Keywords

1 Introduction

There have been numerous attempts to develop nanocarriers, which can not only improve drug solubilisation and delivery to different eukarocytic, but also target the desired intracellular compartments (Treuel et al. 2013). Several cellular barriers, however, need to be crossed before nanoparticles can reach their designated intracellular targets. Accordingly, a carrier may be designed to only enter specific cells in relevant tissues and to enter different cells through a specific endocytic pathway (Wang 2012). Likewise, intracellular transport mechanism of various nanocarriers have also received considerable attention (Treuel et al. 2013; Sakhrani and Padh 2013).

Various methods have been developed to study the mechanisms by which nanocarriers are internalized by cells of different origin and to follow their intracellular trafficking (Vercauteren et al. 2012). Here we describe the most studied endocytic pathways and discuss some of the major barriers for uptake and trafficking of nanocarriers. Advantages and disadvantages of commonly used methods for nanocarrier trafficking studies are also discussed.

2 Endocytosis

For nanocarriers to deliver and release their cargo at an intracellular target site the carriers need to enter the cell by crossing the plasma membrane. Most nanoparticles are believed to be internalized by endocytosis (Canton et al. 2012). Endocytosis is an energy-dependent process where particles are internalized in small vesicles. The mostly studied endocytic pathways are clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis, but more pathways have been identified that includes clathrin- and caveolae-independent endocytosis and phagocytosis (Doherty and McMahon 2009) (Fig. 1).

Fig. 1
figure 1

Schematic overview of intracellular trafficking pathways

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2.1 Clathrin-Mediated Endocytosis

Clathrin-mediated endocytosis (CME) is initiated at clathrin-coated pits in plasma membrane (Maxfield et al. 2004). Once the vesicle is formed in a dynamin-dependent manner (Hinshaw 2000), it is uncoated and followed by fusion with or maturation into early or recycling endosomes (Lemmon 2001; Ma et al. 2002; Maxfield et al. 2004). Clathrin is important for the initiation of invaginations at the membrane level and for the formation of the endocytic vesicles. Other assembly proteins are also involved in the formation of invaginations (Brodsky et al. 2001; Kirchhausen 1999).

The clathrin coated vesicles have been demonstrated to be of various sizes ranging from 50 to 300 nm. Particles reported to be internalized by CME are generally up to 200 nm in diameter (Ehrlich et al. 2004). However, some studies have indicated that larger particles may also be taken up by CME (Moreno-Ruiz et al. 2009; Veiga et al. 2005). CME, however, is important when it comes to cellular uptake and sorting of nutrients, plasma membrane proteins and lipids (Conner et al. 2003).

2.2 Caveolae-Mediated Endocytosis

Caveolae-mediated endocytosis initiates from cholesterol-rich areas on the plasma membrane (Khalil et al. 2006a). Formation of the invaginations is dynamin- and actin-dependent and the vesicles formed are reported to be in 60–80 nm size range (Canton et al. 2012; Hinshaw 2000; Parton et al. 2007). Theses vesicles are coated primarily by caveolin-1, which is responsible for the structure of the endocytic vesicle, and caveolin-2. However, the role of the latter is still not clear (Canton et al. 2012). The endocytic vesicles have been reported to be transported either to the caveosome or to the early endosomes (Parton et al. 2003; Pelkmans et al. 2001). Nanocarriers internalized by caveolae-mediated endocytosis are reported to be transported to endoplasmic reticulum and Golgi apparatus and some nanocarriers are also detected in the nucleus (Harris et al. 2002; Luetterforst et al. 1999; Pelkmans et al. 2001). For gene delivery vectors, caveolae-mediated endocytosis is the preferred route of internalization for efficient delivery and transcription of the exogenous DNA (Gabrielson et al. 2009; van der Aa et al. 2007) as this may substantially avoid lysosomal routing and subsequent lysosomal degradation of the nucleic acids (Harris et al. 2002).

