Advances in Colloid and Interface...

Advances in Colloid and Interface Science xxx (2013) xxx–xxx

CIS-01328; No of Pages 12

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Advances in Colloid and Interface Science

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Exocytosis of nanoparticles from cells: Role in cellular retention and toxicity

Ramin Sakhtianchi a, Rodney F. Minchin b, Ki-Bum Lee c, Alaaldin M. Alkilany d,Vahid Serpooshan e, Morteza Mahmoudi a,e,f,⁎a Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iranb School of Biomedical Sciences, University of Queensland, Brisbane 4072, Australiac Department of Chemistry and Chemical Biology, Institute for Advanced Materials, Devices and Nanotechnology (IAMDN), Rutgers, The State University of New Jersey Piscataway, NJ 08854, USAd Department of Pharmaceutics & Pharmaceutical Technology, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordane Department of Pediatrics, Division of Cardiology, School of Medicine, Stanford University, Stanford, CA, United Statesf Department of Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

⁎ Corresponding author at: Department of NanotechnoE-mail addresses: [email protected], MahmoudURL: http://www.BioSPION.com (M. Mahmoudi).

0001-8686/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.cis.2013.10.013

Please cite this article as: Sakhtianchi R, et alSci (2013), http://dx.doi.org/10.1016/j.cis.20

a b s t r a c t
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Available online xxxx

Keywords:ExocytosisNanoparticlesNanotoxicologyTherapeutic delivery

Over the past decade, nanoparticles (NPs) have been increasingly developed in various biomedical applicationssuch as cell tracking, biosensing, contrast imaging, targeteddrug delivery, and tissue engineering. Their versatilityin design and function has made them an attractive, alternative choice in many biological and biomedicalapplications. Cellular responses to NPs, their uptake, and adverse biological effects caused by NPs are rapidly-growing research niches. However, NP excretion and its underlying mechanisms and cell signaling pathwaysare yet elusive. In this review, we present an overview of how NPs are handled intracellularly and how theyare excreted from cells following the uptake. We also discuss how exocytosis of nanomaterials impacts boththe therapeutic delivery of nanoscale objects and their nanotoxicology.

© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Transportation across the plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Analytical methods for exocytosis detection and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1. Elemental analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Fluorescent microscopy for NP tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Approaches to study exocytosis in cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Factors affecting the exocytosis of NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5.1. Influence of cell type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.2. Effects of the physicochemical properties of NPs on exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5.2.1. Effect of size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.2.2. Effect of shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.2.3. Effects of different surface modifications on exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5.3. Effects of NPs concentration and their cellular incubation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.4. Distribution of NP into organelles: A key factor in exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.5. Acceleration/inhibition of exocytosis process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

6. Translocation of NPs across vital barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 07. Effect of NPs on regulated exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 08. Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

logy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, [email protected] (M. Mahmoudi).

ghts reserved.

, Exocytosis of nanoparticles from cells: Role in cellular retention and toxicity, Adv Colloid Interface13.10.013

http://dx.doi.org/10.1016/j.cis.2013.10.013

mailto:[email protected]

mailto:[email protected]

http://www.BioSPION.com


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2 R. Sakhtianchi et al. / Advances in Colloid and Interface Science xxx (2013) xxx–xxx

1. Introduction

In the past decade, nanoscience and nanotechnology have beenextensively studied in various fields of science such as physics,chemistry, biology, and medicine [1–4]. From the medical point of view,nanoparticles (NPs) demonstrate significant potential applications suchas targeted drug delivery, imaging, gene therapy, and stem cell trackingand/or differentiation [5–8]. However, concomitant with the rapidlygrowing biomedical applications of NPs, there have been increasingconcerns about their adverse side effects and unwanted toxicity [9–17].It is known that the surfaces of NPs are covered by various biomoleculessuch as opsonizing proteins, following their contact with biologicalfluids. Therefore, what the cell directly interacts with is the proteincorona adsorbed onto the surface of NP rather than the originalsurface of the pristine particles [18–23]. Efforts have been made tobetter understand the interactions of NPs with the biological medium[14,15,24–34]. Moreover, the effect of these interactions on traffickingNP–protein complexes into and out of the cells, and their involvementin various signaling pathways have been investigated [34–36].

While the cellular uptake mechanisms of NPs (with and withoutprotein corona [36]) have been extensively explored over last fewyears [37–44], there is little known about NP excretion from the cells.This aspect of NP disposition may be a significant cause of unwantedtoxicity. Once inside the cells, the composition of the protein coronacan undergo significant changes [45]. Thus, following exocytosis,NPs may have the ability to pass through critical in vivo barriers,such as the blood–brain barrier, and cause unpredicted biologicaleffects [46]. A greater understanding of the exocytosis processesand their corresponding effects on the protein corona may lead to thedevelopment of new multifunctional nanomaterials for drug deliveryand imaging applications. For example, specific plasma proteins in theprotein corona can be recognized by specific receptors on target cells[47]. Following NP uptake and exocytosis, a different protein coronamay result in the recognition by other receptors, which may facilitatedifferent targeting outcomes.

In order to achieve a comprehensive understanding of the NPexocytosis process, in this review the biological mechanisms governingexocytosiswill be described followed by the current available techniquesto study the cellular exocytosis of nanomaterials. Finally, the effect ofphysicochemical properties of NPs (e.g. size, size distribution, and surfaceproperties), on the activation of specific exocytosis pathways, and theinvolvement of the NP's corona, will be discussed in detail.

2. Transportation across the plasma membrane

Endocytosis and exocytosis involve processes of complex vesicularsystems, which are strictly dependent on each other. Cells use theseprocesses to add and remove membrane proteins such as transporters,ion channels and receptors and lipids, to take up and excrete essentialmolecules [48], to communicate with other cells in our brain, or torepair the plasma membrane [49]. The important pathways for NPtransport across the plasma membrane are summarized in Fig. 1.

