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Cite This: ACS Appl. Bio Mater. 2019, 2, 3279−3289
In Vitro Blood−Brain Barrier Models for Nanomedicine: ParticleSpecific Effects and Methodological Drawbacks Leopoldo Sitia,†,§ Tiziano Catelani,‡,∥ Daniela Guarnieri,*,†,⊥ and Pier Paolo Pompa*,† †
Nanobiointeractions & Nanodiagnostics, Istituto Italiano di Tecnologia (IIT), via Morego 30, Genova 16163, Italy Electron Microscopy Facility, Istituto Italiano di Tecnologia, via Morego 30, Genova 16163, Italy § Department of Biomedical and Clinical Sciences “L. Sacco″, Università Degli Studi di Milano, via G. B. Grassi 74, Milano 20157, Italy ∥ Piattaforma Interdipartimentale di Microscopia, Università Degli Studi di Milano-Bicocca, Piazza della Scienza 2, Milano 20126, Italy ⊥ Dipartimento di Chimica e Biologia “A. Zambelli”, Università di Salerno, via Giovanni Paolo II 132, Fisciano, Salerno I-84084, Italy
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ABSTRACT: Predicting the therapeutic efficacy of a nanocarrier, in a rapid and cost-effective way, is pivotal for the drug delivery to the central nervous system (CNS). In this context, in vitro testing platforms, like the transwell systems, offer numerous advantages to study the passage through the blood−brain barrier (BBB), such as overcoming ethical and methodological issues of in vivo models. However, the use of different transwell filters and nanocarriers with various physical− chemical features makes it difficult to assess the nanocarrier efficacy and achieve data reproducibility. In this work, we performed a systematic study to elucidate the role of the most widely used transwell filters in affecting the passage of nanocarriers, as a function of filter pore size and density. In particular, the transport of carboxyl- and amine-modified 100 nm polystyrene nanoparticles (NPs), chosen as model nanocarriers, was quantified and compared to the behavior of Lucifer yellow (LY), a molecular marker of paracellular transport. Results indicate that the filter type affects the growth and formation of the confluent endothelial barrier, as well as the transport of NPs. Interestingly, the in situ dispersion of NPs was found to play a key role in governing their passage through the filters, both in absence and in presence of the cellular barrier. By framing the underlying nanobiointeractions, we found that particle-specific effects modulated cellular uptake and barrier intracellular distribution, eventually governing transcytosis through their interplay with “size exclusion effects” by the porous filters. This study highlights the importance of a careful evaluation of the physical−chemical profile of the tested nanocarrier along with filter parameters for a correct methodological approach to test BBB permeability in nanomedicine. KEYWORDS: blood−brain barrier, transwell systems, nanocarriers, nanobiointeractions, transport, porous membranes
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in vivo has not been reported so far.4 The BBB is an active interface between the circulation and the CNS, which strictly controls the molecular and cellular traffic between the blood and the brain, taking an important share in providing a proper environment for neuronal function.14,15 At the same time, the BBB acts as a very selective barrier, hindering the passage of pathogens, xenobiotics, and other exogenous materials to the
INTRODUCTION Disorders of the central nervous system (CNS) are among the most severe diseases from both individual and social points of view;1−3 yet the delivery of drugs to the brain is still a major pharmacological challenge.4,5 In the latest years, nanomedicine has attempted the development of several nanocarriers (NCs), to improve drug delivery to the brain.6−13 However, despite massive efforts, the therapeutic possibilities of CNS diseases are still limited. In fact, potential nanocarriers have a typically poor efficacy because of their weak penetration through the blood−brain barrier (BBB), and direct evidence of transcytosis © 2019 American Chemical Society
Received: April 10, 2019 Accepted: June 25, 2019 Published: June 25, 2019 3279
DOI: 10.1021/acsabm.9b00305 ACS Appl. Bio Mater. 2019, 2, 3279−3289
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Figure 1. Characterization of the different membranes by SEM. Panels show representative top sections of the four different filters F0.4L (A), F0.4H (B), F1 (c), and F3 (D). Scale bar: 5 μm. Panels E−H show the relative pore area distribution across the different membranes evaluated by the analysis of five images per filter type.
