1. Introduction
In the last several decades, the use of nanoparticles (NPs) has widely grown in the biological and biomedical fields, especially for diagnostic and drug delivery purposes. In fact, thanks to their size, shape, and surface properties, NPs can be engineered ad hoc for targeting specific tissues, and they represent an ideal candidate to overcome biological barriers [
1,
2,
3]. Among others, NPs have been proven to be able to cross the blood–brain barrier (BBB) and accumulate in the central nervous system (CNS) [
4,
5,
6]. In fact, due to the presence of tight junctions, the BBB remains the main obstacle to deliver contrast agents and drugs for the diagnosis and treatment of CNS-related pathologies [
7,
8]. Therefore, NPs represent a valid alternative to the invasive techniques currently used to administrate therapies to the brain [
9,
10,
11].
Many efforts have been made for designing and engineering nanomaterials for CNS theragnostic applications, and extensive research has been focused on elucidating the mechanisms used by cells to internalize NPs both in vivo and in vitro [
12,
13,
14]. Nevertheless, the use of NPs in nanomedicine presents some limits. In fact, it is widely reported that most NPs enter the cell by endocytosis [
15,
16]. The endocytic mechanism leads to NP accumulation along the endo-lysosomal pathway [
17,
18]. As a consequence, there is a decrease in the ability of NPs to cross the BBB [
19] and reach the brain parenchyma [
20,
21]. Furthermore, their accumulation in the lysosomal compartment causes, in some cases, cytotoxic effects due to the low pH and degradative environment [
22,
23].
On the other hand, few data are available about the long-term fate of NPs excreted from the BBB. It has been reported that, for the endocytosis process, NP excretion from cells can be affected by several parameters [
24]. For instance, it has been shown that the exocytosis process is size-dependent. In fact, Serda and colleagues showed that the exocytosis of super-paramagnetic iron oxide NPs (SPIONs) trapped in porous silicon carriers from the murine macrophage J774 cell line was significantly greater for carriers containing 15 nm SPIONs than carriers loaded with 30 nm NPs at the same concentration [
25]. Moreover, 100 nm polystyrene NPs were more difficult to excrete than 50 nm polystyrene NPs from human lung cells [
26]. In addition to their size, the NP shape is also important for exocytosis processes. As an example, the fraction of rod-shaped transferrin-coated gold NPs released from the HeLa and SNB 19 cell lines was much higher than sphere-shaped NPs [
27]. Additionally, different surface modifications have important consequences on the exocytosis profile of NPs. In fact, gold NPs, functionalized with a non-targeting or targeting peptide, but with the same physicochemical properties (i.e., a size of ~35 nm and a zeta potential of ~−18 mV), showed different exocytosis profiles. The non-targeted NPs were re-uptaken by the cells after 4 h, while for a period of 6 h, the targeted NPs were progressively exocytosed [
28]. Moreover, it was demonstrated that surface decoration with membranotropic virus-derived peptide gH625 enhanced the BBB crossing of aminated polystyrene NPs in vitro [
29]. Finally, the exocytosis kinetics of NPs is influenced by cell types as well. Recently, Liu and co-workers demonstrated that the exocytosis kinetic parameters strongly depended on the cell types but were insensitive to the initial intracellular concentration of carbon dots [
30].
However, despite many efforts, the findings regarding NPs’ release mechanisms and fate after their experience in the intracellular BBB environment remain unclear and elusive [
24]. In fact, the physicochemical properties of NPs’ surfaces as well as their colloidal stability can change during their uptake, lysosomal, and exocytosis processing [
24,
31,
32]. Therefore, excreted NPs may exhibit a different surface and aggregation state after their cellular release. Understanding the mechanisms of NP cellular release is particularly relevant, not only to predict the efficiency of NPs to cross the BBB, but also to assess the capability of excreted NPs to be internalized by target cells that are in the brain parenchyma. Such investigations may provide important insights into the design of efficient and safe nanoprobes and drug-loaded nanocarriers, thus boosting their theragnostic potential.