In general, foreign material (e.g. NPs) can enter the intercellularcompartment using several well-recognized entry mechanisms suchas clathrin-, caveolae-, RhoA-, CDC42-, ARF6-, and flotillin- mediatedendocytoses, which are comprehensively described elsewhere [50,51].In the early endosomes, some of the endocytosed materials can berecycled back to the plasma membrane or can be delivered to theGolgi apparatus. Other materials that remain in the early endosomesmove slowly along microtubules toward the cellular interior and,subsequently, fuse with late endosomes followed by lysosomes.However, some of the endocytosed particles can escape from thesecomplicated vesicular systems to the cytosol. It is worth mentioningthat some of the lysosomes can undergo exocytosis and release theirundigested content following fusion with plasmamembrane. Althoughthis process occurs under specific situations such as cell stress, some cell

Please cite this article as: Sakhtianchi R, et al, Exocytosis of nanoparticles fSci (2013), http://dx.doi.org/10.1016/j.cis.2013.10.013

types have the ability for fusion of specialized lysosomes with theplasma membrane, which is thought to be involved in membranerepair [52–56].

3. Analytical methods for exocytosis detection and quantification

There are twomain analytical tools for the evaluation of NP exocytosisincluding elemental analysis for the measurement of intracellular andextracellular NP concentrations and fluorescent microscopy for trackingof NP uptake and release.

3.1. Elemental analysis

The basis of this technique is the measurement of massconcentration of internalized or excreted NP elemental compositionand conversion of the mass concentration into the number of NPs.Inductively coupled plasma spectrometry (i.e. ICP-AES, [37,57–61] ICPMS [59,62], and ICP OES [63,64]) is the most widely used method forquantitative assessment of endocytosis and exocytosis [65,66].However, inability to differentiate between the intact NPs and theirbreakdown products (i.e. ions/elements), NPs associated with differentcell types (i.e. within a heterogeneous culture) and also inability todetect NPs composed of materials, which naturally exist in the cells(e.g. carbonaceous NPs) hampered the use of these techniques forendo-/exocytosis investigations. Additionally, since for estimating themass of a single NP to convert the ICP spectroscopy measurement toNP number, some of the NP features (e.g. the atomic weight, the crystallattice unit length, and the well-defined geometry) are needed;reproducibility of the results requires a well-characterized crystallinelattice and NP monodispersity. Consequently, metallic and generallyinorganic NPs can be used as excellent probes for exocytosis evaluationsof the NPs using this method [67].

3.2. Fluorescent microscopy for NP tracking

Fluorescence spectroscopy techniques can be used for bothquantitative and qualitative evaluations of endocytosis, localization,and exocytosis of NPs [37,68–70]. Although the technique is veryprecise, its application is limited to fluorescent NPs, either with intrinsicfluorescent property, such as quantum dots [71–73], semiconductingcarbon nanotubes [74] or with conjugated fluorescent dyes, such asTexas red32 [37], fluorescein isothiocyanate [70,75], rhodamine [69]and 6-coumarin [68]. By using advanced microscopy techniques likeconfocal laser scanning microscope (CLSM) uptake and removal ofNPs can bemonitored in situwith high spatial resolution [72]. However,it should be noted that the labeling of NPswith fluorescence dyesmightchange their surface properties and affect their biological activity.Furthermore, cells act as tremendously efficient filters for elution offluorescent tags with the NPs, including those that cannot be removedby extensive purification, such as dialysis method; in this case, theenzymatic activity inside cells can detach the weak-labeled dye fromthe surface of NPs, causing major error in NP uptake and sub-cellularlocalization [76,77]. In order to overcome these shortcomings,multifunctional NPs, with rigid shell and polymeric gap (i.e. polymerchain between core and rigid shell), were developed [78]. Morespecifically, the fluorescence dye is placed at the polymeric gap, whichis out of reach for proteins and enzymatic activities.

4. Approaches to study exocytosis in cell culture

The most common approach for evaluation of exocytosis is toincubate NPs with cells; then, the cell medium is removed and thecells are washed thoroughly with fresh NP-free medium, followed byincubation of thewashed cells with freshmedium. In order to overcomethese two possible shortcomings, single particle tracking (SPT) hasbeen proposed. Moreover, over-exposure of cells to NPs may induce

rom cells: Role in cellular retention and toxicity, Adv Colloid Interface


Fig. 1. Scheme showing the transportation of biomolecules across plasma membrane (natural exocytosis (red arrows), organelle trafficking (blue arrows), and endocytosis (green arrows)pathways); (a) showing constitutive secretion, in which molecules trapped in secretory vesicles, move directly to the cell surface and immediately leave the cell; (b) showing regulatedsecretion, wherein molecules with appropriate marker, are delivered to the secretory vesicles until extracellular signal triggers their release. (c–i) Illustrating nonconventional secretionsystem, in which some special proteins excreted from the cell without recruitment of routine secretory machine compartments (i.e. without using ER (c–f), Golgi (c–h) or COPII coatedvesicles (c, d, e, f, g, and i)). Following plasma membrane budding and pinching off, the endocytic vesicles fuse with early endosomes, except for phagocytosis entry mechanism; (j) inthe early endosomes some of the endocytosed molecules or macromolecules are retrieved and recycled back to the plasma membrane or are delivered to the Golgi apparatus, otherswhich are remained in early endosomes move slowly along microtubules toward cell interior and, subsequently, fuse with late endosomes, which are located close to the Golgi apparatusand near the nucleus; (k) lysosomes are not necessarily the end of pathway; some of them undergo exocytosis and release their undigested content by fusion with plasma membrane;(l) transcytosis, which involves both endocytosis and exocytosis processes, allows molecules to cross over the cells; it seems that caveosomes participate in endothelial cell transcytosis.Details on the individual pathways are presented in previous reports [56,165–175]. (For interpretation of the references to color in this figure legend, the reader is referred to theweb versionof this article.).