brain.14,16 Therefore, testing the BBB permeability to a nanocarrier is of crucial importance to predict its therapeutic potential, as early as possible in its development pipeline. One of the most accurate methods to measure BBB permeability to a nanocarrier is the use of in vivo models, such as the internal carotid artery single injection or perfusion, intravenous bolus injection, brain efflux index, and intracerebral microdialysis.17,18 These strategies provide truthful information regarding brain uptake and can be complemented with novel imaging techniques, such as magnetic resonance imaging and positron emission tomography.18,19 However, in addition to ethical concerns, in vivo experiments are becoming more and more expensive and time-consuming and are not suitable for medium or high throughput screening.20−22 Therefore, they are rarely used in early stage testing. To address such issues, in vitro models of the BBB have been developed.23,24 Among the others, the transwell systems are the most widely used.24 They are based on porous inserts that separate two compartments, namely, an apical compartment that represents the blood side and a basolateral one that represents the brain parenchyma.25 The cells usually grow on the top of the porous inserts until they form a confluent layer, presenting the typical phenotype of brain endothelium with tight junctions.26 The transwell systems provide several advantages, such as low cost, high reproducibility, easiness of use, and high throughput, that make them useful for large screenings and early stage testing.23 Although with the general limitations of in vitro models, these systems have proven to work well with small biological molecules and drugs.24,27,28 However, their recent and emerging use to study the transport of nanocarriers is raising several concerns.11,29−32 In fact, many works reported on the ability of several nanocarriers to overcome the BBB in vitro.33−36 Nevertheless, the use of different transwell systems and nanocarriers with various physical−chemical properties makes it difficult to assess the nanocarrier efficacy and achieve data reproducibility.29 Notably, the different types of the inserts used for in vitro analysis may influence the quality of the cellular barriers37 and physical interaction with the selected nanocarriers. Consequently, in vitro analyses may often fail to predict in vivo results.
One of the main issues in transport studies of nanocarriers across transwell systems is related to the nanocarrier size.38,39 In fact, contrary to biological molecules or drugs, nanocarriers have sizes comparable to the dimensions of insert pores. Their transport ability may thus be strongly influenced by the type of the porous filter. In addition, the dispersion status of nanocarriers in biological media can further enhance such aspect. Actually, the primary size of nanocarriers can significantly change because of their interactions with biological environments that induce the formation of protein corona as well as particle agglomerates (variable in size and shape).32,40 This depends on nanocarrier physical−chemical properties and can happen before, during, and after the passage through the cell layer. In case of agglomeration in cell culture media, nanocarriers do not act as single particles, possibly leading to misinterpretation of their real BBB permeability. In this framework, the choice of the porous insert plays a crucial role in determining the ability of a nanocarrier to translocate the in vitro BBB and avoiding biased results. However, even though these issues have been suggested in the literature,32 a systematic investigation on their effects in transport studies of nanocarriers across the BBB in vitro still lacks, thus limiting the possibility to predict the real behavior of a nanocarrier in vivo. This work aims at elucidating the role of the widely used transwell systems in affecting the passage of nanocarriers in the presence and absence of a confluent brain endothelial cell layer. In particular, a systematic comparison of four different transwell porous inserts was performed as a function of pore size and density. The transport of carboxyl- and aminemodified 100 nm polystyrene nanoparticles (NPs), chosen as model nanocarriers and showing different stability in biological media, was quantified and compared to the behavior of Lucifer yellow (LY), a molecular marker of paracellular transport. Several particle-specific effects were observed and analyzed.
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RESULTS AND DISCUSSION Filter Characterization. The role of porous inserts in influencing the transport of nanocarriers was studied on four different filter types. In particular, we chose commercially available inserts made of the same material, namely, polyethylene terephthalate (PET), and different nominal pore size, 3280
DOI: 10.1021/acsabm.9b00305 ACS Appl. Bio Mater. 2019, 2, 3279−3289
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Figure 2. DLS and TEM characterization of COOH and NH2 NPs in ddH2O and in DMEM. Size distribution of COOH NPs (A) and NH2 NPs (B) in ddH2O (red line) and complete DMEM (green line). Average size, polydispersity (PDI), and ζ potential (C). Representative TEM images of NPs: in ddH2O, COOH NPs are highly monodispersed (D, E), while NH2 NPs seem less homogeneous (H, I); in complete DMEM, COOH NPs (F, G) remain quite monodispersed, while NH2 NPs (J, K) tend to aggregate, forming larger clusters, likely due to the presence of serum proteins.
