Introduction

Nanoparticles (NPs) are a subcategory of nanomaterials that are currently at the forefront of cutting-edge research in nearly every field imaginable due to its unique properties and tremendous applicability [74].

Clathrin- and caveolae-independent endocytosis occurs in cells that are deprived of clathrin and caveolin. This pathway is utilized by growth hormones, extracellular fluid, glycosylphosphatidylinositol (GPI)-linked proteins and interleukin-2 to enter the cells. For instance, folic acid that employs clathrin- and caveolae-independent pathway to enter the cells [58, 72, 75,76,77,78,79] are conjugated to NPs and polymers used in drug delivery systems and as imaging agents [53, 80, 81]. Macropinocytosis is a type of pinocytosis mechanism in which cells take in high volumes of extracellular fluid by forming a large vesicle (0.5–10 μm) called macropinosomes [82,83,84,85]. Macropinocytosis is a pathway to internalize apoptotic and necrotic cells, bacteria and viruses as well as antigen presentation. This pathway can internalize micron-sized NPs which are not possible to be taken into cells by most other pathways. Macropinocytosis can occur in almost any cells except for brain microvessel endothelial cells [86,87,88,89]. NPs enter into the cell via one of these endocytotic routes as depicted in Fig. 1.

Fig. 1
figure 1

Entry of NPs into cell using different endocytotic pathways. a Macropinocytosis and phagocytosis. b Clathrin-mediated endocytosis, clathrin-caveolin independent endocytosis and caveolae-mediated endocytosis

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Effect of Physicochemical Properties of NP on Cellular Uptake

Studying the effect of physicochemical properties of NPs such as size, shape, surface charge, surface hydrophobicity/hydrophilicity and surface functionalization on cellular uptake is crucial as these parameters directly affect the uptake level, endocytotic route as well as cytotoxicity of NPs. [90, 91]. Physicochemical factors that affect the cellular uptake of NPs are illustrated in Fig. 2. In the following section, the impact of these parameters on cell–NP interactions are discussed.

Fig. 2
figure 2

Physicochemical factors that affect cellular uptake of NP. a Surface charge, b shape, c size and d surface chemistry

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Effect of Size

Size of NP is a key factor in determining the efficiency of cellular uptake [99]. Additionally, NPs ranging in the size of 30–50 nm interacts efficiently with CM receptors and is subsequently internalized via receptor-mediated endocytosis [97]. In drug delivery application of NPs, the main concern is to prevent the NPs from being eliminated by the reticuloendothelial system and to prolong its circulation time in the blood, thus enhancing the bioavailability at the target. In this regard, increasing the size of NPs will lead to an increase in the clearance rate [100,101,102,103,104,105]. Therefore, understanding the role of NP size in cellular uptake is crucial to design effective and safe NPs for medical applications.

Though different studies have investigated the relationship between size of NP and uptake pathways, the revealed results have always been inconsistent [93, 106,107,108,109]. These contradictions can be related to the complexity of controlling other parameters of NP during the process of controlling size. In addition to that, sizes of NPs measured after synthesis may undergo changes during the in vitro and in vivo studies due to agglomeration and aggregation which in turn could affect the cellular internalization pathways [110, 111]. The impact of particle size on cellular uptake pathway in non-phagocytic B16 cells was investigated by employing different sizes of fluorescent latex beads in the range of 50–1000 nm [93]. The results have demonstrated that the internalization mechanism of these beads relies significantly on the particle size. In particular, beads with sizes of 200 nm or less were taken up by clathrin-coated pits whereas larger beads were internalized by caveolae-mediated endocytosis. Lai and co-workers [16] have found that small polymeric NPs with sizes less than 25 nm employs a new mechanism to reach the perinuclear region of the cells via non-degradative vesicle outside the endo/lysosomal pathway. This pathway is non-clathrin and non-caveolae-mediated and cholesterol-independent.

