Apo E-Functionalization of Solid Lipid Nanoparticles Enhances Brain

Article pubs.acs.org/bc

Apo E‑Functionalization of Solid Lipid Nanoparticles Enhances Brain Drug Delivery: Uptake Mechanism and Transport Pathways Ana Rute Neves,† Joana Fontes Queiroz,† Sofia A. Costa Lima,† and Salette Reis*,† †

UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal ABSTRACT: Several strategies have been implemented to enhance brain drug delivery, and herein solid lipid nanoparticles functionalized with apolipoprotein E were tested in hCMEC/D3 cell monolayers. The mean diameter of 160 nm, negative charge of −12 mV, and their lipophilic characteristics make these nanosystems suitable for brain delivery. Confocal images and flow cytometry data showed a cellular uptake increase of 1.8-fold for SLN-Palmitate-ApoE and 1.9-fold for SLN-DSPE-ApoE when compared with the non-functionalized SLNs. Clathrin-mediated endocytosis was distinguished as the preferential internalization pathway involved in cellular uptake and nanoparticles could cross the blood-brain barrier predominantly by a transcellular pathway. The understanding of the mechanisms involved in the transport of these nanosystems through the bloodbrain barrier may potentiate their application on brain drug delivery.

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INTRODUCTION The blood-brain barrier (BBB) is a highly selectable barrier that protects the central nervous system from foreign substances and toxins, thereby limiting the use of drugs for brain diseases.1 Several strategies have been implemented to enhance brain drug delivery, such as the development of nanodelivery systems.2−4 Nanoparticles are solid colloidal particles ranging from 1 to 1000 nm, consisting of natural or artificial polymers, lipids, dendrimers, and micelles in which a drug or biologically active compound can be dissolved, entrapped, or encapsulated. Several administration routes have been exploited to reach the brain, such as peroral, intranasal, intracranial, or intravenous. Some studies have shown increasing progression of the intravenous route by functionalizing the nanoparticle surfaces with specific ligands that can be recognized by receptors overexpressed in the brain, thereby enhancing brain drug delivery.5 In this context, BBB has several transport molecules that can enhance the uptake of nanosystems into the brain, such as growth factors,6−8 biotin binding proteins like avidin,9 insulin,10 albumin,11 leptin,12 lactoferrin,13,14 transferrin,15 and angiopep-2.16 In this study, we have used solid lipid nanoparticles (SLNs) functionalized with apolipoprotein E (ApoE). SLNs are promising candidates for brain delivery of drugs due to their lipid nature, small size, and biocompatibility. ApoE has been attached in albumin nanoparticles for brain delivery since ApoE receptors are predominantly expressed in the brain and bind ApoE with high affinity.17,18 However, the conjugation of SLNs with ApoE ligands is a new approach developed recently in our group.19,20 This functionalization took advantage of a specific recognition between ApoE molecules on the surface of SLNs and the low-density lipoprotein (LDL) receptors that are overexpressed on the BBB. This is a highly regulated and © 2017 American Chemical Society

energy-dependent process, but may allow the whole nanocarrier and the loaded drug to go through the BBB, even passing the drug efflux transporters.21 While ApoE is critical for the regulation of cholesterol homeostasis in the peripheral circulation, its role in the brain appears to involve not only cholesterol transport but also intercellular exchange of metabolites between neurons and glial cells required for maintenance of healthy brain tissue.22 Within the ApoE family, ApoE3 appears to play a major role in the nanoparticlemediated drug transport across the BBB.18 Moreover, SLNs have been recognized as suitable systems for brain delivery due to their biodegradable and biocompatible composition,23−25 as well as the ability to avoid P-gp efflux activity at brain endothelial cells.21,26 Therefore, the main objective was to elucidate cellular uptake profiles, endocytic pathways and transport across the BBB of these promising systems in order to assess the advantage of the functionalization when compared to the unmodified nanosystems. For this purpose, hCMEC/D3 cell line was used as a model of the human BBB that is very useful for the characterization of the mechanisms involved in the internalization of nanoparticles (NPs) by the brain endothelial cells.27,28 Several techniques allowed us to achieve the objective, namely, confocal and electron microscopy, flow cytometry, and the use of different tracers and inhibitors.