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overt toxicity and alter both endocytotic and exocytotic events. However,with SPT, NP transport can be investigated at much lower concentrationsand thus avoid any adverse effect of the NPs. However, conventionalfluorescence based methods experience photobleaching, which hindersaccurate and continuous mapping of NP movement. To resolve thisproblem, Jin and co-workers [74] employed non-photobleaching singlewalled carbon nanotubes (SWNTs), and demonstrated continuousmeasurement of endocytosis, intracellular trafficking and exocytosispathways for DNA–SWNT complexes (see Fig. 2 for details). Unlikethe approach of incubating NPs for different times, which is employedwidely for SPT, binning the total number of real-time processes on thesame cell permits for a precise statistical assessment of each transportstep, together with total material balance [74]. Such an approach isvery useful to cover the whole life span of the NP and to understandexocytosis on the single-cell level.

5. Factors affecting the exocytosis of NPs

5.1. Influence of cell type

Different cells can responddifferently to the sameNPswith the sameprotein corona [27,79–82]. The possiblemechanisms that result in thesevarious cellular responses relate to the numerous detoxificationstrategies that different cells can utilize in response to the same NP.Thus, the uptake and defense mechanisms induced by NPs can beconsiderably different according to the cell type under investigation.


Chu et al. [83] reported that the endocytosis and distribution of50nm silica NPs in human lung carcinoma (H1299), human esophagealepithelial (NE083) and human bronchial epithelial (NL20) cell lineswere similar. However, the excretion profile of NPs was influenced bycell line type. The exocytosis of NPs from NE083 cells was much slowerthan that of NPs from H1299 and NL20 cells. Similarly, the exocytosis ofmesoporous silica NPs (MSNs) from different mammalian cell linesshowed that different retention and excretion properties of the differentcell lines could result in asymmetric cell to cell transfer of MSNs [70].Although a similar behavior was detected forMSMs in human umbilicalvein endothelial cells (HUVECs) and human fibroblast epithelial (HeLa)cells, the exocytosis profiles were very different. To show the diversityof different cell types to retain or release these NPs, they performed aseries of cell co-culture experiments. Interestingly, after co-incubationof two cultures of HeLa cells, one pretreated with FITC–MSNs theother one with TRITC–MSNs, only a small fraction (12.6±1.6%) of thecells were FITC–TRITC positive. However, examination under the samecondition with HUVECs resulted in 89.6 ± 1.2% FITC–TRITC positivecell, which was seven times higher than that observed for HeLa cells.In addition, when the HUVECs were pretreated with a cell-tracer dye(Cell Trace Far Red DDAO-SE), which was not exchangeable betweencells and HeLa cells were pretreated with FITC–MSNs, the co-incubation of both lines led to a little particle transfer (7.5 ± 2.4%).Conversely, when HeLa cells were stained with the tracer dye andHUVECs were pre-incubated with the FITC–MSNs, the extent of particletransfer was significantly increased (74± 2.4%). The observed in vitroretentions of NPs are consistent with the performed in vivo results



Fig. 2. Probing single-walled carbon nanotube (SWNT) exocytosis frommouse embryonic fibroblast cell (NIH-3T3). (a–d) Show near-infrared fluorescence images of a cell that containsSWNTs (bright spots). Over the time period of 32 s exocytosis of an individual SWNT across the cell membrane takes place. Several categories of such SWNT trajectories are shown in thebright field images of one cell (the thick green arrows indicate the perfusion flow direction); (e) indicates adsorption and endocytosis trajectories; (f) indicates exocytosis and desorptiontrajectories in the same cell; (g) indicates confined motion on the membrane and after internalization. (h) A network depicting all of the steps observed in the cellular uptake (mouseembryonic fibroblast cell line (NIH-3T3)) of DNA–SWNT as reconstructed from over 10,000 single particle trajectories for up to 340min: UC, upstream convective diffusion; A, adsorbed;S, surface diffusion; AT, active transport; CD, confined diffusion; E, exocytosed; DC, downstream convective diffusion; AG, intracellular aggregation. The blue line indicates themembranewith blue dots representing internalized particles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).The figure was adapted with permission from ref [74].


[84,85]. Yanes et al. [86] also investigated the effect of the cell type onthe exocytosis rate of phosphonate-modified MSN (~130 nm) fromadenocarcinomic human alveolar basal epithelial cells (A549), breastcancer cell lines (MDA-MB231 and MCF-7), the melanoma cancer cellline (MDA-MB435), the pancreatic cancer cell line (PANC-1), and thehuman embryonic stem cell line (H9). The percentage excretion ofNPs was 87%, 81%, 75%, 61%, 36%, and 4% respectively. Interestingly,the rate of phosphonated-MSN exocytosis was correlated with therate of release of the lysosomal enzyme β-hexosaminidase suggestingthat lysosomal exocytosis plays a key role in the exocytosis of theseNPs. Further investigations revealed that disruption of the Golgiapparatus did not block the NPs exocytosis.

Exocytosis investigation of gold NPs in HeLa, human glioblastoma(SNB 19) and mouse embryonic fibroblast (STO) showed that thefraction of gold NPs exocytosed from the STO cells was considerablyhigher than the other cell types at all NP sizes (14–100nm). However,no significant difference between the extent of exocytosis from HeLaand SNB 19 cell lines was observed [37]. Wang and coworkers [62]showed that gold nanorods (length of 55.6 ± 7.8 nm and width of13.3 ± 1.8 nm) have a significant effect on cell viability of A549 cellswhile posing negligible impact on normal 16HBE cells or mesenchymalstem cells (MSCs). Due to the different organelle distribution of theseNPs in different cell types, as result of different lysosomal membranestability, the cytotoxicity and also exocytosis profile of NPs in cancercells are different from normal and stem cells. Although there was nodistinct difference in the nanorod uptake pathways in the three celltypes, lysosomal membranes of those cells showed different stabilityin the presence of the gold nanorods. In the case of A549 cells, goldnanorods translocated from endosomes/lysosomes into the cytoplasmand mitochondria. Subsequently, they induced mitochondrial damage


and reduced cell viability. However, the lysosomal membranes of16HBE and MSCs remain more intact and gold nanorods were localizedto lysosomes from where they recycled back to the plasma membraneor were retained in the lysosomes.