i.e., 0.4, 1, and 3 μm. Moreover, for a 0.4 μm pore size, we used two inserts with a different pore density. The filters were named hereafter according to their morphological features as F0.4L (low pore density), F0.4H (high pore density), F1, and F3. First, a detailed characterization of the morphology of the filters was carried out by scanning electron microscopy (SEM). Representative top views of all filters are reported in Figure 1. In F0.4L, we measured an average pore diameter (dp) of 0.39 ± 0.06 μm, whereas F0.4H displayed a slightly larger dp and broader size dispersion, namely, 0.54 ± 0.14 μm. The pore sizes measured for F1 and F3 filters were 0.93 ± 0.14 and 2.5 ± 0.68 μm, respectively, in line with the nominal values. In general, most of the pores had circular profiles, though in F0.4H several pores were found to overlap and fuse together in irregular shapes, creating larger membrane openings than the nominal size (up to 1 μm of diameter, Figure 1B,F). The second population of fused pores accounted for more than 25% of the total pores (Figure S1). Although to a minor extent, this was also observed in F3 inserts, where 10% of fused pores
was found. However, due to their lower proportion, the distribution of the pores in F3 was more homogeneous around the average value of 3 μm (Figure 1D,H). The formation of fused species elicited by the random surface distribution of the pores can be ascribed to the stochastic production method followed to fabricate the membranes.41 Important parameters to be considered in filter characterization are also the pore density and the percentage of total pore area over the whole membrane area (AF). In particular, we noticed that the pore density value was the highest for F0.4H (1 × 108 pores/cm2), followed by F0.4L (4 × 106 pores/cm2), and F1 and F3 inserts, which had a similar pore density (2 × 106 pores/cm2). Moreover, on the basis of SEM analyses, we measured an AF of 15.72% for the F0.4H filter, whereas the other membranes had significantly lower values, namely, 8.30% for F3 and only 1.51% and 0.51% for F1 and F0.4L inserts, respectively (Figure 1E− H). Such parameters have to be carefully considered in transport experiments through the BBB, having a direct impact on two aspects: (i) The total surface area available for cell 3281
DOI: 10.1021/acsabm.9b00305 ACS Appl. Bio Mater. 2019, 2, 3279−3289
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Figure 3. Transport studies of NPs and LY through empty filters. Data are expressed as the percentages of transported NPs and LY in the basolateral compartment with respect to the equilibrium concentration between apical and basolateral sides. Data are reported as mean ± standard deviation.
different behavior observed in culture medium for COOH and NH2 NPs, with significant changes in terms of both size and surface charge with respect to the primary particles, might be of great relevance when studying their transport through the transwell systems. In particular, the significant NP aggregation could play a key role in barrier crossing analyses, with filters that have comparable pore sizes. Transport of LY and NPs through Empty Filters. To evaluate if the characteristics of the different filters affect NP transport through the membranes, we first investigated the behavior of NPs in empty inserts and compared them with the transport of LY molecules. To this aim, LY, COOH, and NH2 NPs were incubated in the apical compartment of the filters, and then the fluorescence of the basolateral media was measured (Figure 3) (100% passage indicated an equal distribution of NPs in the apical and basolateral chambers and was referred to as “equilibrium concentration”; for more details, see Materials and Methods). As expected, the transport of LY was not significantly affected in the case of F0.4H, F1, and F3 filters. A more pronounced effect was observed with the F0.4L insert, likely due to its very low AF (0.51%) that partially prevented diffusion and equilibration between the two compartments, with a transport efficiency of 60%. Interestingly, the transport of COOH NPs resembled the molecular transport of LY (especially for F0.4H and F3 inserts, which have a higher AF), thanks to the monodispersion of the particles in the culture medium (Figure 2A,C). For the lower AF filters (F0.4L and F1), NP transport was partially hindered, with F0.4L showing the strongest reduction (27% efficiency). Such latter effect may be due to the combination of reduced pore size and filter porosity. The effect of the inserts was much more evident on the transport of NH2 NPs. Here, only 36% of NPs was transported through the F3 inserts (Figure 3). The hindrance effect of the filters further increased with the lower pore size filters. In fact, we measured as low as 2.5% of transported particles in F1 and F0.4H filters and a negligible fraction (0.2%) for F0.4L. These results were consistent with the tendency of NH2 NPs to agglomerate in complete DMEM (Figure 2B,C), so that the filters exert a size exclusion effect. As a control, we performed the same experiments by dispersing NH2 NPs in ddH2O (where these NPs were more stable according to DLS data), and we found significantly higher transport efficiencies, going up to 60% for F1 and 75% for F3 inserts. Interestingly, these values were similar to what was observed for COOH NPs (Figure 3), suggesting that the particle dispersion status plays a primary role in governing filter