Our hypothesis is that the main limit of NP excretion is represented by the propensity of NPs to accumulate in the endo-lysosome compartment. From this perspective, we previously demonstrated that by bypassing the endo-lysosomal pathway using a pneumatic method, namely the gene gun, it was possible to obtain free NPs in the cytoplasm in several cell lines [
23]. Hence, shot NPs prevalently showed a random walk behavior, typical of non-vesicle confined NPs, and no co-localization with lysosomes. Furthermore, cytoplasmic localization reduced the cytotoxicity of some NPs significantly [
23,
33].
In this work, we analyzed and compared the excretion profile of endocytosed (endo) and shot NPs from human brain endothelial cells cultured in standard and Transwell cell culture systems, as in vitro models of the human BBB, to shed light on the extracellular fate of NPs after their passage through the BBB. In particular, we delivered 100 nm carboxylated polystyrene NPs, chosen as a commercially available nanoprobe model, to the endo-lysosomal compartment by the endocytosis mechanism and directly to the cytosol by the gene gun method in the in vitro model of the BBB, composed of human brain endothelial hCMEC/D3 cells. We analyzed in detail the effects of the different intracellular environments (endo-lysosomes and cytosol) on the excretion profile of the NPs. In addition, we compared the obtained results with those obtained with nanoparticles functionalized with the cell-penetrating peptide gH625 able to escape the endo-lysosomal compartment [
29,
33]. Moreover, we preliminarily characterized the aggregation state and the amount of protein corona of the released NPs as a function of the NP intracellular localization. Furthermore, these parameters were correlated with the capability of excreted NPs to be taken up by the human glioblastoma U87 cell line as a model of brain tumor tissue and, hence, as a possible target for nanomaterials in the CNS.
2. Materials and Methods
2.1. Nanoparticles
FluoSpheres yellow-green carboxylate-modified microspheres (NPs) with a diameter of 100 nm were purchased by Invitrogen (Thermo Fisher, Waltham, MA, USA).
2.2. Cell Cultures
The immortalized human cerebral microvascular endothelial cells/clone D3 (hCMEC/D3) were kindly provided by Dr. P.-O. Couraud (INSERMU1016 UMR 8104, Institut Cochin, Paris, France). The hCMEC/D3 cells were cultured in EBM-2 medium (Lonza) supplemented with ascorbic acid (Sigma Aldrich, St. Louis, MO, USA), hydrocortisone (Sigma Aldrich), basic fibroblast growth factor (Sigma Aldrich), fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), and HEPES (Invitrogen, Waltham, MA, USA). Cells were grown on 100 mm diameter Petri dishes coated with rat tail collagen type-I (BD Biosciences, San Jose, CA, USA) at 37 °C with 5% CO2, 95% air, and saturated humidity. Cells used for the experiments were maintained between passage 23 and 33.
The immortalized human glioma cell line U87-MG (U87) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and 100 units/mL penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2.
2.3. NP Intracellular Shooting by Gene Gun Method
A ballistic system (Gene Gun, BioRad, Hercules, CA, USA) was used to achieve free NPs in the cytoplasm as previously reported [
33]. In brief, NPs were suspended in 30 µL of sterile MilliQ water at the final concentration of 1.8 × 10
12 NPs/mL, deposited and left to dry on a rupture disk under a sterile hood. After solvent drying, 650 psi pressure was used to shoot NPs within cells, formerly seeded in 35 mm culture dishes at a density of 2 × 10
4 cells per cm
2. After shooting, cells were rinsed with PBS five times to remove non-internalized NPs. The cells were then harvested, counted, and reseeded on the opportune substrate for the specific experiments described below.