2.3 Macropinocytosis

Macropinocytosis is an actin-dependent membrane ruffling, which results in the formation of large endocytic vesicles known as macropinosomes (Kerr et al. 2009). Macropinocytosis plays an important role in cellular uptake of fluids (Conner et al. 2003). The reported size of the macropinosomes varies, but have been demonstrated to be up to several micrometres, which is much larger than vesicles from other endocytic pathways (Jones 2007). The size of the formed vesicles further allows an opportunity for internalization of larger particles that cannot enter via clathrin- and caveolae-mediated endocytosis. Indeed, some pathogens use macropinocytosis to facilitate entry to different cells (Mercer et al. 2009).

2.4 Factors Influencing the Endocytic Pathway

Several factors have been reported to influence nanoparticle endocytosis. These include nanoparticle physicochemical properties such as size, shape, surface charge and ligand coating (Canton et al. 2012; Zhao et al. 2011).

A study by Rejman et al. (2004) demonstrated that while uptake of particles up to 200 nm was dependent on microtubule, their corresponding larger particles (500 nm) were not. This study further indicated that smaller particles where mainly internalized by CME, whereas the larger particles entered cells through cholesterol-dependent endocytosis and to a lesser extent were routed to the endo-lysosomal pathway (Rejman et al. 2004). However, the literature is not consistent in correlating nanoparticle size with internalization pathway. For instance, some reports have indicated that particles up to 500 nm can be internalized by caveolae-mediated endocytosis (Georgieva et al. 2011; Rejman et al. 2004), while others report that only particles up to 100 nm can be taken up by this route (Wang et al. 2009). These discrepancies may be due to different experimental setups, nanoparticle surface properties and the cell types examined.

Although the size of the nanoparticles can influence the endocytic pathway, many particles are not uniform in size and have a broad size distribution, which might render them to enter cells by different endocytic pathways, thus complicating the analysis of internalization routes. Others have reported that the shape of the nanoparticles determines the endocytic pathway for internalization (Gratton et al. 2008).

The uptake of particulate carriers of various sizes is also dependent on the cell type (Massignani et al. 2009; Nakai et al. 2003). Zauner et al. (2001) presented a study where they investigated the cellular uptake of microsphere of various sizes by different cell lines. Not all the cell lines were capable of internalizing particles above 1 µm, while all the tested cell lines could take up particles of 20 nm (Zauner et al. 2001). Furthermore, the net surface charge of the particles has also been suggested to influence the internalization pathway. Charged polystyrene or gold particles are reported to internalize to a higher degree than electrically neutral particles (Thorek et al. 2008; Villanueva et al. 2009). Also positively charged particles have been reported to be internalized easier than their corresponding anionic counterparts (Chen et al. 2011). This has been suggested to be due to ionic interaction between the positively charged particle and the negatively charged plasma membrane. In addition to the extent of the particle uptake, the surface charge of the particles may further regulate the pathway of internalisation (Harush-Frenkel et al. 2008; Zuidam et al. 2000). It has been demonstrated that dendrimers coupled to different functional groups enter the cell by different pathways dependent on the functional group. Dendrimers with amine or hydroxyl functional groups enter cells through both CME and caveolae-independent pathways, while dendrimers with functional carboxyl group mainly enter the cells via caveolae-mediated endocytosis (Perumal et al. 2008).

Georgieva et al. (2011) reported that 500 nm nanoparticles coated with either PEI or prion proteins were internalized through different endocytic pathways compared with uncoated particles. The coating of the particles also had an effect on the extent of particles that were co-localizing with recycling endosomes (Georgieva et al. 2011). The percentage of particles removed from cells by exocytosis may also dependent on the size of the particles. For example, the fraction of small 14 nm gold nanoparticles exocytosed was higher than gold particles with a diameter of 100 nm. Even though the percentage of particles exocytosed was reported to be cell-type dependent, the trend is the same and a higher fraction of the small particles being exocytosed (Chithrani et al. 2007). Not only the coating, but also the density of the molecules used for coating can play an important role in determining the endocytic pathway. Coating of liposomes with high density of the cell penetrating peptide R8, shifted the endocytic pathway towards macropinocytosis compared with liposomes coated with low density of the peptide, which were taken up by CME (Khalil et al. 2006b). A study by Li et al. (2011) indicated that PEI/DNA polyplexes are internalized by both clathrin- and caveolae-mediated endocytosis. However, when PEI was chemically modified with the natural polymer cyclodextran, the uptake mechanism was shifted toward caveolae-mediated endocytosis in HEK293T cells, thus demonstrating the role of charge and composition of the carrier in modulating the internalization route (Li et al. 2011).