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RESULTS AND DISCUSSION Cellular Uptake of Nanoparticles. In order to determine the effect of ApoE functionalization on SLNs internalization by Received: December 7, 2016 Revised: March 23, 2017 Published: March 29, 2017 995

DOI: 10.1021/acs.bioconjchem.6b00705 Bioconjugate Chem. 2017, 28, 995−1004

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Bioconjugate Chemistry

Figure 1. Cellular uptake of non-functionalized and functionalized SLNs by hCMEC/D3 cells after 4 h incubation at 37 °C. (A) CLSM images of cell membranes labeled by WGA-Alexa Fluor 633 (red), nuclei stained with DAPI (blue), FITC-labeled NPs (green) and their overlay. Scale bar: 20 μm. (B) Cellular uptake kinetic profiles obtained by flow cytometry. (C) Saturated cellular uptake value (Ymax) and uptake rate constant (k) for each nanosystem studied. Note: Flow cytometry values represent the mean ± standard deviation (n = 3).

Figure 2. hCMEC/D3 cell viability after 4 h incubation with different samples. FITC-loaded NPs (non-functionalized and functionalized SLNs), inhibitors of internalization pathways (4 °C, sucrose, chlorpromazine, filipin, cytochalasin D, and ammonium chloride), and tracers of transendothelial routes (propranolol and lucifer yellow) at the respective concentrations used.

important finding for pharmaceutical drug delivery research, since the nucleus is the target site for several drugs.30 A FITC release study in the same biological fluids as the uptake assays has been previously performed to validate the quantification method. The results revealed that the release of FITC from the lipid nanoparticles up to 48 h was not significant (5−10%), indicating that FITC remains entrapped in SLNs as fluorescent label after 4 h of incubation time with cells. Therefore, the green fluorescence detected can be correlated with NPs themselves and not with the released free probe. Unloaded SLNs were used as negative controls to confirm that there was no fluorescence interference (data not shown). CLSM results indicate a higher uptake of both types of ApoE-functionalized

BBB cells, the cellular uptake of functionalized and nonfunctionalized SLNs by hCMEC/D3 cells was investigated. Therefore, SLNs previously labeled with FITC were incubated with cells up to 4 h. Then, cells were analyzed by flow cytometry and confocal microscopy. CLSM images of cell membranes labeled by WGA-Alexa Fluor 633 (red), nuclei stained with DAPI (blue), and FITC-labeled NPs (green) and their overlay are shown in Figure 1A. The images revealed that NPs were punctually concentrated in intracellular vesicles (endosomes or lysosomes) that can be related to their endocytic mechanism of internalization. 29 Besides their cytoplasmic location, green fluorescence indicates that NPs were also transported to the perinuclear area. This is an 996


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Figure 3. Effect of different inhibitors on hCMEC/D3 cell internalization pathways of non-functionalized and functionalized SLNs after 4 h incubation at 37 °C. Role of energy in endocytosis is tested at 4 °C; sucrose and chlorpromazine inhibit clathrin-mediated endocytosis; filipin inhibits caveolae-mediated endocytosis; cytochalasin D inhibits macropinocytosis; and ammonium chloride is a lysosomotropic agent. Note: Flow cytometry values were normalized against the cells with no inhibitor and represent the mean ± standard deviation (n = 3). The results were analyzed and compared with the cells with no inhibitor. (*) denotes statistically significant differences (P < 0.05).