2.4. Quantification of Internalized Nanoparticles in hCMEC/D3 Cells
2.4.1. Quantification of Endocytosed Nanoparticles
In order to evaluate the number of endocytosed NPs within cells, cells were roughly rinsed with PBS, after incubation of the NPs for 24 h, trypsinized, and counted in a Neubauer chamber. Cells were then centrifuged, and the pellets were lysed with lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris). To measure the amount of internalized NPs, cell lysates were analyzed with a spectrofluorometer (EnSpire, Perkin-Elmer, Waltham, MA, USA). Data were reported as the mean NP number normalized to the cell number.
2.4.2. Quantification of Shot Nanoparticles
To evaluate the number of shot NPs within cells, the cells were shot with NPs by the gene gun, washed roughly with PBS, and then allowed to recover for 24 h at 37 °C. After recovery, cells were roughly rinsed with PBS, trypsinized, and counted in a Neubauer chamber. Afterward, cells were centrifuged, and the pellets were lysed with lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris). The amount of internalized NPs was measured by analyzing cell lysates with a spectrofluorometer (EnSpire, Perkin-Elmer). Data were reported as the mean NP number normalized to the number of cells.
2.5. Cytotoxicity Assay
Alamar blue assay was performed to evaluate cell viability of cells treated with shot and endocytosed NPs. The results of the assay were compared to non-treated cells, which were used as a control. For the assay, 3 × cells, seeded on a 35 mm cell culture dish, were incubated for 24 h with NP suspensions to allow endocytosis. On the other hand, for NP shooting, cells were shot with NPs by the gene gun, washed roughly with PBS, and then, allowed to recover for 24 h at 37 °C. After recovery, cells were trypsinized, counted, and seeded on fresh 35 mm cell culture dishes. Alamar blue assay was carried out according to the manufacturer’s procedure at 24, 48, and 72 h after NP exposure. Absorbance of Alamar blue reagent solution was read at 570 nm and 600 nm by a plate reader (Perkin-Elmer). Data were reported as the percentage of viable cells normalized to non-treated cells.
2.6. Co-Localization with Lysosomes
For co-localization tests, cells were rinsed twice with PBS after NP incubation, to remove non-internalized nanoparticles and fixed with 4% paraformaldehyde at room temperature (RT). After permeabilization and blocking, the lysosomes were localized with rabbit anti-LAMP 2 polyclonal primary antibodies (Abcam, Cambridge, UK) and with 568 goat anti-rabbit secondary antibodies (Molecular Probes, Invitrogen). All samples were then observed using a confocal microscope (SP5 Leica, Leica, Wetzlar, Germany) with a 63× oil immersion objective.
2.7. Excretion of Shot and Endocytosed Nanoparticles
To follow NP exocytosis process, 1 ×
cells were seeded into a 24-well plate and allowed to reach the confluence. Hence, cells were incubated with 100 nm NP suspension at the final concentrations of 3.60 ×
NP/mL in cell culture medium for 24 h at 37 °C. After NP incubation, cells were rinsed twice with PBS to remove non-internalized NPs and a fresh culture medium was added. The exocytosis of NPs was analyzed at different timepoints: 2, 24, 48, and 72 h. The amount of excreted NPs was monitored by measuring the content of NPs in both the cells and the medium as follows: at each time, cells were lysed with lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris) and cell lysate was analyzed by measuring fluorescence intensity (FI) at 488 nm excitation wavelength by spectrofluorometer (EnSpire, Perkin-Elmer). In order to measure the amount of exocytosed NPs, 100 µL of cell culture medium were collected and measured with spectrofluorometer. In order to quantify the amount of excreted NPs allowed to enter the cells by using gene gun method, 1 ×
cells were seeded on a 35 mm cell culture dish. After NP shooting, cells were trypsinized, counted, and reseeded in a 24-well plate. Excreted NPs were measured at the following timepoints: 0, 2, 24, 48, and 72 h. The amount of excreted NPs was measured both in cell and in medium by using a spectrofluorometer as described above. The number of released NPs was expressed as the percentage of the total number of NPs found in cell lysate and cell culture medium according to the following equation:
2.8. Excretion of Shot and Endocytosed Nanoparticles in Transwell Systems
Transcytosis experiments were carried out in order to evaluate the NP excretion profile on a Transwell system. Transwell permeable inserts (6.5 mm in diameter, 3 μm pore size; Corning) were pre-treated with rat tail type I collagen for 1 h at 37 °C in a dry incubator in order to allow the cell adhesion. After 24 h from NP endocytosis and shooting, 7 × 104 hCMEC/D3 cells were seeded on a Transwell filter and incubated for 72 h at 37 °C. To assess the growth of cells on the porous inserts, transendothelial electrical resistance (TEER) was measured by using Millicell1-ERS Voltohmmeter (Millipore, Billerica, MA, USA). After 72 h incubation time, basal medium was collected and the amount of NPs was quantified by using a spectrofluorometer (EnSpire, Perkin-Elmer) at 488 nm wavelength.