3 Methods for Identifying Endocytic Pathways

Two methods are commonly used to study the pathways of nanocarriers internalization by cells. These include exclusion methodologies as well as co-localization studies, where overlap between fluorescently labelled particles and fluorescently labelled proteins are investigated by qualitative fluorescent microscopy.

3.1 Exclusion Methodologies

For exclusion determination, the nanocarrier should be fluorescently labelled so the uptake can be measured by flow cytometry or quantitative microscopy (Vercauteren et al. 2012). Several methods to exclude endocytic pathways have been demonstrated. For instance, siRNA can be used to decrease the expression of proteins required for the specific endocytic pathway in question (Zaki and Tirelli 2010). The use of siRNA may induce less cytotoxicity and be more specific than alternative methods used for the exclusion assays (Spoden et al. 2008). Caveolin-1 and dynamin-2 are among the successful proteins that have been down-regulated with siRNA (Huang et al. 2004). However, knockdown of proteins required for certain endocytic pathways by siRNA technology, may lead to up-regulation of some of the other endocytic pathways to compensate (Damke et al. 1995).

The most commonly used form of exclusion assays entail chemically inhibiting specific endocytic pathways. Table 1 lists some of the most commonly used inhibitors. The endocytosis inhibitors do not show exclusive specificity and many of the inhibitors of CME have been reported to cause reorganization of the actin skeleton (Ivanov 2008). Changes in the cytoskeleton might also affect other endocytic pathways, which are reported to be actin-dependent. Especially hypertonic sucrose as inhibitor for CME has been reported to be non-specific (Ivanov 2008). Also one of the mostly used inhibitors for caveolae-mediated endocytosis, methyl-β-cyclodextrin, which causes acute depletion of cholesterol, has been reported to significantly affect the cytoskeleton and also to inhibit CME and macropinocytosis (Kanzaki et al. 2002; Rodal et al. 1999). Inhibitors of macropinocytosis are also non-specific (Ivanov 2008). Even though, it is difficult to inhibit one endocytic pathway without affecting other pathways, the inhibitors are widely useful when studying internalization of nanocarriers.

Table 1 Chemical inhibitors commonly used for exclusion assays
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Some of the chemical inhibitors have been reported to induce cell type-dependent cytotoxicity. This can result in incorrect interpretation. Accordingly, appropriate concentration of inhibitors must be selected to optimize the conditions prior to testing (Vercauteren et al. 2010). When analyzing exclusion assays by flow cytometry, positive controls such as transferrin and folic acid, which are known to be internalized by clathrin- and caveolae-mediated endocytosis, respectively, should be included (Rothberg et al. 1990; Schmid 1997). However, flow cytometry may not necessarily distinguish between cells with internalized particles and those with surface bound particles (Ogris et al. 2000; Salvati et al. 2011).

In addition, genetically-modified cell lines have been used to exclude endocytic pathways (Ilina et al. 2012; Rejman et al. 2004). For example, Ilina et al. (2012) reported particle uptake studies with genetically-modified cells where dynamin-dependent and clathrin-mediated endocytic pathways were not operative. They further reported that the use of chemical inhibitors and the genetic blockage do not result in the same level of decrease in transfection efficiency (Ilina et al. 2012). These results further indicate that the interpretation of the inhibition assays can be difficult, and several methods should be considered before reaching a conclusion about the uptake mechanism of particles in specific cell lines. One of the problems with inhibition of endocytic pathways by mutant cell lines is the possibility of another pathway becoming increasingly active, which may aid the uptake of nanocarriers (Damke et al. 1995).

When identifying the endocytic pathway important for efficient delivery of pDNA by PEI, many contradicting reports have been published. Von Gersdorff et al. (2006) reported that clathrin-mediated endocytosis is required for efficient transfection, whereas Hufnagel et al. (2009) reported that fluid phase uptake plays an important role for efficient transfection with PEI/DNA polyplexes. However, it has also been demonstrated that blocking of caveolae-mediated endocytosis can inhibit expression of exogenous DNA (Gabrielson et al. 2009).