adhesion, along with the presence of porous features, might influence the endothelium adhesion, differentiation, and functionality. This, in turn, can affect the results of nanocarrier transport studies because of the possible presence of defects in the epithelial layer that provide more probability for paracellular passage of nanocarriers. In this respect, evidence about the effect of membrane pore size on brain endothelial cell growth and barrier formation has been already reported.41 (ii) The occurrence of a different pore size and AF can likely affect the transport efficiency of nanocarriers, due to the physical size of primary particles and particle agglomerates in culture media, both in the absence of a cell layer and in the case of NP transcytosis through the epithelial barrier, where particles may undergo further agglomeration in endolysosomal vesicles. Nanoparticle Characterization. Fluorescent polystyrene NPs functionalized with carboxyl (COOH NPs) or amine (NH2NPs) groups were selected as model nanocarriers for transport studies. Both particles had a nominal diameter of 100 nm. The presence of two different surface functionalizations conferred a diverse behavior to nanoparticle suspensions, when varying the dispersion media, as demonstrated by dynamic light scattering (DLS) and ζ potential analysis (Figure 2A−C). In fact, in ddH2O, both COOH and NH2 NPs were stable and monodispersed, with a mean hydrodynamic diameter (HD) of 119.2 ± 28.2 and 123.5 ± 47.9 nm, and a ζ potential of −55.9 ± 9.6 and +56.2 ± 8.8 mV, respectively. COOH NPs exhibited a quite narrow size dispersion (with 0.019 polydispersion index (PDI)) and a homogeneous distribution with a spherical shape, as shown by transmission electron microscopy (TEM) analysis (Figure 2D,E). NH2 NPs had higher polydispersity, with a measured PDI of 0.114 (Figure 2C) as also visually confirmed by the TEM analysis (Figure 2H,I). In complete cell culture medium (DMEM supplemented with 10% fetal bovine serum (FBS)), COOH NPs remained quite stable and monodispersed, with a slight increase in size and PDI values, due to their interaction with serum proteins (Figure 2A,C,F,G). Conversely, NH2NPs exhibited a significantly different behavior, forming heterogeneous agglomerates up to 1 μm in size. This was confirmed by TEM analyses, which showed the presence of significant particle agglomeration in culture medium (Figure 2J,K). Furthermore, the presence of serum proteins changed the surface charge that became similar for both NPs (−23.5 ± 8.2 and −26.2 ± 8.5 mV for COOH and NH2 NPs, respectively) (Figure 2C). This behavior can be ascribed to the formation of protein corona around the particles, in agreement with previous reports.40,43,44 The 3282
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ACS Applied Bio Materials permeability (for COOH NPs, similar values were obtained in ddH2O or complete DMEM). Confocal microscopy analysis of empty membranes after NP incubation confirmed this observation. For instance, in the case of F3 inserts, the presence of big NP aggregates was observed on the filters incubated with positively charged particles (Figure S2B), while limited NP accumulation occurred with COOH NPs (Figure S2A). Moreover, fluorescence imaging confirmed the presence of NPs in the basolateral media and indicated a higher amount of COOH NPs than NH2 NPs in this compartment (Figure S3), in qualitative agreement with the results of Figure 3. Taken altogether, these data indicate a significant effect of the filters on NP transport, depending on the NP dispersion status in the culture medium combined to insert characteristics, unlike the LY molecule that is quite insensitive to these experimental parameters. In particular, while monodispersed NPs have a predominant “molecule-like” behavior (except for the smallest size and porosity filter), unstable/aggregated NPs undergo strong physical/chemical interaction with the membranes, leading to significantly underestimated results in terms of transport efficiency. Such particle-specific issue, which will be further analyzed in the following in the presence of cell layers, constitutes