Nanomaterials are proven to affect the biological activity of mammalian and microbial cells profoundly. It has been revealed that the shape of the nanomaterial plays a crucial role. This aentrticley reviews the influence of nanomaterial shape on various biological activities of mammalian and microbial cells, such as proliferation, differentiation, and metabolism.
1. Introduction
Nanomaterials are widely used in medicine as platforms for developing advanced drug delivery systems with controllable drug loading efficacy, biodistribution and cell/tissue targeting, therapeutic actions, cytotoxicity, selectivity, imaging ability, blood circulation time, half-life, and excretion. It is widely thought that all these properties are connected only with nanomaterial surface chemistry, total surface area, hydrodynamic size, the loaded drug, etc. The phenomenon of nanomaterial shape is usually considered in the context of systemic toxicity, biodistribution, and blood circulation time. However, its influence on the biological activities of mammalian and microbial cells has been reported in many articles, such as shape-induced directed differentiation
[1], cellular death via apoptosis
[2], necrosis
[3], gene transfection and transfer
[4], metabolism alteration
[5], and other processes. These effects arise from different surface areas, uptake level, protein corona, physical disruption of the cell membrane, and particles’ wettability and surface curvature
[6]. Moreover, the shape control of particle morphology demonstrates the importance of the ratio of the different crystal facet surface areas, which have a different dissolution rate in solution
[7].
The shape control of nanomaterials represents a major field in materials science, and a large number of approaches exist towards the production of sphere-
[8], ellipsoid-
[9], dumbbell-
[10], cube-
[11], polyhedra-
[12], rod-
[13], urchin-
[14], star-
[15], chain-
[16], ribbon-
[17], hollow
[18], prism-
[19], and hexagon-shaped
[20] nanomaterials. Nanomaterial shape control stems from effects such as selective adsorption growth of reactive facets; spontaneous aggregation and agglomeration; seeded growth on particularly-shaped templates; controllable crystal fusion via orientation attachment; self-assembly via selective strong interactions, e.g., chemical and hydrogen bonds; and Ostwald ripening directed at free surface energy minimization. Several synthetic approaches exist toward synthesizing nanomaterials with controlled morphologies, e.g., using polymeric additives, surface-capping surfactants, solvo/hydrothermal approaches, etc.
We focus on the mechanisms involved in nanomaterial shape control, synthetic approaches toward its production, and the influence of nanomaterial shape on various biological activities of mammalian and microbial cells, e.g., proliferation, differentiation, and metabolism, as well as reviewing the prospects of this emerging field.
2. Biological Effect and Application
2.1. Mammalian Cells
2.1.1. Cell Death
Apoptosis
Using nanoparticles to kill cells is one of the most common tasks. Nanoparticles are usually considered for use as drug carriers and heat or light conductors. However, even in the case of drug delivery and retention, the shape is of great importance
[75][21]. Nevertheless, nanoparticles themselves can cause cell death, and not because of their toxicity. The following are studies showing the difference in the cellular response due to the various nanoparticle shapes.
Gold nanoparticles were among the first to be used in medicine. Widely developed methods for controlling the shape of gold nanoparticles have made it possible to obtain various shapes and sizes. However, when considering gold nanoparticles, it is challenging to separate shape effects from the size or surface functionalization effects. Thus, one of the most common methods of shape control is using surfactants. In
[2], gold spheres, rods, and stars were compared. Anisotropic rod and star nanoparticles were obtained using CTAB, which is known for its cytotoxicity. The authors recorded a significant increase in the level of pro-apoptotic protein (Bax) and a decrease in that of anti-apoptotic protein (Bcl-2) for star and rod shapes. The authors also recorded the induction of autophagy in tested cells. In general, osteosarcoma MG-63 and 143B cells were more susceptible to nanoparticles than normal osteoblast hFOB 1.19 cells.
In most cases, increased toxicity (and subsequently, induction of cell death) is associated with “sharp” shapes
[76][22]. However, some authors report opposite results. For instance, in a work by Favi et al., commercially purchased citric-stabilized gold nanospheres were found to be more toxic than gold nanostars. In that work, a star-looking shape was obtained using HEPES buffer as a reducing agent. On the other hand, Enea et al. reporting that nanospheres and nanostars capped with 11-mercaptoundecanoic acid (MUA) had similar cytotoxic properties
[77][23]. In this case, the aspect of the shape fades into the background. However, in a work by Sultana et al.
[78][24], flower-like gold nanoparticles and gold nanospheres with the same PEG shell and size were found to have different uptake and cytotoxic profiles. Authors associate the increased toxicity of gold nanoflowers with high surface roughness, mediating uptake and disturbance of the cytoskeleton.