Uptake of fluorescently-labeled nanoparticles is usually measured by flow cytometry at various time points after addition of particles. Not all fluorophores are suitable for use in investigating cellular uptake through endocytosis. An example is fluorescein which has been reported to show decreased brightness when pH is below 9 and almost fully quenched at pH 6, which could result in inaccurate interpretation of the results when the particles have reached the acidic environment of endosomes and lysosomes (Geisow 1984).

3.2 Co-localization Microscopy Studies for Uptake Mechanism

Co-localization studies with fluorescence microscopy depend on the availability of fluorescently labeled nanocarriers and fluorescently labeled markers for the specific endocytic pathways that are being investigated (Vercauteren et al. 2012). A commonly used method is to add the nanocarrier to living cells, which is followed by fixation at various time points after addition. After fixation, immunostaining of the specific endocytic marker is performed to detect possible co-localization between nanocarrier and the marker for the endocytic pathway. Several fixative agents have been reported to cause artifacts, which need to be considered when analyzing the data (Lundberg et al. 2001; Pearson 2007). To avoid such artifacts live-cell imaging may be applied.

To study co-localization by live-cell imaging, the marker protein has to be tagged with an appropriate fluorescent marker. This may include stably transfected cell lines where the marker proteins are coupled to a fluorescent tag such as the Green Fluorescence Protein (GFP) or the Red Fluorescent Protein. However, it is important to control the expression of the fluorescently-labeled marker proteins, since their overexpression might increase the activity of an endocytic pathway, which does not have the same activity in the parental cell line. Furthermore, co-localization between two molecules acquisition, pre-processing of the image and also sample preparation are very important steps to overcome possible artifacts (Abraham et al. 2010; Bolte et al. 2006).

3.3 Other Methods to Study Internalization

Other methods have also been used to analyze the internalization of particles in the nanometer range. Scanning electron microscopy, transmission electron microscopy and atomic force microscopy have been applied to detect reorganization of the plasma membrane (Georgieva et al. 2011; Leroueil et al. 2007). The re-organization of the membrane when endocytosis is initiated varies between the different pathways and the electron density at the invagination sites can be distinguished by electron microscopy thus making it possible to distinguish between endocytic pathways (Doherty and McMahon 2009). Dynamic surface enhanced Raman spectroscopy has also been used to investigate nanoparticle (e.g., gold nanoparticles and carbon nanotubes) internalization and intracellular transport pathways (Ando et al. 2011).

4 Intracellular Transport

From the point when the endocytic vesicles are detached from the membrane the carrier has to be transported to the targeted cellular compartment. Several mechanisms of how the cargo is transported from early endosomes through late endosomes to lysosomes or recycling endosomes have been suggested (Gruenberg et al. 2004; Luzio et al. 2007; Saftig et al. 2009). Both the possibility of gradual maturation and fusions of compartments have been suggested (Stoorvogel et al. 1991). It has further been demonstrated that endosomes and lysosomes can communicate via small vesicles in cell free systems, which indicate that it is not only gradual maturation that takes place (Vida et al. 1999).

Nanoparticles have been reported to accumulate in the peri-nuclear region shortly after internalization (Bae et al. 2012; Bregar et al. 2013; Fichter et al. 2013; Kim et al. 2012; Suh et al. 2003). The movement of carriers has in several cases been demonstrated to be dependent on polymerization of either actin or microtubule (Suh et al. 2003). To investigate the role of microtubule or actin in the cytosolic transport of nanocarriers chemical inhibitors of polymerization of tubulin or actin have been reported to interrupt the transport. PEI/DNA polyplexes have been reported to be dependent on actin during cellular uptake, whilst the intracellular trafficking is suggested to be microtubule dependent. In addition, a lactosylated derivate of PEI was reported to be transported along microtubule (Grosse et al. 2007). Similarly, liposomes have been reported to be transported along microtubule during cellular transport. However, it has been demonstrated that the transfection efficiency is enhanced both when disrupting or stabilizing microtubule, because liposomes no longer are transported to the lysosomes. R8-coated liposomes only reached the periphery of the nuclear when microtubule was not disrupted by nocodazole (Hasegawa et al. 2001; Khalil et al. 2008). On the other hand, PEI-mediated transfection was almost eliminated when disrupting microtubule with nocodazole treatment in COS-7 cells (Grosse et al. 2007). Also free plasmid DNA has been reported to be dependent of microtubule-mediated transport on the route to the nucleus (Vaughan and Dean 2006). Suh et al. (2003) reported that PEI/DNA polyplexes were transported along microtubule. The transport was directed towards the nucleus and carried out by motor protein along microtubule (Suh et al. 2003). However, it should be considered that when destabilizing the microtubule and actin network the cell morphology is highly affected. This can lead to changes in both cell uptake and intracellular transport of nanoparticles (Dos et al. 2011).