a first important step to be considered when using in vitro models to evaluate NP efficacy in crossing biological barriers. Cell Monolayer Growth and Characterization. Brain endothelial cells seeded on transwell inserts are able to grow and form a confluent layer, with features similar to BBB endothelium in vivo.45 Therefore, to study the transport of NPs across the cell barriers, brain endothelial bEnd.3 cells were cultured on the different membranes. The growth of the cells was monitored by trans epithelial electrical resistance (TEER) measurements over time every 24 h. As reported in Figure S4A, TEER values progressively increased for all inserts, indicating cell proliferation and monolayer formation, though with a different rate depending on the filter type. In general, after a fast increase of TEER observed during the first week of culture, the electrical resistance typically reached a plateau. After 9 days of culture, TEER values were around 38 Ω cm2 for F1, 28 Ω cm2 for F3 and F0.4L, and 15 Ω cm2 for F0.4H inserts (Figure 4A). Then, as cells were left growing up to 17 days, TEER did not increase further (Figure S4A). In the case of F1 inserts, we measured the highest TEER among all of the filters. This value was comparable with those reported with this cell line.42 The TEERs obtained for F3 and F0.4L were lower than the F1 membranes, suggesting a looser monolayer. However, such values were still considered acceptable in the literature.45 On the contrary, in our experimental conditions, the TEER value obtained for F0.4H inserts was surprisingly low, even after 17 days of culture. In parallel with TEER, the tightness of the monolayers was also analyzed through permeability (Papp) to LY. The trend of the Papp confirmed TEER observations, with the lowest values of permeability obtained with F1 inserts (Figure 4B). Furthermore, to characterize in more detail the cell monolayers, the morphology of the cells and the formation of tight junctions was studied by confocal microscopy. To this aim, we studied the expression of claudin 5 (CL5), a validated marker of tight junctions in BBB models.24,45 Confocal imaging of CL5 revealed that bEnd.3 cells grown on F0.4L and F3 inserts did not seem to show the typical cell morphology of a confluent endothelium, with most of the cells appearing spread and randomly oriented (Figure S5). On
Figure 4. Characterization of the cell monolayer grown on different inserts in terms of TEER (A) and Papp to Lucifer Yellow (LY) (B). Transport studies of LY and NPs through the endothelial barriers (C). Data are reported as mean ± standard deviation of three independent experiments.
the contrary, the cells grown on F0.4H and F1 membranes were more elongated, and their spatial orientation was in line with what expected from highly differentiated endothelial cells forming a polarized monolayer. However, even if both filters seemed to give rise to polarized monolayers from a morphological point of view, this was not in accordance with the different TEER and Papp measured between F0.4H and F1 membranes. To better elucidate this inconsistency, we analyzed the cellular distribution of CL5. We found that for F0.4H, CL5 was both expressed along cell membranes and in the cytoplasm (Figure S6A,B), indicative of an incomplete formation of tight junctions.46 On the contrary, CL5 was found only along cell membranes in F1 filters, confirming the correct formation of tight junctions in these inserts (Figure S6C,D). These data were in agreement with Wuest and co-workers reporting a similar behavior in CL5 expression of bEnd.3 cells as a function of the insert type.42 Overall, our findings indicate that the morphological parameters of the filters are crucial to define the characteristics of the cell monolayers, with both pore size and density significantly affecting cell adhesion and correct polarization.47,48 In particular, the irregular geometry of the inserts with the presence of nano/microsurface features and many edges and crests on their surface can create an unfavorable surface pattern for the formation of a tight monolayer. For instance, for the same filters with 0.4 μm nominal size, the higher density and porosity of F0.4H resulted in a much lower TEER of the cell barrier. This suggests that the presence of a high fraction of porosity, with porous features up to 1 μm, partially prevented correct cell adhesion in several points, with the consequent formation of defects in the barrier. Similarly, 3 μm pores, although with a lower density, seem not 3283
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Figure 5. TEM micrographs of endothelial barriers, cultured on F0.4L (A and E), F0.4H (B and F), F1 (C and G), and F3 (D and H) inserts, after incubation with COOH (A−D) and NH2 NPs (E−H). Scale bar: 200 nm.
