Though nanomaterials are very useful in many important applications, research has shown that cells and tumors do not respond to every type of nanoparticle in the same way. Nanoparticle variations such as size, shape, and surface chemistry make a difference in the basic mechanisms of cellular uptake and responses as well as their usefulness in different biological applications (Panariti et al. 2012). Understanding the way specific nanoparticles interact with specific cell types is crucial for determining the usefulness of the nanoparticles as well as their effects on cells and the environment. The following review sections illustrate the nature of nanoparticle cellular uptake as it relates to HER2-positive breast cancer. Here we can see that differences in particle and cell type results in differences in the nature of the cellular uptake and localization of the nanomaterials. This section also illustrates various techniques that can be used to study the cellular uptake process of different nanomaterials in HER2-positive breast cancer cells. The following studies involve polystyrene particles in different cell types, gold nanoparticles with differing shapes, iron oxide nanomaterials, gold particles with surface modifications, dextran–spermine nanoparticles, gold nanospheres, and dendrimers. The focus of this section is strictly to understand cellular uptake in the different cancer cell models.
Cellular uptake of polystyrene microparticles in different human breast cell lines
There are multiple examples of the work being done using polystyrene particles (Sutapa Barua 2020; Martínez-Jothar 2020), but the following work highlights an example of the work being done specifically in the area of cellular uptake as it relates to HER2-positive breast cancer. Patiño et al. (2015) examined the impact of nanoparticle surface modifications on uptake efficiency for various cell types, as well as the mechanism of internalization. Polystyrene microparticles were functionalized with a fluorescently active antibody. Two types of polyethylenimine (PEI) differing in structure and molecular weight were used to coat the particles, which were then studied with two types of human breast cell lines. This study revealed that cancer cells and normal cells use different types of endocytosis pathways when interacting with different surface modifications. The tumor cell SKBR-3 showed favorable uptake to positively charged microparticles using the macropinocytosis machinery. On the other hand, the non-tumor MCF-10A cells favored a negatively charged microparticle and did not seem to favor a dominant endocytosis mechanism.
In this example, we see differences in uptake based on cell type. The mechanisms used by the two cell types differ with the same type of nanoparticle. The SKBR-3 breast cancer cells responded in a different way than the non-tumor cells MCF-10A to the same polystyrene microparticles in terms of the cellular uptake process. This study was conducted using fluorescence microscopy techniques, which is a good way to study cellular uptake and mechanisms for non-metal nanomaterials. This is a good example of how all cells do not respond the same way to the same particle type. The next example examines the cellular uptake process of gold nanoparticles with different shapes in SKBR-3 cell lines.
Cellular uptake of gold nanoparticles of different shapes in breast cancer cell lines
Gold nanoparticles are very commonly studied in all areas of nanotechnology (Ghosh et al. 2008; Sperling et al. 2008; Dreaden 2012). In this example, we see how shape can influence cellular uptake behavior. Here, we also see how optical signatures of gold can also be used for studying cellular uptake of gold nanomaterials in breast cancer cells using UV–Vis and how this method compares to the more common Inductively Coupled Plasma Mass Spectrometry method or ICP-MS. Cho et al. (2010) introduced a method for studying the cellular uptake of gold nanoparticles with two different shapes using SKBR-3 cells. The authors showed that due to the different shapes of gold nanospheres and nanorods, their distinctive optical signatures could be exploited to measure their individual concentrations in a single sample with UV–Vis absorbance spectroscopy. Using this method, the study investigated the role of shape in the cellular uptake process. This method was compared to inductively coupled plasma mass spectrometry and proved to be useful for quantifying the uptake of nanoparticles in HER2-positive breast cancer cells. Interestingly, the results showed differences in the number of particles taken up by the cells depending on nanoparticle shape and whether they were added together or independently. Nanoparticle uptake was also dependent on the surface modification of the gold particle, e.g., polyethylene glycol (PEG) and targeting antibody anti-HER2. Not only do we see that shape makes a difference in gold particle cellular uptake, we also see here that surface coating such as PEG influences this process as well. The next example in this section looks at iron oxide nanoparticle uptake in different cell types.