SLNs compared to non-functionalized SLNs (Figure 1A). Moreover, flow cytometry data showed a time-dependent accumulation and the cellular uptake follows Michaelis− Menten kinetics (Figure 1B). A comparative evaluation of Ymax and k parameters can reflect the differences in cellular uptake (Figure 1C). SLN-Palmitate-ApoE exhibited a 1.8-fold and SLN-DSPE-ApoE a 1.9-fold higher Ymax than nonfunctionalized SLNs. In terms of k, a smaller value reflects a quicker cellular internalization. Therefore, we can conclude that SLN-Palmitate-ApoE and non-functionalized SLNs presented a similar uptake speed, while SLN-DSPE-ApoE showed a slower but higher uptake by cells, displaying a more controlled and sustained internalization profile. Since Ymax of SLN-PalmitateApoE and SLN-DSPE-ApoE were roughly the same, we can assume that the increased cellular uptake was probably induced by the presence of ApoE molecules on the surface of SLNs and was based on a mechanism of receptor-mediated endocytosis. To further verify this assumption, we have investigated the internalization pathways involved. Internalization Pathways. There are distinct internalization pathways for particles to enter cells.31 Generally, they can be divided into active and passive pathways. The influence of temperature on cellular uptake is often tested to evaluate the active transport that is reduced under 4 °C.32 Hence, NPs can be internalized by cells via energy-dependent transport (endocytosis) or energy-independent mechanisms (nonendocytic pathways). Endocytosis can further be subdivided into phagocytosis and pinocytosis (clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis).33 Therefore, we have used several specific endocytic inhibitors to identify the internalization pathways involved in the cellular uptake of functionalized and non-functionalized SLNs by hCMEC/D3 cells. The potential cytotoxicity of NPs and inhibitors on hCMEC/D3 cells was investigated by MTT assay and Figure 2 shows that no significant cytotoxicity was detected in the tested conditions. In Figure 3 it is possible to see the effect of each inhibitor on the NP internalization. The fluorescence of cells incubated with FITC-labeled NPs in the absence of any inhibitor was considered to be 100%, while the fluorescence after incubation

in the presence of inhibitors was expressed as a relative percentage compared to the cells with no inhibitor. We can observe that the uptake of all NPs was almost completely inhibited at 4 °C, suggesting that their uptake was mediated by endocytosis. To assess the role of clathrin-mediated endocytosis in the uptake of functionalized and non-functionalized SLNs, sucrose and chlorpromazine were used to disrupt the formation of clathrin-coated vesicles on the cell membrane.34,35 These inhibitors reduced cellular uptake by 30−40% for all three types of SLNs studied, thereby suggesting this is the preferential pathway involved in cellular uptake of non-functionalized and ApoE-functionalized SLNs. We further investigated the involvement of the caveolae/lipid rafts on the internalization of SLNs, using filipin to disrupt cholesterol domains responsible for the caveosome formation,36 but a reduction of only 10% of non-functionalized SLNs was obtained and no effect was detected on ApoE-functionalized SLNs. This result suggests little or no involvement of caveolae-mediated endocytosis for SLNs uptake. Macropinocytosis was also investigated by using cytochalasin D which depolymerizes actin filaments necessary for this endocytic pathway,37 but no effects were observed in any type of SLNs studied. Therefore, the main mechanism involved in the SLN uptake by hCMEC/ D3 cells was found to be clathrin-mediated endocytosis. This might be related to the NPs size, since clathrin-coated vesicles are known to mediate the internalization of particles with a size between 100 and 200 nm,38,39 which is precisely the size range of the NPs tested. In the case of ApoE-functionalized SLNs, we also took advantage of a mechanism of LDL receptor-mediated endocytosis which is probably responsible for the cellular uptake of these ApoE-modified NPs. In fact, LDL receptor was found to be associated with clathrin-coated pits on the cell surface, which when bound to ApoE-coated particles form clathrin-coated vesicles in the cell.33,40 The involvement of the endosomal/lysosomal compartments in the NPs trafficking inside hCMEC/D3 cells was studied by using the lysosomotropic agent ammonium chloride.41 An uptake reduction of 30% was observed for all functionalized and non-functionalized SLNs. Therefore, NPs may partially follow lysosomal and endosomal trafficking inside cells, which is important for drug 997


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Figure 4. Transcytosis of non-functionalized and functionalized SLNs across hCMEC/D3 cell monolayer. (A) CLSM images of cell monolayer grown on transwell porous membrane after 1 h incubation at 37 °C of FITC-labeled NPs (green). Cell membranes were labeled with WGA-Alexa Fluor 633 (red) and nuclei stained with DAPI (blue). Images were collected at xy planes with a step length of 0.5 μm in z-axis from 0 to 12 μm. Scale bar: 20 μm. (B) Intracellular fluorescence distribution curves of FITC-labeled NPs during different incubation times at 37 °C, at different depths of cell monolayer.