2.9. Transmission Electron Microscopy (TEM)
In order to precisely localize the intracellular nanoparticles in hCMEC/D3 cell line on Transwell, TEM analysis was performed. The samples were prepared as described in the following: after NP exposure, cells were trypsinized, reseeded on Transwell filter and fixed after 72 h. Afterward, Transwell containing confluent hCMEC/D3 cell monolayer was rinsed with PBS and fixed with a solution of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2 h at RT. Then, cells were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 4 °C, and dehydrated in graded concentrations of ethanol (30%, 50%, 70%, 95%, and 100%). Cells were then embedded in epoxy resin. Ultrathin sections were cut with an ultramicrotome (UC7, Leica). Sections of 90 nm were collected on copper grids and stained with lead citrate and uranyl acetate. The grids were visualized using a TEM Tecnai G2 (FEI Company, Hillsboro, OR, USA).
2.10. gH625 Peptide Synthesis, Nanoparticle Conjugation, and Characterization of gH625-NPs
gH625 peptide (Ac-HGLASTLTRWAHYNALIRAFGGG-COOH) were synthesized using the standard solid-phase-9-fluorenylmethoxycarbonyl (Fmoc) method as previously reported [
29]. Good yields of 30–40% in purified peptide were obtained.
Peptide conjugation to orange-fluorescent amine-modified polystyrene, 100 nm, nanoparticles (NPs) (Sigma-Aldrich) was performed as described previously [
29]. Briefly, a solution of the peptide, EDC (1-Ethyl-3(3-dimethylamino-propyl)-carbodiimide, hydrochloride) and NHS (
N-Hydroxysuccinimide) in a molar ratio of 4:4:1 was prepared in PBS buffer at pH 7.4, at RT under stirring for 30 min. NPs were conjugated with the preactivated-peptide in MES 0.1 M buffer at pH 5.5 for 3 h at RT in the presence of Tween 20. The yield of the reaction was higher than 90%. The peptide-NPs were purified from the unconjugated NPs by exclusion chromatography on a 1 × 18 cm Sephadex G-50 (Amersham Biosciences, Amersham, UK) column pre-equilibrated in PBS buffer at pH 7.4. The fluorescence spectra of peptide-NPs and unconjugated NPs were measured in a Cary Eclipse Varian fluorescence spectrophotometer (Varian Medical Systems, Palo Alto, CA, USA) in the same conditions. For 100 nm NPs, 35% of functionalization degree was used.
The gH625-NPs were characterized by circular dichroism (CD) and measurements of size and zeta-potential. CD spectra were recorded using a Jasco J-715 spectropolarimeter (Jasco, Hachioji-shi, Tokyo, Japan) in a 1.0 or 0.1 cm quartz cell at RT. The spectra were an average of 3 consecutive scans from 260 to 195 nm, recorded with a band width of 3 nm, a time constant of 16 s, and a scan rate of 10 nm/min. Spectra were recorded and corrected for the blank. Measurements of zeta potential and size of gH625-NPs were made with a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). The measurements were conducted at 25 °C using a 3.6 × 1010 NP/mL suspension in MilliQ water at pH 7 (ionic strength 6 × 10−5 M). All measurements were performed in triplicate for each sample.