Mesoporous silica nanoparticles (MSNs) have long been used for drug delivery and theranostics applications
[79][25]. Little attention is usually paid to their shape since widespread protocols allow one to obtain mostly spherical particles. It was shown that silica nanorods with a length of about 450 nm (NLR450) are absorbed by cells much faster than rods with a length of 240 nm (NSR240) or spherical 100-nm particles (NS100)
[80][26]. Consequently, NLR450 caused a greater cytotoxic effect, disturbances in the cytoskeleton structure and an increase in the number of apoptotic cells by 10% compared to other tested shapes. The authors also noted the influence of nanoparticles with various shapes on the migration and adhesion of cells, which may be associated with a cytoskeleton disruption.
Another widespread material, titanium dioxide, was also found to cause cytoskeleton damage in a shape-dependent manner
[81][27]. Spherical TiO
2 particles were discovered to penetrate the blood–brain barrier, probably through the disturbance of F-actin fibers. However, in addition to the shape, these particles also had a different crystalline phase.
When mentioning the crystalline phase, it should be noted that some authors equate the effect of the shape of particles and their crystalline phase
[21][28]. It can be sometimes true; however, that the crystalline phase has more to do with surface morphology. For instance, TiO
2 is known to have two crystalline phases—anatase and rutile—in addition to an amorphous form. Anatase was shown to have pronounced cytotoxic effects that were associated with the density of defect sites
[82][29]. The anatase phase was also more toxic than rutile in a similar work, even though rutile particles were rod-like and sharp
[83][30]. This observation suggests the necessity of the careful consideration of shape-dependent effects, especially in the context of inorganic particles.
Another example, in which a dependence of one kind can be mistaken for a dependence of another kind, is given in the work of Xu et al.
[84][31]. Hydroxyapatite particles (HAP) with four different shapes were tested for general cytotoxicity manifestations (MTT assay), ALP activity, apoptosis assay (p53 and cytochrome C expression), and ROS generation. Almost all effects were in close correlation with particle-specific surface area but not with shape.
Usually, the shape of nanoparticles is discussed in the context of inorganic particles since a large number of approaches have been developed for them to create spatially anisotropic shapes. However, organic particles also have variations in shapes other than spherical. Biocompatible PLGA and PEG are some of the most common polymers used to create particles. However, it was shown that changing the shape of a particle from spherical to elongated leads to a significant increase in the particles’ cytotoxicity and induction of apoptosis
[85][32]. The primary mechanism is assumed to be the rupture of lysosomes, followed by the launch of a cascade through caspase-3 and DNA damage.
Another example of organic particles with shape-dependent cytotoxicity is poly(3,4-ethylenedioxythiophene) (PEDT) polymer
[86][33]. Oh et al. compared three types of PEDT nanomaterials with an average diameter of 55 nm. PEDT-1 was sphere-like, PEDT-2–rod-like, and PEDT-3 took the form of 1350 nm-long tubes. It was found that all samples caused a dose-dependent increase in LDH release in normal cells (IMR90 and J774A.1) by 5–50%. The ATP amount decreased in the same manner by 5–70%. The highest effect was observed for the smallest sphere-like particles. Furthermore, they caused a significant increase in the number of apoptotic cells (by 30%). However, the longest particles lead to considerable elevation of IL-1, IL-6, and TNF-α levels after 24 h of incubation.
Caspase-3 levels were elevated upon incubation of PC12 cells with graphene nanostructures of different dimensions
[87][34]. The authors compared graphene layers (G) and carbon nanotubes (SWCNT), showing that SWNCT leads to LDH leakage, generation of ROS, and subsequent induction of apoptosis. The sharp shape of SWCNT was listed as the main reason for increased cytotoxicity.
Aluminum salts have long been widely used as an adjuvant for vaccines, whereas aluminum oxide (alumina) was used as an adsorbent. At the same time, methods for the synthesis of alumina usually include hydrothermal and ultrasonic-assisted approaches, in which precise control of the shape is not always possible
[88][35]. Dong et al. managed to produce two types of alumina nanoparticles in the shapes of flakes and rods and studied them in detail using a metabolomics approach
[89][36]. The main focus of the work was the study of brain cells, including primary cultures, which is especially important in the context of the medical use of alumina. Rod-like particles have been shown to have greater uptake ability, connected with increased cytotoxicity in astrocytes. Apoptotic markers and pro-inflammatory cytokine levels were increased in a dose-dependent and shape-dependent manner (similarly to the cytotoxic results). A careful analysis of 66 metabolites showed that nanorods cause more significant metabolic changes (21 unique differential metabolites (DMs) vs. 15 for nanorods and nanoflakes, respectively). These DMs mainly included amino acids, lipids, and carbohydrates.