The exocytosis of 100 nm fluorescent nanodiamonds was inves-tigated in various cell lines and the results showed that, after 6 days,15% or less of nanodiamonds were released from either HeLa or multi-potent stromal cells [87]. However, exocytosis of these NPs from thepre-adipocyte cell line (3T3-L1) was notably higher (up to 30%).Comparison of these results with exocytosis data of carboxyfluoresceindiacetate succinimidyl ester, a widely used dye for cell tracking, revealedthat these NPs can be a promising alternate probe for evaluation of theexocytosis process.

On the basis of the above results, one can conclude that theexocytosis of NPs with the same physicochemical properties fromvarious cells is significantly different. Therefore, every assessment ofexocytosis or related properties like toxicity has to be performed forevery specific cell type and general predictions are not possible. Onlyphysiologically motivated combinations of cellular systems and NPswill have in vivo implications in terms of endocytosis/exocytosis andtoxicity.

5.2. Effects of the physicochemical properties of NPs on exocytosis

It has been shown that the size, [37,75,88] and surfaceproperties [61,86,89] of NPs can influence the exocytosis process andthe retention time of these particles in cells. However, there are fewreports on the impact of other properties, such as shape, [37] presenceor absence of surface-bound biomolecules, or surface roughness/smoothness. Thus, further investigations are needed to clarify the




impact that various physicochemical properties of NPs may have ontheir exocytosis processes.

5.2.1. Effect of sizeThe amount and rate of NP uptakes are dependent on the size of the

NPs [51,90–99]. Interestingly, it has been shown that the exocytosisprocess is also size dependent [37]. For example, the exocytosis ofsuperparamagnetic iron oxide NPs (SPIONs) trapped in porous siliconcarriers showed that the amount of iron excreted from murinemacrophages J774 cell line pretreated with carriers containing 15 nmSPIONs, was significantly greater (p b 0.03), than carriers loaded with30 nm NPs at same concentration [88]. In agreement with this report,Hu et al. [75] investigated the exocytosis of mesoporous silica NPswith different sizes in liver hepato-cellular HopG2 cells. After first fewhours, the percent exocytosis for 60, 180, 370, and 600 nm particleswas 63%, 67%, 58%, and 38%, respectively. These results illustrate thecomplex effect of size on the extent of excreted NPs and showed thatsmaller NPs are more easily released from HepG2 cells. The sametrendwas observed for transferrin-coated gold NPs [37]. The exocytosisrate of 14 nm transferrin-coated gold NPs was two and five timesfaster than 74 nm and 100 nm transferrin-coated NPs, respectively.Interestingly, the same trend was observed in three different cell lines(HeLa, SNB and 19STO cells). One explanation for this observationcould be the existence of ligands on the surface of the NPs that interactwith specific receptors affecting release. Another possibility for theeffect of NP size on the exocytosis rate was proposed by Panyam et al.[68,100]. Based on their proposed recycling process, the cell has atleast two possible intracellular compartments (endosomes andlysosomes/cytoplasm). Following endocytosis, a fraction of the NPs isdelivered to the lysosomes or translocates from the recyclingendosomes into the cytoplasm. Other NPs remain in the endosomes orare recycled back to the surface of the cell (see Fig. 3). It appears thatat least two intracellular compartments are involved in the exocytosisof NP, one with a rapid turnover and the second with a much slowerturnover. The size of different NPs could affect their delivery into thefast or slow recycling compartments [101,102]. For instance, exocytosisof poly(lactic-co-glycolic acid) (PLGA) NPs in human vascular smoothmuscle cells (VSMCs) showed that the excretion rate of the fluidphase marker Luciferase yellow was higher than that for the NPs [68].It was also shown that the distribution of different marker moleculesinto the rapid and slow turnover compartments is size-dependent.Therefore, one can expect to see differences in the exocytosis profile ofLucifer yellow and PLGA NPs based on their differences in size. Cartieraet al. [69] demonstrated that PLGA NPs appear to be localizedin the early endosomes, Golgi apparatus and ER (slow recyclingcompartments). However, the localizing patterns were different, inhuman bronchial epithelial HBE and opossum kidney epithelial OK celllines. This suggests that the NPs interact with different proteins withinthe different cells leading to changes in their intracellular localization.However, specific experiments addressing this possibility are needed.Fang et al. [87] found that exocytosis of fluorescent nanodiamonds issignificantly slower than exocytosis of D-penicillamine coated CdSe/ZnS QDs from HeLa cells [71]. They proposed that this difference wasthe combined effect of the larger particle size and the aggregation ofthefluorescent nanodiamondparticles in slow turn over compartments.Under these conditions a larger number of proteins and receptors arerequired for their exocytosis, as compared to the smaller NPs withoutagglomeration [103]. Jin et al. studied the size dependence of SWNTexocytosis using SPT techniques. The exocytosis rates of SWNTs inNIH-3T3 cells, followed the same trend as above: smaller SWNTs canbe exocytosed faster than larger SWNTs [91].

Both theoretical and experimental studies of endocytosis confirmedthat there is an optimal NP size (40–60nm) for efficient entry into cells,which can also be affected by several other factors such as membrane-NPs binding energy, protein or ligand density on the surface ofnanoparticles, and nanoparticle curvature [90–92,104,105]. Despite


the fact that the same factors are expected to regulate/affect exocytosisextent and dynamics, little is reported on these important issues.

5.2.2. Effect of shapeIn addition to size, NP shape is also important for both endocytosis

[37,51,90,106] and exocytosis processes [37]. It has been shown thatthe uptake of gold NPs by HeLa cells is highly dependent on the shapeof the NP [90]. In these studies, the uptake of rod shaped gold NPs wasless than spherical NPs due to the different membrane wrapping time[37,90]. Chithrani et al. [37], specifically investigated the effect of NPshapes on both endocytosis and exocytosis rates of transferrin-coatedgold NPs in HeLa, SNB 19 and STO cell lines. In the case of exocytosis,the fraction of rod-shaped NPs released from HeLa and SNB 19 celllineswasmuch higher than spherical-shaped NPs. However, the releaseof NPs from STO cells was the same for both rod- and spherical-shapedNPs. The observed differences between various cell lines are related tothe cell type effect (the exocytosis pathways might be different invarious cells), which is recognized as crucial factor that should beconsidered for the safe design of any type of NPs. Further study wouldbe necessary in the future to explore the effect of shape on theexocytosis of NPs.