The uptake of gold (Au) NPs of different sizes (2 to 100 nm) conjugated with Herceptin-AuNPs by SK-BR-3 cells was shown to be size dependent. The highest cellular internalization was observed for NPs in the size ranges of 25–50 nm [63]. In this entry route, the size of NP was found to be the determinant in the binding and activation of membrane receptors and the eventual expression of the proteins. The effect of variation in the size and shape of colloidal AuNPs on the intracellular uptake was assessed [112]. AuNPs of 14-, 50- and 74-nm size with spherical and rod shape were incubated with HeLa cells. It was found that the NP uptake strongly depends on its size and shape and those particles with 50 nm size showed the highest uptake rate. Moreover, the uptake of spherical AuNPs was 500% more than rod-shaped NPs of similar size. Shan et al. [113] investigated the size-dependent force of endocytosing AuNPs with diameters of 4, 12 and 17 nm by HeLa cells. The results revealed that both the uptake and unbinding force values increase by the size of AuNPs. The uptake of SiO2 NPs of different sizes (50, 100 and 300 nm) by A549 cells (lung epithelial cells) has studied by means of combination of flow cytometry, fluorescence and electron microscopies. These researchers had shown that the uptake of SiO2 NPs has decreased by size [114].

Effect of Shape

In addition to size, the shape of the NP also plays a pivotal role in the uptake pathway as well as trafficking of NPs. Chithrani et al. [112] studied the effect of the shape of colloidal AuNPs on the uptake of HeLa cells. The result revealed that spherical AuNPs had five-fold higher uptake than rod-shaped AuNPs. In another work, same researchers investigated the uptake level of spherical and rod-shaped transferrin-coated AuNP on three different cell lines; STO cells, HeLa cells and SNB19 cells [94]. They observed that spherical AuNPs were internalized by all the cell lines at a higher rate than rod-shaped AuNPs.

In order to establish the effect of shape in vivo, Geng and coworkers [115] employed filomicelles to evaluate the differences in transport and trafficking of flexible filaments with spheres in rodents. The results revealed that filomicelles remained in the circulation about ten times more than spherical counterparts. Moreover, the sphere filomicelles are internalized by the cells more readily than longer filaments. Gratton and co-workers [106] demonstrated the effect of the shape of monodisperse hydrogel particles on uptake into HeLa cells. They have found that rod-like-shaped NPs had the highest internalization rates compared to spheres, cylinders and cubes. In another study, the impact of the shape of NPs on cell uptake was investigated by employing disc-shaped, spherical and rod-shaped polystyrene (PS) NPs on Caco-2 cells. The result demonstrated that the rod and disc-shaped NPs were internalized twofold higher than spherical NPs. They concluded that NP-mediated drug delivery can be advanced by considering the shape of NPs [116].

Xu and co-workers [117] studied the impact of shape on cellular uptake by preparing layered double hydroxide (LDH) NPs with fluorescein isothiocyanate (FITC) in different morphology such as hexagonal sheets (50–150 nm laterally wide and 10–20 nm thick) and rods (30–60 nm wide and 100–200 nm long). All morphologies were taken up via clathrin-mediated endocytosis. LDH-FITC nanosphere were retained in the cytoplasm, whereas LDH-FITC nanorods were moved towards the nucleus by microtubules. Dasgupta et al. applied [118] a simulation to probe the role of the shape of NPs on cellular uptake. They have simulated membrane wrap** of the nanorod- and nanocube-shaped NPs. For rod-like particles, they found stable endocytotic states with small and high wrap** fraction; increment in aspect ratio was undesirable for complete wrap**. Nangia and Sureshkumar [119] have computerized the effect of shape on the translocation rate of NPs by applying advanced molecular dynamics simulation techniques. A major revelation of the study is the significant variation in the translocation rate of cone-, cube-, rod-, rice-, pyramid- and sphere-shaped NPs.

Effect of Surface Charge

Another critical factor which influences cellular uptake of NPs is surface charge. In the recent decade, nano surface modification has been employed to engineer the surface charge of NPs to be either cationic or anionic [139].