a significant role. On the other hand, the transport of COOH NPs was consistently reduced, compared to LY. The highest decrease was measured on the tight F1 inserts, where only 2.6% of COOH NPs were found in the basolateral side. In this case, it is likely that the NP passage through the barrier mostly occurred by transcytosis, with a negligible fraction of paracellular transport. For the other filters, the transport efficiency of COOH NPs was higher, due to the leakier characteristics of the cell monolayers. The higher value was obtained in F0.4H, the filter with the lowest TEER, where ca. 10% of particles were found to cross the barrier. While in the case of the empty filters we observed a molecule-like behavior for COOH NPs, due to their monodispersion in culture medium, in the presence of the cell barriers, the particles did exhibit a certain degree of interaction with the filters, which significantly decreased their passage efficiency. Actually, when internalized by epithelial cells through classical endocytosis pathways,49,50 COOH NPs underwent intracellular confinement in endolysosomal vesicles. As clearly shown by TEM analyses (Figure 5A−D), this resulted in NP agglomeration and formation of structures larger than primary particles,
to be ideal candidates to promote homogeneous monolayers, likely because of the large physical dimensions of the pores, which is comparable to cell bodies. In our experimental conditions, the F1 insert allowed the formation of the tightest monolayer, in terms of resistance, permeability, and cell polarization. Transport Studies of NPs through Cell Barriers. After the characterization of cell monolayers, transport experiments with LY and NPs were performed after 9 days of growth, namely, at the plateau values of TEER for all of the transwell systems. As a preliminary control, we verified that NPs were not cytotoxic to bEnd.3 cells at the concentration used for the experiments (50 μg/mL) and did not alter the integrity of the barriers (Figure S4B,C). As expected, upon incubation of LY molecules with the endothelial barriers, we observed that their diffusion primarily depends on the tightness of the formed monolayer (Figure 4C). The lowest molecular permeability was observed in F1 inserts, with a measured LY passage of ca. 13%, while transport exceeded 30% efficiency with the leaky F0.4H monolayer. In agreement with Figure 3, for the molecular marker, the filter morphology did not play per se 3284
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Figure 6. TEM micrographs showing NH2 NP uptake in F3 cell monolayers. NPs seem to interact with membranes already as big aggregates, forming micrometric invaginations (A and B), and are uptaken inside the cells by macropinocytosis (C). Scale bar: 500 nm.
Figure 7. Confocal microscopy characterization of the bEnd.3 cell barriers after 24 h incubation with COOH and NH2 NPs (A−H). COOH and NH2 NPs are visible in green. Cell nuclei were stained with Hoechst 33258 (blue). Scale bar: 20 μm. Panel I: semiquantitative evaluation of NP uptake in cells, evaluated as the average NP surface area over the total cellular area. Panel J: semiquantitative analysis of the average size of intracellular NP aggregates.