5.2.3. Effects of different surface modifications on exocytosisDifferent surfacemodifications have decisive effects on the exocytosis

profile of NPs. For instance, gold NPs with the same physicochemicalproperties (size ~35nm and zeta potential~−18mV), but functionalizedwith non-targeting or targeting peptide, show different endocytosispatterns. The no- targeted NPs were taken up by a non-specific routewhile the targeted NPs were mostly taken up via a specific NRP-1receptor mediated pathway [107]. These NPs also exhibited differentexocytosis profiles suggesting different pathways for release [61]. Morespecifically, after incubation of targeted NP-treated-HUVECs with NPfreemedium for 2h, nearly 25% of theNPswere excreted and a significantportion of the particles (approximately 10%) was re-entering the cellsafter 4 h. By contrast, while approximately 20% of non-targeted NPswas excreted, no re-uptake was observed. Importantly, there were nomeasurable variations in the hydrodynamic diameters or zeta potentialsbefore and after the exocytosis process for either NP indicating that theexocytosed NPs retain their colloidal stability, independent from theiruptakemechanism [61]. In a separate study, Yanes et al., [86] investigatedthe effect of different surface modification of MSNs (phosphonate, folate,and polyethylenimine (PEI)) on the release of NPs from A549 cells.After 6 h, 84%, 66%, and 49% of the phosphonate-MSNs, folate-MSNsandPEI-MSNshadbeen released, respectively. In another study, the effectof crosslinking on the endocytosis and exocytosis of micelles composedof poly(methyl methacrylate)-block-poly(polyethylene glycol methylethermethacrylate) from human ovarian cancer OVCAR-3 cell line wasstudied [108]. According to the results, shell crosslinking did not showany considerable effect on endocytosis. By contrast, about 25% of shellcrosslinked micelles were released from the cells over 2h whereas only3% of non-crosslinked NPs was released.

Recently, it was shown that the binding stability between transferrin(Tf) and the transferrin receptor (TfR) could affect the cellular retentiontime of Tf modified carbonated Cd/Se QDs in HeLa cells and A549xenograft tumor models. Three types of transferrin conjugated carboxylQDs (iron saturated and desaturated with carbonate as the synergisticanion in transferrin, and iron saturated with oxalate as the synergisticanion) with size range of 22 to 25nm, along with bovine serum albuminQDs (18 nm) as control were investigated and the results showedthat the mean fluorescence intensity of oxalate-Tf-QDs in HeLa cellswas approximately 2 times higher than that of carbonate-Tf-QDs andfour times higher than that of albumin QDs and iron desaturatedcarbonate-Tf-QDs. In addition, the half-maximum fluorescence intensityof the oxalate-Tf-QDs in vivo was 4 times higher than that of thecarbonate-Tf-QDs. However, Tf synergistic anion did not show any effecton intracellular trafficking route of Tf-QDs in HeLa cells [89]. Tf attaches




to the TfRwith the help of 2 ferric ions. In the acidic pHof endosomes, theiron is released and the resulted desaturated-Tf/TfR complexes arerecycled back to the cell surface and dissociate in the higher extracellularpH. Therefore, it seems that the delay in Tf iron release can increasethe binding between Tf-QDs and TfR leading to the extension of the


intracellular NP retention. The prolonged intracellular retention timecould provide an opportunity to enhance the function of the NPs, suchas increased drug delivery or better tissue imaging.

All these studies show that the surface chemistry of NPs plays acrucial and perhaps the decisive role for endocytosis/exocytosis.




Unfortunately, there are not many analytical tools available that canvisualize the organic corona around NPs. Therefore most of thestudies rely on functional proofs. For example, in most cases thefunctionalization scheme cannot be verified by analytical means butthe different biological outcomes of cell experiments with differentfunctionalized NPs can serve as an indirect proof. Before leaving thissubsection, we would like to highlight the importance of evaluatingand understanding changes of surface chemistry inside cells (afterendocytosis and before exocytosis). This topic is highly unreportedin the literature where most efforts are dealing with NP surfacefunctionalization before dosing to cells. Lynch and co-workers took anexcellent initiative in this regard and they studied protein coronacomposition for nanoparticles incubated with plasma fluid and thenfor the same nanoparticles incubated in cytosolic fluid (simple modelfor intracellular media). The authors found that there were significantdifferences between the protein corona in both media that may alternanoparticle fate, distribution, and behavior [45].

5.3. Effects of NPs concentration and their cellular incubation time

The rates of endocytosis and exocytosis, which are dynamic andsimultaneous processes, are dependent on the concentration of NPs,both inside and outside of the cell. The maximum exocytosis rate hasbeen reported when the number of NPs in the cells is at its highest orsaturated level [59,63,83,109]. For example, the amount of silver NPsin the supernatant of human glioblastoma U251 cells is depended onincubation time and NP concentration, and the amount of the releasedNPs from the cells, was three times greater for NPs at 400 μg/mlcompared to the NPs at 100 μg/ml [63].

Panyam et al. [68] investigated the effect of pre-incubation time anddose on NP removal from the culture medium on PLGA NP exocytosisfrom VSMCs. They found that the fraction of NPs that was retained inthe cells increased 2.5 fold by increasing the NP concentration from100 to 200 μg/ml in the medium. In addition, the pre-incubation timeof NPs before exocytosis was measured showed no significant effect.In contrast with Panyam's data, Park and coworkers [110,111] showedthat the amount of exocytosed chitosan NPs from HeLa cells decreasedwith increasing pre-incubation time, from 10 min to 6 h. Theseconflicting results may be due to the different protein coronas thatform within different cell-types and around different NPs as describedbefore. In addition, it has been shown that the amount of CuO NPsexpelled from A549 was enhanced by increasing the exposure time(12–24 h) and NP concentration (5–15 mg/ml) [112]. In contrast tothe CuO NPs, exocytosis of Cu from Cu2+ pretreated cells was notdependent on exposure time and showed fluctuation over time.Therefore, it seems that by increasing the amount of NPs and pre-incubation time, NPs probably have more chance to be transported tothe slow recycling compartment. Furthermore, evaluation of the uptakeand removal of Si-QDs (aggregate size of 65 nm) with three different