Figure 5. Blood-brain barrier permeability of non-functionalized and functionalized SLNs across hCMEC/D3 cell monolayer. (A) Transendothelial pathway study across hCMEC/D3 cell monolayer using tracers of paracellular (lucifer yellow) and transcellular (propranolol) routes. (B) Particle size, polydispersity index, and zeta potential from apical and basolateral sides of hCMEC/D3 cell monolayer after 4 h incubation with NPs. (C) apparent permeability (Papp) values of SLNs recovered in the basolateral side after 4 h of transport across the hCMEC/D3 cell monolayer. (D) TEM images of hCMEC/D3 cell monolayer with no treatment or after 4 h incubation with NPs (arrows). Scale bar: 300 nm. Note: all values represent the mean ± standard deviation (n = 3).

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Bioconjugate Chemistry delivery or drug transport through barriers.39 In fact, the lysosomal pathway is responsible for particle degradation and release of drug content inside cells, while the endosomal trafficking may be involved in the transport of intact drugloaded NPs from one side to the other of the cell barrier. BBB Permeability Studies. Several techniques were used to verify the transcytosis of ApoE-functionalized and nonfunctionalized SLNs across hCMEC/D3 cell monolayer, namely, CLSM, TEM, transendothelial tracer flux, and DLS analysis of the apical and basolateral media. In Figure 4A it is possible to see CLSM images of hCMEC/D3 cells grown on porous membrane to form a compact monolayer. The transcytosis of FITC-labeled NPs can be followed as green fluorescence, while cell membranes are stained in red (WGAAlexa Fluor 633) and nuclei in blue (DAPI). In agreement with the studies discussed already, green fluorescence intensity of cells incubated with SLN-Palmitate-ApoE and SLN-DSPEApoE is much stronger than that of non-functionalized SLNs, suggesting that ApoE modified nanocarriers not only enhanced cell uptake but also increased the transcytosis through BBB cells. This outcome probably happened from the interaction between ApoE ligand and LDL receptors overexpressed in BBB. Additionally, cell monolayer was scanned at xy planes with a step length of 0.5 μm in the z-axis from 0 to 12 μm and the intracellular fluorescence was recorded at different depths after 15 and 60 min (Figure 4B). The transport of NPs through the cell membrane increased as time went on, and after 60 min the amount of green fluorescence in cells incubated with ApoEfunctionalized SLNs was around 2 times higher than that in cells incubated with non-functionalized SLNs. The higher and faster transport of ApoE-functionalized nanocarriers was consistent with the previous results demonstrated above. Moreover, the maximum fluorescence migrated from the apical to basolateral side of membrane over time, which further demonstrated the dynamic permeation of SLNs through BBB barrier. The transendothelial flux of ApoE-functionalized and nonfunctionalized SLNs through the hCMEC/D3 cell monolayer was investigated. Hence, the transport rates of paracellular (lucifer yellow) and transcellular (propranolol) markers were tested in the absence and in the presence of all NPs. Lucifer yellow was used to assess viability of the cell layer, since this tracer can only cross the BBB by a paracellular route. Therefore, hydrophilic compounds such as lucifer yellow, which flux by a paracellular pathway, are very sensitive to tight junctions integrity. However, hydrophobic compounds cross cell barriers by transcellular pathway, being insensitive to tight junction integrity.42 The effective permeability of lucifer yellow and propranolol alone was found to be (1.32 ± 0.07) × 10−3 cm/min and (4.63 ± 0.12) × 10−3 cm/min, respectively, which is in agreement with previous tests.43 MTT results presented in Figure 2 showed that these tracers were nontoxic to the hCMEC/D3 cell line, at the concentrations used in this study. Therefore, after incubation of ApoE-functionalized SLNs and non-functionalized SLNs with each tracer in the apical side of the monolayer, we could evaluate the preferential transendothelial route for each NPs (Figure 5A). The presence of all three different types of SLNs had no detectable effect on the paracellular flux of lucifer yellow, suggesting no apparent adverse effects on the integrity of hCMEC/D3 monolayer which was also confirmed by no changes in the transendothelial resistance values. In this way, it is clear that the previously reported enhancement of ApoE-functionalized SLNs trans-