Cellular uptake of magnetic iron oxide nanoparticles in human breast cell lines
Truffi et al. (2018) examined the cellular uptake process of SKBR-3 and MDA-MB-453 cell lines. Fluorescently labeled particles targeting HER2 (half chains of trastuzumab) conjugated to magnetic iron oxide nanoparticles (MNP-HC) were used to assess the mechanism of cellular uptake as well as the cellular localization. The authors showed that in the first hour, these nanoparticles accumulated at the cell membrane, but much faster in the HER2-overexpressing cell line SKBR-3. The signal was detected in the cytoplasm at 4 and 24 h of incubation, and at 48 h, the signal decreased, indicating a reduced rate of cell membrane interaction. In addition, the intracellular trafficking of the iron oxide core was evaluated using transmission electron microscopy (TEM). In SKBR-3 cells, nanocrystals were detected outside the cells and attached to the cell membrane after 1 h of incubation. After 4 and 24 h of incubation, nanoparticles were seen in endosomal vesicles and later in lysosomes, suggesting an endocytosis pathway (Fig. 2).
Here, we see a few different methods for studying cellular uptake, trafficking, and localization of iron oxide nanoparticles in two different breast cell types. Iron oxide nanoparticles can easily be labeled with fluorescence and tracked using fluorescent microscopy techniques. In addition, transmission electron microscopy or TEM is an excellent way to study the internalization and localization of nanomaterials inside cells. The authors show that iron oxide particles follow the endocytic pathway in a time dependent manner for internalization in both breast cancer cell types (SKBR-3 and MDA-MB-453) using a combination of these techniques. While many works have been published in work using iron oxide (Shakil et al. 2019) the example reviewed here was based on the details found on the cellular uptake process, which many published works may not cover.
The next section deals with aspects of cellular uptake of surface modified gold nanorods.
Cellular uptake of conjugated gold nanorods in breast cancer cells
Previously we saw that the shape of a gold nanoparticle has an effect on cellular uptake. In this study, we see how modifying the surface of gold nanorods for targeting plays a role in the cellular uptake process. We also see another example of how cell type influences this process as well and the use of TEM to study these processes. Kang et al. (2017) studied some aspects of cellular uptake of gold nanorods conjugated with porphyrin and trastuzumab using BT474 and SKBR-3 breast cancer cell lines. The cells were incubated with the conjugated nanorods for 48 h before TEM was performed. In BT474 cells, TEM showed that the conjugated gold nanorods accumulated in gaps between closely growing cells and inside the cytoplasm of the cell. Further analysis using higher magnification identified multivesicular bodies encapsulating the nanorods. Endocytic bodies with nanorods were also observed in the nucleus of the BT474 cells. In SKBR-3 cells, a few nanoparticles were observed, which led the researchers to conclude that greater uptake occurs in BT474 than SKBR-3. This was mainly due to the conjugated gold nanorods localizing on the surface of the BT474 cells, which resulted in more efficient cellular uptake.
In this example, we see how surface modification can affect the mechanics of cellular uptake. It is also important to note that the difference in cell type is very much a factor to consider as well. This is an excellent example of how small variations can influence the mechanism of cellular uptake and how important it is to investigate the cellular uptake behavior in each and every model.
In the next example of this section, we look at the cellular uptake behavior of dextran–spermine nanoparticles that have been modified for targeting.
Cellular uptake of magnetic dextran–spermine nanoparticles functionalized for targeting HER2
Cellular uptake changes with particle type, cell type, surface modification, etc. Here we see how SKBR-3 breast cancer cells respond to magnetic dextran–spermine nanoparticles. We also see another technique for studying cellular uptake, which is the Prussian blue method. Avazzadeh et al. (2017) conducted a cellular uptake assay with HER2-positive breast cancer cells using magnetic dextran–spermine nanoparticles. The nanoparticles were conjugated with anti-HER2 antibody, and the cellular uptake and targeting process was evaluated using the Prussian blue method and microscopy techniques with and without the targeting antibody. The results showed differences in cellular uptake between the SKBR-3 and fibroblast cells. A large number of antibody-conjugated nanoparticles entered the SKBR-3 cells, but none were observed in the control fibroblast cells, and no differences were seen in cellular uptake with the non-targeting nanoparticles. This study illustrates the significance of using a targeting antibody to help facilitate the cellular uptake of nanoparticles. This example highlights the nature of this specific particle type. It also shows the effect of conjugating a targeting antibody, which is discussed more in the section on targeting HER2 breast cancer.
In the next example, we describe gold particles that are spherical and look at how shape as well as size affects the cellular uptake process.