Fig. 3. (a) Scheme showing brief cellular excretion processes ofNPs (red arrows) togetherwith tcells. After being endocytosed, NPs usually delivered in early endosomes, which are themain soreceptor mediated entry mechanisms, fuse with early endosomes. In the early endosome ssubsequently excreted by cells (i.e. (see 1,4)) others, which are remained in early endosomeendosome. Finally late endosomes turn/fuse with lysosomes, which are not necessarily the enby fusion with plasma membrane (5,6). On the pathway to late endosomes or even in lysosomsome NPs from the beginning enter to the cytoplasm via diffusion or unspecific mechanisms (1mitochondria, ER, and Golgi apparatus via unknownmechanisms. TheNPs that enter to the ER othat colocalized in the cytoplasm can leave the cells via re-entry to vesicular system or directly v(e.g. endothelial cells), are responsible for endocytosis and exocytosis (translocation) of NPs asunknownmechanisms. (b) TEM images correspond to numbers in (a); (1) exocytosis of single Nmembrane wrapping, and internalization via clathrin mediated endocytosis, (scale bars are 50wrapping and internalization via clathrin mediated endocytosis (scale bars are 500 and 100 nand exocytosis of NPs to extracellularmatrix (scale bar 500nm); (5) blue arrows indicate releasrelease of NPs from vesicles via membrane translocation (scale bar 500 nm); (7) endocytosis o(scale bars are 200 and 100nm, respectively); (11) transcytosis of NPs across the cell (scale barmitochondria and nucleus, respectively (scale bar 1000 nm). (For interpretation of the referenThe figures were adapted with permission from references [36,37,60,90,115,116,176].


concentrations (50, 100, and 200μg/ml) by HUVECs were in agreementwith previous observations that both endocytosis and exocytosisprofiles of Si-QDs depend on exposure time and particle concentrationin cell culture [72].

5.4. Distribution of NP into organelles: A key factor in exocytosis

Following cellular uptake, NPs are enclosed into vesicles (see Fig. 3),which are known as early endosomes. The newly formed vesiclesmature into late endosomes or multivesicular bodies, and then intolysosomes, which have an acidic pH. When these vesicles contain NPs,they are transported along microtubules from the periphery to theperinuclear region by dynein motors [64,71]. These internalized NPscould then be actively transported to the periphery and exocytosed bycells, or fused with lysosomes and other cytoplasmic compartmentsthat have a slow turnover [68,69,100].

In general, organelle distribution and NP fate in cells are influencedby their uptake pathways (clathrin-mediated endocytosis, caveolae-mediated endocytosis, macro-pinocytosis, and clathrin- and caveolae-independent endocytosis). In addition, the physico-chemicalcharacteristics of NPs, such as size, shape and surface properties, andalso the nature of the target cell could influence the organelledistribution pattern and removal profile of NPs [69,83,113–117]. Forexample, Wang et al. [112] measured the intracellular uptake andexcretion of CuO NPs in A549 cells and found that a portion of NPs,whichwere located in mitochondria and nucleus, could not be excretedby the cells. Similarly, based on findings by Chu et al. [83], clusters ofsilica NPs in lysosomes are more easily exocytosed by H1299 cellscompared to single NPs in the cytoplasm. In a typical exocytosis process,the NPs are initially trapped in lysosomes before their transportation tothe cell membrane for excretion. Stayton et al. [59] showed that NPsthat leave the endocytotic vesicles or lysosomes and translocate intothe cytoplasm have greater difficulty in exocytosis. Moreover, NPs thatare trapped in the slow turn-over compartments such as the lysosomes,need a larger number of proteins and receptors for their exocytosis[103]. For example, Lucifer yellow (MW of ~450 Da) trapped in rapidrecycling compartment had a higher exocytosis rate compared to largermolecules (horseradish peroxidase or PLGANPs), whichmostly enteredthe slow recycling compartments [68,69,101,102].

Crystallinity can affect organelle distribution of amorphous andcrystalline silica NP. Unlike amorphous NPs, which were mostly foundin lysosomes, crystalline NPs had a much higher chance to be foundin the cytoplasm without encapsulation by membranes [83]. Forexocytosis, only the amorphous NPs were examined. Interestingly,they have found that most of the NP aggregates in the lysosomesmoved out of the cells, but single NPs remained in the cells. Thissuggested that NPs that are encapsulated by a membrane in thecytoplasm are more easily excreted by cells in comparison with NPs incytoplasm.

heir intracellular trafficking (blue arrows), and entrance (greenarrows)mechanisms to therting station in endocytosis process (e.g. TEM images of 2,3); even vesicles related to non-ome of the NPs are transported along with receptors to the recycling endosomes ands move slowly along microtubules toward cell interior and, subsequently, fuse with lated of the pathway; some of them undergo exocytosis and release their undigested contentes, fractions of NPs escaped from vesicular compartments to the cytoplasm; in addition,

2,13). NPs that are located in the cytoplasm or trapped in vesicles can enter to the nucleus,r Golgimay leave the cell via vesicles related to the conventional secretion system. The NPsia unspecific mechanism. It seems that caveolae, which are abundant on specific cell typeswell as plasma proteins (11); the “question marks” in the scheme correspondence to theP to extracellularmatrix (scale bar 100nm); (2) single NP binding to the surface receptors,0 and 200 nm, respectively); (3) NP cluster binding to the surface receptors, membranem, respectively); (4) movement of vesicles containing NP cluster toward cell membranee of NPs from vesicles viamembrane rupture (scale bar 500nm); (6) blue arrow indicatesf NPs via caveolae (scale bar 200 nm); (8–10) localization of NPs in the vesicular system;5000nm); (12) and (13) black, blue and red circles indicate NPs' location in the cytoplasm,ces to color in this figure legend, the reader is referred to the web version of this article.).