cytosis was not due to a paracellular route or loss of cell integrity. However, the presence of SLNs significantly changed the permeability of propranolol across the hCMEC/D3 cell monolayer, indicating the involvement of transcellular pathways on the NPs permeation. Non-functionalized SLNs decreased the effective permeability of propranolol showing that these NPs may compete with propranolol for the same transcellular mechanisms. However, ApoE-functionalized SLNs could enhance propranolol permeability through the cell monolayer, probably because they activated LDL-receptor mediated mechanisms of internalization which could also facilitate cellular uptake of propranolol simultaneously with the NPs. This might be related to clathrin-coated pits associated with LDL receptors that form clathrin-coated vesicles in the cells when ApoE-coated nanoparticles bind to these receptors.33,40 In fact, the results obtained using different inhibitors of the endocytosis pathways corroborate with this evidence, since clathrin-mediated endocytosis is the exclusive mechanism involved in the internalization of the functionalized NPs, whereas non-functionalized NPs can be internalized preferably by two different mechanisms involving caveolae and clathrincoated vesicles. Thus, non-functionalized NPs may compete with propranolol for the caveolae and clathrin-mediated endocytosis. On the contrary, ApoE-functionalized nanoparticles may stimulate LDL receptor-mediated mechanisms driven solely by clathrin-coated vesicles facilitating the internalization of propranolol by this pathway and reversing the competition for caveolae-mediated mechanisms. Therefore, one can assume that NPs will possibly cross the blood-brain barrier predominantly by a transcellular pathway. Indeed, TEM images support this statement, since in Figure 5D it is possible to observe the formation of endocytic vesicles engulfing NPs (see black arrows), reinforcing the idea that NPs may enter cells by an endocytic mechanism, being therefore trancytosed through a transcellular pathway. Besides that, NPs were observed in the basolateral medium after 4 h of incubation for all types of particles tested. Their size, polydispersity index, and zeta potential resembled that of the apical side prepared in the beginning (Figure 5B), supporting the suggestion that NPs could cross the BBB monolayer from one side to the other without being degraded inside cells. Finally, the apparent permeability coefficients (Papp) revealed a significant increase of permeation across a monolayer of ApoE-functionalized SLNs (1.5-fold higher) when compared to non-functionalized ones, as shown in Figure 5C. This result is in agreement with previous work developed recently in our group.19 The difference between the two strategies of functionalization was not statistically significant, suggesting that both ApoE-functionalized SLNs could successfully increment permeability, actually resulting in potential carriers for active targeting drug delivery to the brain. Overall Conclusion. Several strategies have been developed for brain drug delivery, trying to overcome the limitations of the current therapies applied.24,44,45 Some previous studies describe the adsorption of ApoE molecules on the surface of nanoparticles.46,47 For instance, polysorbate 80-coated poly(lactide) nanoparticles have been developed as promising tools, providing the adsorption of ApoE molecules onto the nanoparticle surface throughout the blood circulation.48 Besides that, covalent attachment of ApoE has also been reported regarding the use of human serum albumin nanoparticles.17,18 In fact, ApoE-enriched nanoparticles can mimic LDL lipoproteins as carriers for brain-specific delivery.49 However, the 999


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Figure 6. Schematic representation of the proposed mechanism of ApoE-functionalized SLNs uptake and transcytosis through the blood-brain barrier. Black arrows indicate pathways that were determined in this study; black dashed arrows indicate less pronounced pathways; gray arrows indicate trafficking pathways already described; and red arrows indicate recycling pathways. ER − endoplasmic reticulum; AEE − apical early endosomes; CE − common endosomes; LE − late endosomes; BSE − basolateral sorting endosomes; ARE − apical recycling endosomes.

nanosystems will clarify and potentiate their application in the drug delivery through this challenging barrier.