Cellular uptake of HER2 targeting gold nanospheres in breast cancer cell lines
Cruz and Kayser (2019) analyzed cellular uptake of targeting gold nanospheres using a combination of ICP-MS and TEM. Trastuzumab was conjugated to gold nanospheres, and the uptake quantification and localization in SKBR-3 cells was compared to non-trastuzumab gold nanospheres. Two sizes of nanoparticles, 20 nm and 50 nm, were tested. The results found that both sizes of trastuzumab-targeting nanoparticles had enhanced cellular uptake compared to the non-targeting nanoparticles, but in both cases, cellular uptake did occur. TEM analysis showed that these particles localized in vesicular structures inside the cells (Fig. 3).
As shown in Fig. 3a, based on quantitative ICP-MS data, it is clear that there is a differential uptake of the particles based on their size. Larger particles (50 nm) show more uptake than smaller particles (20 nm) without targeting antibodies. However, both 20 nm and 50 nm particles show almost similar fold increase in particle uptake following targeted antibody conjugation as compared with the respective particles without the targeting antibody. This study shows how the size of the nanoparticles has an effect on both accumulation and localization in this particular cell type. Here we have the same cell type, same targeting mechanism, and same shape particle, but the size of the particles being used differ and have a different cellular uptake result. This study also shows how TEM and ICP-MS used together serves as a great technique for studying gold nanoparticles in breast cancer cells.
Lastly, we review a nano-design involving drug-loaded dendrimers for its cellular uptake behavior.
Cellular uptake of targeting and drug-loaded dendrimers in HER2-positive BC cell lines
Dendrimeric nanoparticles have also been used to target HER2-positive breast cancer cells (Miyano 2020; Chan 2020; Rameshwer Shukla 2020) However, we found the study by Kulhari et al. (2016) significant and worth reviewing in this section due to the focus on cellular uptake. Kulhari et al. (2016) studied the cellular uptake of trastuzumab-grafted, docetaxel-loaded dendrimers conjugated to fluorescein isothiocyanate (TZ–Dend–FITC). The authors used the HER2-positive breast cancer cell line MDA-MB-453 and compared it with the HER2-negative MDA-MB-231 breast cancer cell line. Combinations of targeting and non-targeting nanoparticles were used for comparison of uptake efficiency. Uptake was observed in both MDA-MB-453 (HER2-positive) and MDA-MB-231 (HER2-negative) cells. In the HER2-positive cell line, both targeting and non-targeting nanoparticles showed fluorescence after 1 h of incubation. The intensity of the fluorescence was higher in the targeting nanoparticles, indicating more uptake occurred due to the targeting mechanism. The results indicated that cellular uptake of the non-targeting nanoparticles was 11.4%, compared to 23.5% for the HER2-targeting nanoparticles. Results also indicated that the cellular uptake process is time dependent. After 4 h of incubation, cellular uptake increased for both particle types, but the targeted nanoparticles still showed higher uptake efficiency. In the non-HER2-positive cells, no difference in cellular uptake efficiency was observed between the targeted and non-targeted nanoparticles. However, in a mixed culture of the two cell types, selective targeting was observed. The mixed cell culture assay showed inhibition of uptake by the HER2-negative cell line and selective uptake by the HER2-positive cell line (Fig. 4).
The studies above show that not all cell and nanoparticle combinations result in the same type of cell/particle interaction. Changes in the model—e.g., cell type, particle type, surface modification, functionalization for targeting, etc.—can result in changes in cellular uptake mechanics as cells interact with different particle sizes and surfaces differently. This suggests that with every model the cellular uptake process must be evaluated. Nanoparticle cellular uptake cannot be generally defined due to the changes seen throughout the various models. For example, we see that a cell type may employ receptor mediated endocytosis for gold nanoparticles while another cell type uses macropinocytosis for the same type of particle. In some cases, we see more quantity being taken up due to size in a certain cell type, etc. Another thing that is important to consider is the behavior of the particles themselves in terms of clumping and aggregation affecting the size and how cells or tumors may respond differently as a result. In this section, we have also seen that a wide range of techniques are available for studying the cellular uptake process. For gold nanoparticle analysis, we see that TEM and ICP-MS work very nicely when used in combination to study internalization, quantification, and localization of nanomaterials in breast cancer cells. For non-metal nanomaterials fluorescent labeling and TEM work well to study these processes.
In the next section, we look at some of the research being conducted in the area of screening and detecting HER2-positive breast cancer using nanotechnology.