochondria [39, 40]. (5) Disturbance of lysosome functioning [51]. (6) Generation of reactive oxygen species [61]. (7) Disturbance of membrane protein functions [172]. (8) Synthesis of inflammatory factors and mediators [54, 55]

Full size image

Although there are numerous mechanisms of NP toxicity, it is necessary to determine and classify the type and mechanism of each particular toxic effect of NPs as dependent on their physical and chemical properties.

Relationships of Nanoparticle Toxicity with Their Physical and Chemical Properties

The toxicity of NPs is considered to depend on their physical and chemical characteristics, including the size, shape, surface charge, chemical compositions of the core and shell, and stability. In particular, Oh et al., using the data meta-analysis of 307 papers describing 1741 cell viability-related data samples, recently analyzed the CdSe quantum dot toxicity. It has been shown that the QD nanotoxicity is closely correlated with their surface properties (including shell, ligand, and surface modifications), diameter, toxicity assay type used, and the exposure time [58]. Which of these factors is the most important is determined by the specific experimental task and model; therefore, we will now consider each factor separately.

Nanoparticle Size and Toxicity

The NP size and surface area play an important role, largely determining the unique mechanism of NP interaction with living systems. NPs are characterized by a very large specific surface area, which determines their high reaction capacity and catalytic activity. The sizes of NPs (from 1 to 100 nm) are comparable with the size of protein globules (2–10 nm), diameter of DNA helix (2 nm), and thickness of cell membranes (10 nm), which allows them to easily enter cells and cell organelles. For example, Huo et al. have demonstrated that gold NPs no larger than 6 nm effectively enter the cell nucleus, whereas large NPs (10 or 16 nm) only penetrate through the cell membrane and are found only in the cytoplasm. This means that NPs several nanometers in size are more toxic than 10 nm or larger NPs, which cannot enter the nucleus [59]. Pan et al. have traced the dependence of the toxicity of gold NPs on their size in the range from 0.8 to 15 nm. The NPs 15 nm in size have been found to be 60 times less toxic than 1.4-nm NPs for fibroblasts, epithelial cells, macrophages, and melanoma cells. It is also noteworthy that 1.4-nm NPs cause cell necrosis (within 12 h after their addition to the cell culture medium), whereas 1.2-nm NPs predominantly cause apoptosis [60]. These data suggest not only that NPs can enter the nucleus, but also that the correspondence of the geometric size of NPs (1.4 nm) to that of the major groove of DNA allows them to effectively interact with the negatively charged sugar–phosphate DNA backbone and block the transcription [61, 62].

In addition, the NP size largely determines how the NPs interact with the transport and defense systems of cells and the body. This interaction, in turn, affects the kinetics of their distribution and accumulation in the body. The review paper by [63] presents both theoretical considerations and numerous experimental data demonstrating that NPs smaller than 5 nm usually overcome cell barriers nonspecifically, e.g., via translocation, whereas larger particles enter the cells by phagocytosis, macropinocytosis, and specific and nonspecific transport mechanisms. An NP size of about 25 nm is believed to be optimal for pinocytosis, although this also strongly depends on the cell size and type [63, Nanoparticle Shell and Toxicity

Application of a shell onto the surface of NPs is necessary for changing their optical, magnetic, and electrical properties; it is used for improving NP biocompatibility and solubility in water and biological fluids by decreasing their aggregation capacity, increasing their stability, etc. Thus, the shell decreases the toxicity of NPs and provides them with the capacity for selective interaction with different types of cells and biological molecules. In addition, the shell considerably influences the NP pharmacokinetics, changing the patterns of NP distribution and accumulation in the body [94].

As noted above, NP toxicity is largely related to the formation of free radicals [40, 57, 95, 131].

Even QDs containing heavy metals are often found to be nontoxic. One of the possible explanations is that QDs are coated with the protein crown upon entering the living body; this crown shields their surface and protects cells against damage [132]. Usually, the proteins that are included in the NP molecular corona are major serum proteins, such as albumin, immunoglobulin G (IgG), fibrinogen, and apolipoproteins [133]. Molecular corona also can influence on the interaction of NPs with cells. Zyuzin et al. have demonstrated that, in human endothelial cells, the NP protein corona decreases the NP nonspecific binding to the cell membrane, increases the residence time of NP in early endosomes, and reduces the amount of internalized NPs [134].

However, even in the absence of direct signs of intoxication in experimental animals, it remains unclear whether the use of QDs in medicine is safe for humans. In some cases, the QD toxicity was not detected in mice because the NPs were neutralized by the liver and accumulated in it [135]; in other cases, QDs coated with phospholipid micelles exhibited reduced toxicity owing to the shell [129]. Despite the extensive in vivo studies on QD toxicity, their use in biomedicine remains an open question. One of the main reasons is that all the delayed effects of QDs cannot be monitored in experimental animals, because their lifespan is as short as a few years, which is insufficient for complete elimination or degradation of NPs.

Conclusions

The potential toxicity of NPs is the main problem of their use in medicine. Therefore, not only positive results of the use of NPs, but also the possible unpredictable negative consequences of their action on the human body, should be scrutinized. The toxicity of NPs is related to their distribution in the bloodstream and lymph stream and their capacities for penetrating into almost all cells, tissues, and organs and interacting with various macromolecules and altering their structure, thereby interfering with intracellular processes and the functioning of whole organs. The NP toxicity strongly depends on their physical and chemical properties, such as the shape, size, electric charge, and chemical compositions of the core and shell. Many types of NPs are not recognized by the protective systems of cells and the body, which decreases the rate of their degradation and may lead to considerable accumulation of NPs in organs and tissues, even to highly toxic and lethal concentrations. However, a number of approaches to designing NPs with a decreased toxicity compared to the traditional NPs are already available. Advanced methods for studying the NP toxicity make it possible to analyze different pathways and mechanisms of toxicity at the molecular level, as well as reliably predict the possible negative effect at the body level.

Thus, it is obvious that designing NPs that have small or no negative effects is impossible unless all qualitative and quantitative physical and chemical properties of NPs are systematically taken into consideration and a relevant experimental model for estimating their influence on biological systems is available.