2.11. BCA Assay
The amount of adsorbed proteins on NP surface was determined by BCA assay (Sigma Aldrich). Briefly, 5 × 104 cells were seeded on a 35 mm cell culture dish and incubated for 24 h with NP suspensions to allow endocytosis. Conversely, for NP shooting, cells were shot with NPs by gene gun method. After 24 h, endo and shot cells were washed roughly with PBS, trypsinized, counted, and seeded at a density of 7 × 104 cells/cm2 on a Transwell filter with a pore size of 4 µm and incubated for 72 h at 37 °C. The Transwell filters were pre-treated with rat tail type I collagen for 1 h at 37 °C in a dry incubator in order to allow the cellular adhesion. Afterward, the basal medium was recovered, centrifuged, and the pellet was rinsed twice with PBS in order to remove cellular debris. Next, the pellet was suspended in 300 µL of sterilized water and was analyzed using a spectrofluorometer (EnSpire, Perkin-Elmer) in order to quantify the amount of recovered NPs. BCA assay was performed using the same number of NPs for endo and shot NPs according to the manufacturer’s procedure (Sigma Aldrich). Absorbance of BCA assay reagent solution was read at 560 nm by a plate reader spectrophotometer (EnSpire, Perkin-Elmer). Data were reported as µg/mL of protein normalized with non-conditioned NPs.
2.12. Quantification of Nanoparticles in U87 Cell Line
In order to evaluate the number of endocytosed NPs in brain tumor cells, 2 × 103 U87 cells were seeded on a 96-well plate and incubated with NPs recovered from the basal media of hCMEC/D3 cell layers on Transwell systems after endocytosis and shooting at 4 × 106 NPs/mL concentration. After incubation of the NPs for 72 h, cells were roughly rinsed with PBS and lysed with lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris). Cell lysates were analyzed using a spectrofluorometer (EnSpire, Perkin-Elmer) to measure the amount of internalized NPs. Data were reported as the number of NPs per cell. For confocal microscopy images, cells were seeded on 12 mm glass coverslips and incubated with 4 × 106 NPs/mL for 72 h. Then, cells were washed with PBS to remove non-internalized NPs, fixed with 4% paraformaldehyde, and stained with red fluorescent wheat germ agglutinin (WGA) (Thermo Fisher Scientific) and DAPI (Sigma) to localize cell membranes and nuclei, respectively. All samples were then observed using a confocal microscope (SP5 Leica) with a 63× oil immersion objective.
2.13. Statistical Analysis
Quantitative results were reported as mean ± standard deviation (SD). Statistical comparisons were performed with a Student’s unpaired test. p values < 0.05 were considered statistically significant.
4. Discussion
The fulfillment of the great potential of NPs for biomedical application, as nanosensors and nanocarriers, needs a complete knowledge of how they interact with cells, tissues, and organs. Despite numerous technological advancements and very interesting properties, very few nanomaterials have been approved by the Food and Drug Administration (FDA) for use in medical applications. Therefore, new efforts must focus on a detailed understanding of the interaction of nanomaterials with the cells at the fundamental level. There is proof that nanoparticles are indeed taken up by cells with an extent that depends on their physical–chemical properties and subsequent interactions. However, their subsequent release and/or intracellular localization/degradation, transfer to other cells, and/or translocation across tissue barriers are still unclarified and under investigation. In this work, we aimed at elucidating the behavior and fate of NPs as a function of their different intracellular localization, i.e., confined into endo-lysosomal compartment or free in the cytosol. We chose commercially available 100 nm carboxyl-modified polystyrene NPs as a model of fluorescently labelled nanoprobes. To obtain a different intracellular localization of NPs, we incubated human brain endothelial hCMEC/D3 cells with NPs dispersed in cell culture media or shot by gene gun method according to a procedure that we previously optimized and used for different cell types [
23].