membranes already as heterogeneous agglomerates, entering the cells through large invaginations typical of macropinocytosis processes (Figure 6) and leading to further intracellular accumulation and aggregation inside the vesicular compartments. This limits their possibility of transcytosis and overall transport to the basolateral side of the membrane, as it is very unlikely that such big aggregates can be transported through the pores of the filters. The interaction of positively and negatively charged NPs with the cell monolayers was also evaluated by confocal microscopy. The uptake efficiency and intracellular distribution of the two particles were quite different (Figure 7). COOH NPs accumulated in cells in small vesicles, mainly localized in the perinuclear region, while NH2 NPs appeared as large aggregates of variable size, in line with TEM data. In particular, a semiquantitative analysis of confocal images indicated a
although some single particles could also be observed. Such agglomerates can then interact with the filter membranes after transcytosis, with consequent size exclusion effects and a decrease of their overall transport efficiency. This particlespecific mechanism was even more pronounced in the case of positively charged NH2 NPs. We already noticed that this is an important issue in empty filters, where NH2 NP transport was strongly limited, due to extensive particle agglomeration in culture medium. In the presence of cell monolayers, NP transport was found to be very low for all of the filters (1 μm size). Due to their behavior in DMEM, NH2 NPs interact with cell 3285
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interaction of the large particle aggregates with the porous filters). In particular, considering the large intracellular bioaccumulation of NH2NPs, despite the agglomeration already observed in the apical compartment, it is difficult to understand the different contributions of large vesicles limited intracellular trafficking and exocytosis with the physical size exclusion effects of the filters, in hindering the transport efficiency of the particles. Detailed studies on the intracellular trafficking and fate of nanocarriers in the BBB barrier (e.g., fusion with sorting vesicles, characterization of particle exosomes) will significantly help in clarifying such aspects. However, in our study, preliminary confocal microscopy observations revealed that the few NPs observed in the basolateral side were mostly present as single particles, indicative of a key role of size filtering by the porous membranes. Such issues are much less evident with small molecules, such as LY, so that classical experiments only required careful assessment of the monolayer characteristics and tightness,42 unlike nanoparticulate formulations, which raise novel problems. In conclusion, while in vitro models of BBB constitute a great opportunity for early and large-scale screenings of drugs, substantial attention should be paid when testing nanoparticles/nanocarriers in nanomedicine applications, as deep characterization of all the underlying nanobiointeractions is strongly required.
higher NP bioaccumulation for NH2 NPs as compared to COOH NPs (Figure 7I), despite the opposite behavior observed in terms of transport efficiency. The size exclusion effects discussed above were confirmed by the significant difference found in the average size of intracellular vesicles containing the NPs, with a value of ca. 0.6 μm for COOH NPs versus 1.3 μm for NH2NPs (Figure 7J). In this latter case, large size heterogeneity was found, with several vesicles > 3−4 μm. Hence, confocal experiments supported the transport processes described above. Taken altogether, these observations indicate that the transport experiments performed with the transwell systems can be significantly affected both by the filter characteristics, in terms of pore size and density, and by the dispersion status of the nanocarriers. The transport efficiency of NPs through cell monolayers can be only partially compared to small molecules, due to the particle hindrance and interaction with the porous membranes. Actually, the overall transport process of NPs through the endothelial barriers in vitro can be framed in 3 subsequent nanobiointeraction steps, each dictated by the particle chemical−physical profile. Such frames govern the in situ dispersion status of NPs and eventually their barrier passage efficiency: (i) NP interaction with a cell culture medium in the apical compartment, which defines the nanobioentities seen by the cells of the barrier51 and contributes to establish their molecule-like or particle-specific behavior. (ii) NP transcytosis through the barrier, which involves cellular uptake of NPs and their intracellular distribution, as well as NP trafficking and exocytosis toward the inserts. Such process is strongly affected by step 1 and likely promotes further agglomeration in the endolysosomal vesicles. (iii) Interaction of the exocytosed nanobioentities with the porous filters, with possible size exclusion effects. Step 1 is thus fundamental for the whole transport process and is strongly related to the particle properties and medium characteristics.43,52 Accurate characterization of such step is crucial for in vitro studies on BBB permeability to NPs, taking into account that similar interactions also take place in vivo, e.g., in human serum, so that these data can be useful and predictive of the in vivo behavior. Moreover, trafficking and transcytosis of NPs and NP agglomerates are complex processes, not yet fully elucidated, which can depend on the size of vesicles containing particle agglomerates. Further studies are necessary to clarify this issue. As discussed, also the intracellular distribution of NPs strongly depends on the physical/chemical profile of the NPs, so also this step may be influenced by particle-specific effects. As a final consequence, porous filter crossing by NPs in in vitro BBB studies is strongly modulated by the characteristics of the nanobioentities interacting with the inserts and by the specific parameters of the filters employed in the experiments, eliciting size exclusion effects, unlike molecular species. Remarkably, this frame can significantly perturb the measurements of the transport efficiency of NPs. In particular, strong underestimations can be recorded in vitro. This is fundamental, since in vivo BBB does not contain any artificial porous membranes. For instance, once we have carefully characterized the whole behavior of positively charged NH2NPs, along with their large cellular uptake in the epithelial barriers, it is still very difficult to draw reliable conclusions about their capability to cross the BBB barrier. This is because the negligible transport efficiency observed in our experiments (