Caveolae-mediated endocytosis has in many cases been reported to be an endocytic pathway where delivery to endo-lysosomal pathway can be avoided and trafficking occurs from the caveosome to endoplasmic reticulum and Golgi apparatus (Badizadegan et al. 2000; Lencer et al. 1999). Currently, this transport mechanism has not been completely elucidated. Particles can also be transported between ER and Golgi apparatus. This event has been demonstrated to take advantage of the normal cellular trafficking between the two organelles. Glycofect particles has been demonstrated to be transported in COP I coated vesicle from Golgi to ER (Fichter et al. 2013).

Liposomes internalized by macropinocytosis have also been reported to avoid lysosomal degradation and to be present in the cytosol after endocytosis. When the liposomes where internalized by macropinocytosis the transfection efficiency was significantly higher than when internalized through CME (Khalil et al. 2006b).

4.1 Endosomal Escape

To avoid degradation in the acidic lysosome environment nanoparticles have to escape from early endosomes. One suggested mechanism is the proton-sponge hypothesis and applicable to cationic species. This hypothesis was first suggested in 1997 by Behr and colleagues. Here, polycationic species were suggested to induce influx of negatively charged ions (e.g., chlorides) into the endosomes. This influx is followed by water due to change in osmotic pressure. This may result in endosome swelling and eventual rupture, resulting in cytosolic release of the polycation (Behr 1997). A recent work, however, reported that the lysosomal pH does not change after treating cells with PEI, thus suggesting that the proton sponge hypothesis may be an inadequate explanation for endolysosomal release (Benjaminsen et al. 2013).

Polycations have previously been described to be able to cause perturbations of lipid bilayers, which may be an alternative mechanism by which nanocarriers may escape endo-lysosomes (Bieber et al. 2002). Addition of cationic lipids to lipid carriers has resulted in endosomal escape probably due to destabilization of the lipid bilayer of endosomes (Wasungu et al. 2006). Akita et al. (2010) reported that a pH-sensitive fusogenic peptide could be modified for endosomal escape. One example is the GALA peptide, which can destabilize the lipid bilayer resulting in successful endosomal escape (Akita et al. 2010).

4.2 Cytosolic Transport

After release from the endosomes nanoparticles must reach their designated intracellular targets. For example, if pDNA is delivered the release should happen in the periphery of the nucleus so the DNA is not degraded before it reaches the nucleoplasm (Lechardeur et al. 2005). For cytoplasmic dissociation biodegradable carriers have been designed. The biodegradable carriers will be gradually degraded in the cytoplasm resulting in release of the cargo (Gosselin et al. 2001).

To reach the cellular compartment where the cargo is going to be released an increasing amount of research has been put into targeting the organelle of interest (Sakhrani and Padh 2013). For instance, the ER retention signal consisting of four amino acids (KDEL) has been used to target ER. By targeting ER the endo-lysosomal pathway might be bypassed and lysosomal degradation can be avoided (Acharya et al. 2013; Wang et al. 2013). Mitochondria play an important role in initiation of apoptosis, regulating calcium homeostasis, removal of reactive oxygen species and in ATP synthesis. Hence, targeting of mitochondria through mitochondrial targeting signal peptide can be very important in treatment of various conditions (Heller et al. 2012; Sakhrani and Padh 2013).

4.3 Nuclear Entry

If the cargo of the nanocarrier requires transport to the nucleus, passing the nuclear envelope is a major barrier for efficient delivery. On the nuclear envelope, nuclear pore complexes are widely distributed (Grossman et al. 2012). Molecules that enter the nucleus have to pass through the channel of this complex. Small molecules up to 9 nm in diameter can freely diffuse through the pore channel (Paine et al. 1975), whereas molecules up to 39 nm are transported through the complex by an energy-dependent transport (Pante et al. 2002).