It is known that carboxyl-modified polystyrene NPs of different sizes did not exert cytotoxic effects on several cell types after internalization by endocytic mechanism [
39,
40]. Our results indicated that these NPs were not cytotoxic for human brain endothelial hCMEC/D3 cells in our experimental conditions, which is in agreement with the already reported observations on other cell types [
39,
40]. Moreover, the gene gun method did not affect cell viability as well, thereby demonstrating the feasibility of this method to deliver NPs directly inside the cytoplasm, and to allow in vitro fundamental studies of nano–bio interactions.
Previously, we demonstrated that, when exposed to the cell surfaces, amino-modified, 100 nm polystyrene NPs were taken up by mouse brain endothelial bEnd.3 cells through endocytic mechanisms and tended to accumulate in lysosomes [
23,
29]. Conversely, when NPs were allowed to bypass endocytic mechanisms, they were able to remain free in the cytoplasm and to not co-localize with lysosomes [
23]. Similarly, in this work, we observed that when incubating carboxyl-modified 100 nm NPs with human brain endothelial hCMEC/D3 cells, most of them were internalized by endocytosis. Indirect immunofluorescence against the endo-lysosomal marker LAMP2 indicated co-localization of endocytosed NPs with these intracellular compartments. Conversely, shot NPs were free in the cytoplasm, as evidenced by TEM images.
The endocytosis of nanomaterials has been widely investigated in recent years. However, only a limited number of reports show the exocytosis of NPs. In general, exocytosis processes occur via the fusion of vesicular membranes with the plasma membrane, leading to the release of the vesicular content into the extracellular environment [
41]. Exocytosis can follow two pathways in the cell: constitutive and regulated [
42,
43]. Constitutive exocytosis is present in all cell types; the regulated pathway is observed in cells that are specialized in secreting their products on demand. It has been reported that endocytosis and exocytosis of nanomaterials are coupled and can influence each other by stimulating or compensating [
41]. Moreover, exocytosis occurs more slowly than endocytosis and it is affected by several parameters such as cell type, NP properties (i.e., size, surface charge, surface functionalization, etc.), NP dose, and exposure time [
24].
Our results indicated that endocytosed NPs were excreted by human brain endothelial cells. Their release was slower and fewer than positive NPs used in previously reported works [
41]. These observations confirmed that the excretion of nanomaterials depends on NP type. Furthermore, our data indicated that cytoplasmic nanoparticles (shot NPs) were expelled faster than the same endocytosed NPs. In particular, the percentage of endocytosed NPs that were able to leave the cells was lower than shot NPs at 24, 48, and 72 h, demonstrating that internalized NPs that bypassed the endo-lysosomal compartment were excreted more efficiently. After 48 h, the release percentages of both endocytosed and shot NPs reached a plateau, probably due to a balance between NP excretion and the cellular re-absorption of released NPs. Taken altogether, these results showed that the intracellular localization of NPs affects their retention. The hypothesis is that NPs enclosed in endo-lysosomal vesicles are less able or slower to leave the cells compared to cytosolic NPs because the exocytic vesicles (with cargo) must be formed/separated from multivesicular bodies, which are formed following the fusion of the endosomes, and transported to the cell membrane to be excreted. In line with our observations and in spite of the current sparse knowledge on the mechanisms of NP exocytosis, Liu et al. reported that the lysosomal pathway is not the main exocytosis mechanism used by NPs to be excreted from several cell types despite the prevalent NP accumulation into the lysosomes [
30].