Necrosis
In some cases, the process of cell death upon interaction with nanomaterials can follow the necrosis pathway
[90][37]. From the therapeutic point of view, such an outcome is unfavorable, leading to inflammation
[91][38]. However, in the context of screening potential nanomaterials for biomedical applications, it is imperative to conduct additional research for signs of necrotic cell death.
One of the most frequently used approaches to distinguish apoptotic and necrotic pathways is staining with FITC-labeled annexin. Using this technique, Huang et al. made a distinction between four shapes of hydroxyapatite nanoparticles (HAP)
[3]. The authors found that cytotoxicity (and, similarly, the percentage of necrotic cells) rose in the order of plate > sphere > needle > rod. The difference between HAP plates and rods in the number of necrotic cells was almost twofold (17.13% vs. 9.67%, respectively). The impact of HAP on mitochondrial membrane potential and lysosome integrity followed the same shape-dependent trend. The authors concluded that the primary influence on cytotoxicity was exerted by the surface area, conductivity, and zeta potential of nanoparticles. However, cellular uptake of HAP was higher for the most toxic shapes—a fact that should be kept in mind.
The aggregation state of nanomaterials plays an essential role in cytotoxicity
[92][39]. Lee et al.
[93][40] showed that iron oxide rods that remain stable under physiological condition were more toxic than aggregated nanospheres. The number of necrotic cells was increased after incubation with rod-shaped particles by about 20% at a 200 μg/mL concentration. However, considering important observations connected with ROS generation and aggregation state, the straight correlation between shape and cytotoxicity is not clear.
Ferroptosis
In discussing the possible effect of nanoparticles on cell viability, the phenomenon of ferroptosis cannot be ignored. This form of cell death exists in many cancer cells and therefore attracts particular interest in cancer treatment
[94][41]. The main object of studies in nanotechnology has become iron-containing nanoparticles, since it is iron that participates in the Fenton reaction, leading to ferroptosis. The primary mechanism involves the release of iron ions Fe
2+ or Fe
3+ after the nanoparticles enter the lysosomes or an acidic environment. The ion release process is directly related to the surface area of the nanoparticles, which is sometimes associated with the shape. Smaller particle sizes are preferable since they tend to generate more iron ions. Ferumoxytol is used as a contrast for MRI
[95][42]. It is a stabilized magnetite (Fe
3O
4) nanoparticles with a size of about 50 nm
[96][43]. This size range is greatly suited for cellular uptake. It has been shown that ferumoxytol can cause the polarization of macrophages in cancerous tumors, reducing their growth
[97][44]. Tumor size was halved on day 21 in the ferumoxytol-treated group. To note, the polarization of macrophages plays an essential role in the treatment and diagnosis of cancer, which was the subject of our recent review
[97][44]. Amorphous forms of iron also cause Fe
2+ release and can be used to trigger the Fenton reaction
[98][45]. The advantage of the amorphous form over the crystalline one was that, under the action of an acidic environment, the release of ions from the amorphous form was significantly higher. In the first 6 h, the release from the amorphous form was 100%, whereas for the crystalline form, it was only 20%.
Combined strategies, such as drug loading, are successfully implemented along with iron-oxide nanoparticles
[99][46]. The delivery of hydrogen peroxide can be considered one of the relevant strategies
[100][47]. A complex structure, consisting of a PLGA shell incorporated with magnetite nanoparticles and loaded with H
2O
2, was developed. The release of H
2O
2 was carried out using ultrasound. Tumor size was reduced by eight times compared with the control group on the 22nd day of the experiment.
Non-iron-containing engineered nanomaterials have also been reported to cause ferroptosis. A relatively large amount of papers have been published recently on manganese oxide (MnO
2) particles
[101,102,103][48][49][50]. Though no shape-dependent activity has been reported, the investigated particles possessed developed surfaces (nanoflowers or nanobubbles). Another example is two-dimensional transition metal dichalcogenides. WS
2 and MoS
2 nanosheets were shown to cause ferroptosis in epithelial (BEAS-2B) and macrophage (THP-1) cells
[104][51]. However, cell death was connected with surface vacancy, not the 2D nature of the nanomaterials.
2.1.2. Disturbance of Cell Function
Alterations of cell proliferation are not always accompanied by cell death and should be considered separately. In this section, we describe and discuss the shape-assisted alteration of cellular metabolism, genotoxicity, mammalian cell differentiation, as well as the influence of external stimuli on these processes ().
Figure 31.
Different mechanisms of nanoparticle action on cells.