DNA needs to be transported through the nuclear pore complex, but the mechanism behind this still needs further investigation. Some reports suggest that only during mitosis DNA can enter the nucleus or that the transfection efficiency is dependent on the cell cycle, but microinjection of DNA in the cytoplasm has shown that the exogenous DNA can be transcribed in non-dividing cells (Brunner et al. 2000; Dean et al. 2005; Pollard et al. 1998).

It has been reported that a nuclear localization sequence added to the nanocarriers can increase the transport to the nucleus. Also a DNA sequence coding for a binding site for NFkB can increase the transport of DNA to the nucleus resulting in an increase in transfection (Breuzard et al. 2008; Dean et al. 2005).

Entry to the nucleus has been under intense investigation and many contradicting reports have been published. For example, PEI has been suggested to be transported to the nucleus, only to be present in the nucleus after mitosis, whereas other studies have indicated the inability of PEI to reach the nucleus (Brunner et al. 2000; Dowty et al. 1995; Larsen et al. 2012). Not many studies have evaluated the mechanism by which a nanocarrier enters the nucleus, but it has been suggested that polycations might rupture the membrane to access the nucleoplasm (Godbey et al. 1999). The size of the nanocarriers can also be a major obstacle in the transport through the NPC, since only molecules with a diameter of less than 40 nm can be transported through the nuclear pore complex and most nanocarriers have been reported to be above 100 nm (Pante et al. 2002).

5 Techniques to Investigate Cellular Localization of Nanocarriers

Investigations of intracellular trafficking of nanocarriers are in many cases carried out by fluorescence microscopy. However, it is important to interpret the microscopy data with care, since artifacts may be generated and the interpretation of microscopy images can be very subjective (Bolte et al. 2006).

Fixation of cells before staining is widely used, but as previously mentioned the fixation can induce artifacts. Live-cell imaging decreases the amount of artifact that usually can be observed after a fixation procedure. However, to be able to detect which organelles the nanocarrier is transported to it is necessary to use cell permanent organelle specific dyes or to label organelle specific proteins with GFP or similar species (Vercauteren et al. 2012).

There exist several types of fluorescence microscopes. When using confocal microscopes the end product is an image where only light from the focal plane is emitted. For wide-field microscopes also light from the out of focus planes is visible in the final image (Bolte et al. 2006). Images from wide-field microscopes require deconvolution of the images to eliminate the out of focus blur. Especially, if quantifying the co-localization of nanocarriers with organelle markers wide-field images need to be deconvolved but it can also be an advantage to deconvolve confocal images (Bolte et al. 2006; Scriven et al. 2008). Deconvolution requires an accurate point-spread-function, which can be determined with use of various algorithms calculating point-spread-function (PSF) in various ways. The PSF should also be determined for the optical conditions used in experiments (McNally et al. 1999). Co-localization can also be determined visually. An overlap of two channels where the detected light is represented in two different colors will result in color change if there is an overlap between the fluorophores. This, however, is a subjective way of determining overlaps between channels and is only qualitative (Bolte et al. 2006; Dunn et al. 2011). In recent years quantitative algorithms have been developed to calculate the degree of co-localization.

The degree of co-localization can be calculated according to various coefficients. The most commonly used are the Pearson co-localization coefficient (PCC) and the Manders overlap coefficient (MOC). The PCC identify co-localization when a pixel from two different has the same intensity, whereas MOC detect overlap when fluorescence in a pixel from two channels is detected (Bolte et al. 2006; Manders et al. 1992). When using PCC it is important that the same instrument settings are used every time and when imaging nanocarriers their might still be a different in intensity in both the nanocarrier and also in the protein labeled from cell to cell and especially between experiments (Dunn et al. 2011).

For live-cell imaging MOC is the most commonly used coefficient since intensity from organelle markers and the nanocarrier itself can vary between experiments (Dunn et al. 2011). It is important when using MOC that a background threshold is set to avoid false positive read-out, which is a major risk when calculating MOC to analyze the degree of overlap (Bolte et al. 2006; Dunn et al. 2011; Fletcher et al. 2010).