5.5. Acceleration/inhibition of exocytosis process

Calcium (Ca2+) can trigger the exocytosis process of naturalcomponents in mammalian cells [118–122]. The physiological con-centration of extracellular Ca2+ for mammalian cells is approximately2 mM [123]. Recent findings suggest that the extracellular calciumconcentration can influence the release of NPs from cells. It has beenreported that the concentration of excreted 10 nm gold NPs fromhuman colonic adenocarcinoma HT-29 cell line was directly related tothe extracellular calcium concentration. By increasing extracellularcalcium, the amount of exocytosed gold NPs was enhanced. Calciumcould also trigger lysosomal exocytosis by changing the conformationof the transmembrane protein synaptotagmin VII which allowssynaptotagmin VII to bind to the SNARE complex on the plasmamembrane enhancing the fusion of the lysosomal membrane with theplasma membrane [124,125]. Yanes et al. [86] studied the effect ofincreased intracellular calcium concentration on phosphonated MSNexocytosis. Treatment of A549 cells with the calcium ionophoreionomycin resulted in a 2-fold increase in the rate of phosphonatedMSN exocytosis.

In the exocytosis process, vesicles fuse with the plasma membraneand discharge their content into the extracellular environment.Lipid rafts and, more particularly cholesterol, are involved in theregulation of exocytosis. Recently, it was shown that lipid rafts,cholesterol and sphingolipid-rich microdomains enriched in theplasma membrane play an important role in the regulation ofexocytosis [58,126,127]. Furthermore, cholesterol depletion couldinterfere with NP exocytosis [128]. For instance, the exocytosis ofmaltodextrin NPs was completely blocked after filipin treatment,which sequesters membranous cholesterol.

Since exocytosis is an active process, metabolic inhibitors shouldinhibit exocytosis of NPs. Panyam and coworkers [68] showed thatsodium azide and deoxyglucose could reduce the exocytosis of PLGANPs by 40%. These inhibitors did not show any effect on the initialdecrease of NP levels during the first hour, which is in agreement withthe fact that at least 1 h time lag is needed for metabolic inhibitors toblock cell processes [100]. The effects of bovine serum albumin (BSA)and platelet derived growth factor on the exocytosis of PLGA NPs werealso examined. The exocytosis of NPs was almost completely blockedusing serum free medium condition. However, addition of 1% BSA inthe medium caused exocytosis of NPs similar to that in serum positivemedium. By contrast, addition of platelet derived growth factor alongwith 1% BSA did not show any significant effect on the exocytosisprofile, and was similar to 1% BSA alone [68].

It is noteworthy that NPs are not just transported via unspecificprocesses in the cell. As shown above, exocytosis of NPs can bekinetically changed by adjusting the molecular machinery of cells suchas calcium concentrations. Therefore, the picture of NPs that appear asmore or less inactive particles in cells is wrong. Instead, these particlesbecome part of the complex chemical environment of the cell andmight even use themachinery for exocytosis in a non-intended fashion.Conversely, using NPs to alter physiological exocytosis events might bea useful application for NPs.

6. Translocation of NPs across vital barriers

For administration of NPs via the gastrointestinal or respiratoryroutes, NPs have to cross barriers such as endothelial and epithelialcells, which are polarized and are responsible for the transcytosis ofdifferent substance (see Fig. 3) [129–132]. There are two possiblepathways across these types of barriers. The first is the transcellularroute, which involves both endocytosis and exocytosis processes andallows NPs to pass through the cell membrane and is termedtranscytosis. The second is the paracellular route which allows NPs topass through the tight junctions between individual epithelial orendothelial cells. In this case, there is often a need for specific surface


coating on the NPs to open the tight junctions [133]. However, to date,few investigations have been done on NP translocation across cellbarriers and the physicochemical effects of theNPs on their transcytosis.

As a step forwards understanding the effect of surface chemistryon NP translocations, Lin et al. [134] recently investigated thecellular transport pathways of gold NPs, with different polymercoatings, through human epithelial colorectal adenocarcinoma(Caco-2 cell) mono-layers. Both polyethylene glycol and poly 2,3-hydroxy propylacrylamide coated gold NPs transport across cellbarriers via the transcellular route, but with different endocytosispathways. Using colchicine to differentiate non-microtubularassisted endocytosis from microtubular assisted endocytosis, theyshowed that microtubules participate in translocation of PEG coatedgold NPs. On the contrary, other pathways involving cytoskeletalcomponents contributed to the transportation of poly 2,3-hydroxypropylacrylamide coated NPs. Furthermore, in the case of poly N-isopropylacrylamide coated NPs, either the microtubule assistedendocytosis pathway or the paracellular pathway was responsiblefor translocation depending on temperature.

In another study, charge effects on the transcytosis of poly(ethyleneglycol)-D,L-polylactide (PEG-PLA) NPs by epithelial canine kidneyMDCK cells were investigated [135]. The results showed that bothcationic and anionic NPs are delivered to the basolateral site. However,only a small fraction of anionic NPs co-localized in degradativelysosomes. Hence, NP surface charge not only impacts uptake efficiency[136,137], but also can influence intracellular trafficking [135,137].

The blood–brain barrier is an important site for NP developmentboth for drug delivery and imaging within the CNS. Early studiesfrom Kreuter's laboratory showed that polymeric NPs could traversethe blood–brain barrier [138]. Importantly, apolipoproteins present inthe protein corona were primarily responsible for this transcytosis[139]. Furthermore, they showed that NPs could be specificallyengineered with surface bound apolipoprotein to target the CNSin vivo [140,141]. Georgieva et al. [142] used different surfacecoatings on silica-based NPs to demonstrate transcytosis via differentmechanisms. While uncoated NPs were taken up by caveolarendocytosis, polyethyleneimine-coated NPs were accumulated byadsorptive endocytosis and prion-coated NPs were internalized byreceptor-mediated endocytosis (Fig. 3). Other surface modificationssuch as transferrin have been shown to promote transcytosis acrossthe blood–brain barrier [143,144].