QDNPs interactions with mixed lipid/polymer membranes were assessed by changing the hydrophobicity surface of NPs. It was observed that hydrophobic NPs have located within the polymer domains in a mixed lipid/polymer monolayer of the membranes, whereas hydrophilic QDNPs adsorbed onto the monolayers and spread throughout, indicating higher effect on the molecule packing at the air/water interface [140]. Incorporation of functionalized AuNPs with mixed hydrophobic and hydrophilic ligands into liposome walls was studied. The result demonstrated that hydrophobic ligands interact with the hydrophobic core of the bilayer, while hydrophilic ligands interact with the aqueous solution [141].

Effect of Surface Modification

In biomedical applications of NPs, surface chemical modification of NP is a critical step utilized to decreases toxicity, increase stability and to control and modulate cellular internalization of NPs, hence their biological fate [142]. Surface functionalization of NPs predominantly comprises of PEG, the negative carboxyl (–COOH) group, neutral functional groups like hydroxyl (–OH) groups, and the positive amine (–NH2) group. The increment in the amount of (–NH2) lead to an enhanced positive surface charge, and hence raise the uptake of NPs into cells [143,144,145,146]. Similarly, –COOH functional groups increase the negative charge of NPs and accordingly enhance its uptake [144].

Tao et al. [147] have designed polydopamine functionalized NP-aptamer bioconjugate for tumour targeting. They have reported that the functionalized NPs have better targeting efficacy compared to non-functionalized NPs, indicating higher cellular uptake rates for functionalized NPs which translates into enhanced therapeutic effect. In another research, folic acid-functionalized NPs demonstrated higher efficacy in the targeting of cervical cancer cells than non-functionalized NPs [148]. The impact of surface coating on toxicity and cellular uptake of AuNPs were studied by Qiu and co-workers [90]. They have revealed that surface coating is a key factor in determining the cellular uptake rate since poly (diallyldimethyl ammonium chloride)-coated AuNRs showed a higher efficiency in internalization by the cells.

The differences in the cellular uptake of pristine polystyrene (PS-NPs) and amino-functionalized polystyrene NPs were investigated by Jiang and co-workers [149]. The results have demonstrated that amino-functionalized polystyrene NPs have a higher uptake rate than PS-NPs, and the former were internalized mainly via clathrin-mediated pathway and the latter via clathrin-independent endocytosis. This remarkable difference highlights the key role of surface chemical modification in cellular interactions with NPs. Surface-modified fullerene, C60(C(COOH)2)2 NPs were internalized by the cells predominantly via endocytosis in a time-, temperature- and energy-dependent manner. Clathrin-mediated endocytosis was found to be the preferred pathway for the internalization of C60(C(COOH)2)2 NPs [150].

Effect of Elasticity

The elasticity of NPs plays is an intrinsic factor in influencing its internalization by cells. The elasticity of NPs can be explained by its resistance to changes when forces are applied on it. Stiffness, hardness and rigidity are some of the terms that are synonymous in describing the elasticity of NPs. A measurement index that is being used to gauge the elasticity of NPs is Young’s modulus and the unit of measurement is Pascal (Pa). Based on this measurement, a higher Young’s modulus value denotes higher NPs elasticity and vice versa. Examples of the analytical devices or instruments that are used to measure this value on NPs are atomic force microscope, rheometer and nanoindenter. NPs that have higher elastic values are called hard NPs and examples of these are gold NPs, quantum dots and magnetic NPs. NPs that have lower elastic values are called soft NPs and examples of these are hydrogels, liposomes and biodegradable polymers.

Numerous studies that have focused on this parameter of NP with respect to cellular uptake have reported on the preference of cells to internalize stiffer NPs more efficiently compared to softer NPs [151, 152]. Evidently, this observation is attributed to lesser overall energy expenditure by membranes in wrap** stiffer NPs compared to softer NPs even though the deformational energy required to wrap the NPs varies throughout the internalization process. Furthermore, computational modelling of membrane wrap** of NPs with varying elasticity conducted using coarse-grained molecular dynamics (CGMD) simulation concurs with the experimental observation regarding deformational energy changes involved in internalizing stiff and soft NPs [153]. However, there are also other studies that have reported on softer NPs being internalized more efficiently than stiffer NPs [154, 155] and intermediate elastic NPs internalized more efficiently compared to either stiff or soft NPs [156]. Hence, tuning the elasticity of NPs for better cellular internalization could be a valuable tool in biomedical applications such as drug delivery. A potential application was demonstrated by Guo and coworkers, whereby accumulation of nanolipogels in tumour cells were enhanced primarily by controlling this parameter of NP [157].