Suh et al. (2003) have performed real-time multiple particle tracking when reporting that intracellular transport of PEI/DNA polyplexes to the periphery of the nuclei is dependent on motor proteins along microtubule (Suh et al. 2003). The same group also reported that polyplexes were actively transported to the early and late endosomes and transport between the endosomes was also observed when performing real-time multiple particle tracking (Suh et al. 2012).

Single-particle tracking is another powerful tool to study the route by which one single nanocarrier is transported through the cell (Ruthardt and Brauchle 2010). This method requires a fast and sensitive camera and the efficient laser excitation. Single-particle tracking is following the trajectory for each separately visible particle. Co-localization of particles are defined as significant correlation between the trajectories (Braeckmans et al. 2010). This method provides the possibility to investigate if the single particles are transported in the same direction and to the same organelles and will provide new insights as how a population of nanoparticles behaves when added to cells (Ruthardt and Brauchle 2010). Other microscopy methods have been applied to study the intracellular distribution of nanocarriers. Among them Raman microscopy and Fluorescence recovery after photo-bleaching have been used to investigate interaction between nanoparticles and mitochondria (Chernenko et al. 2009; Hemmerich et al. 2013; Phizicky et al. 2003).

5.1 High-Resolution Fluorescence Microscopy

Many of the traditional microscopes have a detection limit around 200 nm in the XY-plane and 500 nm in the Z-plane for optimal settings. This gives a problem when imaging nanocarriers with few hundred nanometers in diameter and single particles are not possible to image (Bolte et al. 2006).

An optimized hardware is necessary to perform high-resolution microscopy. Each step from the laser excitation to the camera acquisition has to be optimized compared with conventional microscopy. When performing high resolution microscopy on live cells there is a risk of inducing cell death due to high laser power. Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are two of the most described high resolution microscopy techniques (Henriques et al. 2011). The super resolution techniques create a dataset by excitation of few fluorophores at a time and an image is the reconstructed based on the fluorescent particles. PALM and STORM are two very similar setup but they vary in the fluorophores that are used for the experiments. PALM uses genetically attached fluorophores such as GFP coupled proteins, whereas STORM take advantages of fluorescent dyes (Henriques et al. 2011).

5.2 Transmission Electron Microscopy

Transmission electron microscopy (TEM) is also widely used for co-localization studies. Gold labeled antibodies against a specific antigen are a specific method to investigate the cellular localization of the protein of interest. By TEM it is possible to distinguish between various organelles and cellular compartment and the endocytic vesicles (Georgieva et al. 2011). However the interpretation of electron microscopy can be difficult and may generate artifacts during sample preparation. Sample preparation for electron microscopy includes embedding, slicing and staining of the sample before imaging and this is very time consuming compared to fluorescent imaging (Henriques et al. 2011). Nanocarriers can often be visualized on their own by negative staining.

5.3 Cellular Fractionation

Cellular fractionation has also been used to study the intracellular localization of nanocarriers. Centrifugation steps are carried out to separate the various organelles (He et al. 2013; Shi et al. 2013). For instance, Shi et al. (2013) labeled their polyplexes with [3H]-DNA and [14C]-PEI before adding the polyplexes to the cell growth medium. They separated the lysed cells into nuclear, light mitochondria, heavy mitochondria, microsomes and cytosolic fractions. Quantification of the radioactivity indicated that a large fraction of both PEI and DNA was detected in the nuclei fraction. This method, however, was not sufficient to demonstrate if polyplexes entered the nucleus intact or separately as DNA and/or PEI (Shi et al. 2013). A major disadvantage of this method is the risk of contaminating the different cellular compartments with parts from the other fractions.

6 Conclusion

Uptake and intracellular trafficking of nanocarriers is currently under intense investigation. Many factors can influence the endocytic pathways and need to be taken into consideration when designing assays and analyzing the data. Quantification of co-localization between nanocarriers and the marker protein can be determined in a very subjective manner by looking at the overlay images from two or more channels. However, several algorithms have been developed to give a more objective quantification of the co-localization. Also exclusion assays when studying the uptake of nanocarriers can be difficult to interpret with certainty, since inhibiting the selected pathways can result in unwanted cellular responses such as toxic responses or undesired increased activation of alternative pathways.