Our growing understanding of NP delivery across the blood–brainbarrier has led to important practical developments. For example,dual-modality NPs that can be visualized optically, by positive emissiontopography or magnetic resonance imaging have proved useful inidentifying tumor margins in animal models [145–147] and in humans[148]. This application of NPs promises to significantly enhance theaccuracy of surgical removal of different brain tumors.

7. Effect of NPs on regulated exocytosis

NPs can affect many fundamental cellular functions, including thecell cycle and normal exocytosis [27]. They can also regulate cellsignaling pathways, which could be important in cellular responses todifferent NPs [149]. For instance, the uptake of some NPs by cells isstrongly dependent on the phase of the cell cycle [150]. Due to lack ofanalytical techniques, the effects of NPs on normal excretion processof cells have not been extensively investigated [151]. Haynes et al.[151–155] used a carbon-fiber microelectrode amperometry technique,which can detect chemicalmessengers at the single-granule level in realtime, to investigate the influence of gold, silver, TiO2 and SiO2 NPs onregulated exocytosis. Following pre-incubation of murine peritonealmast cells with 1nM gold NPs (28nm) for 48h, the amount of excretedserotonin per granule and the rate of intra-matrix expansion increasedmore than 20% and 30%, respectively. In addition, more than 30%decrease in the number of successful granule transportation and fusion




was observed [151]. Moreover, they investigated the effect of exposuretime and concentration [152], surface area, composition [154] and zetapotential [155] on the regulated exocytosis of serotonin frommast cells.According to their results, serotonin secretion from mast cells treatedwith gold NPs increased at early exposure times (24 h) but decreasedat later times (72 h). Furthermore, after 24 h of exposure, differentconcentration of NPs did not show any effect on the secretion ofserotonin and kinetics of release. However, after 72 h of NP exposure,serotonin secretion decreased with increasing NP concentration [152].Pre-incubation of murine peritoneal mast cells with three differentmetal oxide NPs (porous, nonporous SiO2 and nonporous TiO2 withsize of 24±3, 25±4, and 11±5nm, respectively) at 100μg/ml revealedthat SiO2 NPs, both nonporous and porous, decreased the number ofserotonin molecules released per granule. In addition, nonporous SiO2

exposure also caused a decrease in the frequency of release. As acomparison, pre-incubation with nonporous TiO2 only slowed thekinetics of secretion.

Exposure of murine adrenal medullary chromaffin cells to gold andsilver NPs [153] (with size of 28 and 61 nm, respectively) resulted invesicular matrix disruption, similar to that seen in mast cells reportedin previous studies [151,152]. Furthermore, the NPs did not interferedirectly with the recycling machinery and cellular function was unableto recover following vesicle content expulsion [153].

The effect of PLGA NPs on antigen-induced exocytosis of β-hexosaminidase and histamine from rat basophilic leukemia RBL-2H3cells was investigated by Tahara et al. [156]. Unmodified and chitosanmodified PLGA NPs (with size of 240 ± 4.7 and 338 ± 14.4 nm,respectively) at a concentration of 2.5mg/ml were pre-incubated withRBL-2H3 cells for 2 and 20 h. Experimental data showed that NPexposure only inhibited the release of histamine, and not β-hexosaminidase. This phenomenon was observed in cells pre-incubated with NPs for 20 h, but not for 2 h. After 20 h, some of theendocytosed NPs may have escaped from recycling endosome com-partment into the cytoplasm. Alternatively, some of the NP containingvesicles may have interacted with cytoplasmic compartments such asER, Golgi apparatus or intracellular secretory granules where theysubsequently alter the exocytosis profile of histamine. In cellsoverexpressing SNAP-23 recovery of histamine release was seen,which NP containing vesicles may compete with histamine containingvesicles for t-SNARE. Since β-hexosaminidase exists in both vesiclesthat contain NPs or histamines, its release profile should be unchanged.Other groups have also investigated the effects of various NPs onexocytosis. For instance, carboxyl-modified 30nm polystyrene NPs cantrigger calcium-dependent recycling of inflammatory granules inmastocyte like rat basophilic leukemia RBL cells [157]. Interestingly,mesoporous silica NPs could perturb intracellular vesicle traffickingand induce MTT formazan exocytosis, [158] similar to β-amyloidpeptides and cholesterol [159–161]. As another example, NPs canabsorb the proteins and nutrition components of the cell mediumleading to the toxicity of new modified medium to the cells [162]. Thisphenomenon suggests that the conventional toxicological evaluationof nanomaterials may contain several shortcomings [79,162,163].Thus, the modification of conventional toxicity assays (e.g. using hardcorona coated NPs instead of pristine coated one [164]) and theconsideration of the cell type effect are crucial matters to obtainreliable and reproducible nanotoxicology data [79]. These new conceptsoffer a suitable way to obtain a deep understanding on the cell-NPinteractions. In addition, by consideration of these ignored factors,the conflict of future toxicological reports would be significantlydecreased.

All these findings indicate that there are possible toxicologicalchallenges when using NPs for biomedical applications. But from adifferent perspective these processes could be used to change thesecretion from cells on purpose. Secretion plays an important rolefrom neurotransmitter release to insulin release and is therefore apotential pharmaceutical target.


8. Summary and outlook

This review highlights the central role of exocytosis processes on NPexcretion by cells. We presented a wide variety of examples of NPswithdifferent functionalities, made from different materials with varyingcharges, sizes, and morphologies, and discussed their intercellularpathways that lead to cellular release. Due to the variety of cell types,biological environments, and types of NPs, there are stillmany indistinctpathways that can result in exocytosis. This knowledge will be essentialto exploit the full potential of nanomaterials in both in vitro and in vivoapplications and to evaluate their toxicities. A better understanding ofexocytosis and related cellular functions and their dynamics will shedmore light on the design of NPs in-demand for different medicalapplications. Prolonged intracellular retention could provide a betterchance for NPs to exert their desired application more efficiently incells. In addition, alteration in the composition of the protein coronawhich can occur following uptake and exocytosis of NPs [84,85], maychange the biological fate of NPs. Therefore, a greater understandingof the altering corona may offer a way to predict the dosage of NPsrequired in various therapies and in the studies of nanotoxins.

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