Intracellular Trafficking of NPs

In the previous sections, different possible uptake pathways of NPs and the parameters that affect the efficacy of uptake has been discussed. Following uptake, the next crucial matter is the intracellular trafficking of NPs which determines its final destination within cellular compartments, its cytotoxicity and its therapeutic efficacy [158, 159]. After NPs are internalized by the cells, they will first encounter membrane-bound intracellular vesicles called early endosomes. Endosomes formed at the plasma membrane are categorized into three types; early endosomes, late endosomes and recycling endosomes [106, 160,161,162,163].

Early endosome ferries the cargo to the desired cellular destination. Part of the cargo is recycled to the plasma membrane via recycling endosomes. Early endosomes transform into late endosomes via maturation and differentiation process. The late endosomes will then integrate with lysosomes to form endolysosomal vesicles and hydrolytic enzymes contained within these vesicles degrade the trapped NPs [18, 164,165,166]. However, some NPs are able to escape this pathway and are released into the cytoplasm therefore bypassing the lysosomal degradation process [167,168,169]. Another intracellular degradation pathway which plays important role in the intracellular fate of NPs is an intracellular process called autophagy [170,171,172]. In this process, cytoplasmic contents will be surrounded by autophagosome and delivered to the lysosome to be broken down and recycled [173]. In addition, aggregated proteins and dysfunctional organelles are degraded by autophagy to maintain cellular homeostasis. It is necessary to consider this pathway since recent studies demonstrated that several NPs are capable of inducing autophagy [174,175,176,177,178].

The intracellular trafficking of Tat peptide-conjugated quantum dots (Tat-QDs) in live cells was studied by Ruan and co-workers [179]. Dynamic confocal imaging showed that Tat-QDs interacted with negatively charged CMs leading to its internalization by macropinocytosis. The QD containing vesicles were observed to be actively transported by molecular motors towards the perinuclear region known as the microtubule-organizing center (MTOC). Tat-QDs bind to cellular membrane structures such as filopodia and vesicle shedding results in releasing QD-containing vesicles from the tips of filopodia.

The uptake and intracellular fate of fluorescent carboxylated polystyrene particles (20 nm and 200 nm in diameter) were evaluated by applying it on hepatocyte [180]. It was found that the particles were internalized by hepatocytes in size, time and serum-dependent manner. The fate of the particles was studied and they were not observed in early endosomes or lysosomes, but only in the mitochondria of the hepatocyte. Particles accumulated inside bile canaliculi show that NPs can be eliminated within bile. A study on the uptake and intracellular fate of silver NPs into human mesenchymal stem cells demonstrated that they agglomerate in the perinuclear region [181]. It was observed by using fluorescent probes that particles are contained within endo-lysosomal structures but not in the cell nucleus, endoplasmic reticulum or Golgi complex. Confocal imaging of FITC conjugated titania nanotubes in mouse neural stem cells revealed that they have crossed the karyotheca entering the cell nucleus [182]. Single-walled carbon nanotubes were observed to enter the cytoplasm and localize in the cell nucleus leading to cell mortality [183]. Translocation of AuNRs towards the nucleus has also been reported [184].

Conclusions

The application of NPs in the modern world is growing at an exponential rate as the scientific enterprise is looking for novel ways to address current problems. NPs can be found as active ingredients in many formulations intended for human consumption, from cosmetics to processed foods. As its application increases in consumer products, so does human exposure to NPs. Hence, more research should be carried out to understand its potential hazards to humans and other living beings. In this review, we have looked at the current knowledge on the effects of NPs at a cellular level. Some of the topics discussed include cellular pathways of NPs and the influences of physiochemical properties of NPs on the uptake rate and uptake mechanism.