Transwell experiments were carried out with the aim to work in a more physiologically relevant in vitro model of BBB and to achieve information about BBB crossing by NPs. Results obtained in Transwell systems demonstrated that the intracellular localization of NPs also influenced NP transport and release across the BBB layer. The endocytosis process of carboxylated NPs unavoidably leads to the accumulation into lysosomal compartment as widely described in literature [
39]. Lysosomes are a terminal degradative compartment of the endocytic pathway, hence, transport into lysosomes is considered mainly unidirectional [
44,
45]. Even if lysosome fusion with the plasma membrane is a process present in several specialized cell types [
46], the accumulation in lysosomes reduces the ability of NPs to reach the basolateral side of the BBB in vitro [
19,
47], thus diminishing the transport of NPs to the CNS. On the contrary, by bypassing the lysosomal compartment, we demonstrated that NPs are able to reach the basal compartment more efficiently. Therefore, using strategies that help NPs to escape the endo-lysosomal compartment ameliorates NP transcytosis. In this context, we previously reported that the functionalization with the virus-derived cell-penetrating peptide gH625 affected the intracellular behavior of aminated polystyrene NPs, reduced endo-lysosomal accumulation, and improved BBB crossing in bEnd.3 cells [
23]. Furthermore, by avoiding lysosomal storage, the presence of gH625 peptide reduced the cytotoxic effects of 50 nm aminated polystyrene NPs in several cell types [
33]. Based on these results, in this work, we used gH625-functionalized NPs as a positive control to confirm the role of different intracellular localization on NP excretion from hCMEC/D3 cells (
Figure 5 and
Figure 6). Our preliminary results showed that the gH625 functionalization allowed NPs to be released by the hCMEC/D3 cells more efficiently compared to the blank (non-functionalized) NPs, starting from the same initial NP intracellular content. In fact, after 24 h, the percentage of released gH625-NPs was two times higher than blank NPs and comparable to shot NPs. In addition, experiments carried out in Transwell systems indicated that the number of gH625-NPs transported to the basal compartment was four times higher than blank NPs. The functionalization with gH625 peptide determined the same effects in terms of hCMEDC/D3 cell excretion as shot NPs, thus confirming that reducing the endo-lysosomal accumulation of NPs improves their extracellular release.
Uptake and lysosomal processing can modify NP surface, thus likely changing the surface properties of the exocytosed NPs. As a consequence of such modification, exocytosed NPs may recognize different targets or receptors on the cell membrane influencing their long-term fate [
24,
31]. In this framework, we analyzed the uptake of endocytosed and shot NPs after their release from hCMEC/D3 cells in human brain U87 cells. Our data indicated that endocytosed NPs were internalized to a lesser extent in U87 cells than shot NPs. This result was probably due to the propensity of endocytosed NPs to leave the cells as large aggregates (as demonstrated from DLS analysis of excreted NPs). The aggregation of NPs because of the uptake mechanism unavoidably led to a physical hindrance and, hence, a greater difficulty to be internalized by target cells. Moreover, as shown by BCA assay, released NPs showed a different amount of adsorbed proteins depending on the different internalization mechanism (
Figure 7). In particular, endocytosed NPs adsorbed a greater amount of proteins upon hCMEC/D3 release than shot NPs. These observations suggest that the different intracellular compartments experienced by endocytosed and shot NPs also affected NP surface in terms of protein corona formation and, likely, composition. These findings are in agreement with Xiao et al., showing that BBB crossing changed the composition of the protein corona of transferrin (Tf)-functionalized NPs as well as their targeting capabilities [
32]. Because the NPs used in this work were not functionalized with targeting moieties and analyzing confocal images of
Figure 7, we hypothesized that exocytosed NPs were likely internalized by U87 cells through endocytosis and, hence, accumulated into the endo-lysosomal compartment. This aspect should be considered to design effective NPs for relevant clinical applications by creating multistage NPs (e.g., with a pH-sensitive shell functionalized with molecules that allow BBB targeting and crossing—by avoiding endo-lysosomal confinement—and a drug-loaded core functionalized with specific targeting moieties).
The results of this work open interesting new questions about the nano–bio interactions to identify the underlying mechanisms that regulate NP exocytosis and about the nanomaterial design to boost their theragnostic potential. In this direction, further investigations are necessary to elucidate biological mechanisms involved in the excretion of NPs not confined in the endo-lysosomal compartments.