conjugation of SLNs with ApoE ligands is a new approach developed recently in our group.19,20 Therefore, in the present work, we have successfully validated the recently developed ApoE-functionalized SLNs with proven efficacy in drug delivery to the brain, taking advantage of a specific recognition between ApoE molecules on the surface of SLNs and the LDL receptors overexpressed on the BBB.19 The mean diameter of around 160 nm as well as the negative charge of −12 mV and their lipophilic characteristics make these nanosystems suitable for brain delivery, since they can cross BBB without affecting its integrity.50,51 CLSM images and flow cytometry data showed a timedependent accumulation and a cellular uptake increase of 1.8fold for SLN-Palmitate-ApoE and 1.9-fold for SLN-DSPEApoE when compared with the non-functionalized SLNs. The study using specific endocytic inhibitors identified clathrinmediated endocytosis as the preferential internalization pathway involved in hCMEC/D3 cellular uptake of these NPs. LDL receptor has been associated with clathrin-coated pits on the cell surface, which is in agreement with this finding.40 Moreover, results showed that NPs could cross blood-brain barrier predominantly by a transcellular pathway and they appear in the basolateral side of the monolayer, suggesting an efficient and specific drug delivery into the brain. In Figure 6, it is possible to see a schematic representation of the proposed mechanism of ApoE-functionalized SLNs uptake and transcytosis through the blood-brain barrier. The understanding of the mechanisms involved in the transport of these

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MATERIALS AND METHODS Materials. Lipid nanoparticles were prepared using cetyl palmitate supplied by Gattefossé SAS (Nanterre, France), polysorbate 80 (tween 80) provided by Merck (Darmstadt, Germany), sodium deoxycholate, avidin, palmitic acid Nhydroxysuccinimide ester (NHS-palmitate), N-ethyl-N′-(3(dimethylamino)propyl)carbodiimide hydrochloride (EDC), and apolipoprotein E3 provided by Sigma-Aldrich (St. Louis, USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethylene glycol)-2000] (DSPE-PEG-NH2) purchased from Avanti Polar Lipids (Alabama, USA) and the biotinylation reagent supplied by Thermo Scientific (Waltham, Massachusetts, USA). Immortalized human cerebral microvascular endothelial (hCMEC/D3) cell line until passage number 35 was obtained from the Institut National de la Santé et de la Recherche Médicale (INSERM, Paris, France) and maintained in Endothelial Basal Medium-2 (EBM-2) purchased from Lonza (Basel, Switzerland), supplemented with Fetal Bovine Serum (FBS) “Gold” provided by PAA The Cell Culture Company (Cansera, Canada), chemically defined lipid concentrate and penicillin−streptomycin (PenStrep) obtained from Gibco (Carlsbad, CA, USA) and human basic Fibroblast Growth Factor (bFGF), ascorbic acid and hydrocortisone purchased from Sigma-Aldrich. Cultrex Rat Collagen I was provided by R&D Systems (Minneapolis, USA). Trypan blue, 1000


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reason, the kinetics of cellular uptake were analyzed and curve-fitted to the Michaelis−Menten equation:

thiazolyl blue tetrazolium bromide (MTT), phosphate buffered saline pH 7.4 (PBS), paraformaldehyde (PFA), sucrose, chlorpromazine hydrochloride, filipin, cytochalasin D, ammonium chloride, propranolol, lucifer yellow, and fluorescein isothiocyanate (FITC) were obtained from Sigma−Aldrich, while wheat germ agglutinin (WGA) Alexa Fluor 633 conjugate and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Gibco. Preparation and Characterization of Nanoparticles. Non-functionalized and functionalized SLNs composed of cetyl palmitate (10%) and Tween 80 (3%) were produced according to an already described method of high-speed stirring in UltraTurrax followed by sonication.52−54 The functionalization process with ApoE molecules was developed and described in a previous report19 and took advantage of the strongest and most stable noncovalent interaction (Kd of 10−15 M) between avidin and biotin.55 ApoE molecules were first biotinylated and NPs activated with avidin molecules on their surface by using either a phospholipid (DSPE-PEG-Avidin) or palmitic acid (Palmitate-Avidin) as the linker.19 Avidin-conjugated NPs were then incubated with the biotinylated ApoE, producing two different ApoE-functionalized SLNs: SLN-DSPE-ApoE and SLN-Palmitate-ApoE.19 All NPs (functionalized and nonfunctionalized) were labeled with 2 mg of FITC which was added to the lipid phase during the SLNs preparation. We have previously performed the same studies to ensure the suitability of this quantification method. The quantification of the loaded dye was assessed by measuring the free FITC still present in the aqueous phase by using centrifugal filter devices followed by quantification in a spectrofluorometer at 495/521 nm. A loading percentage of 90% was found for FITC which is satisfactorily high for its use as the fluorescent dye. Moreover, free FTIC was excluded by dialysis against PBS, overnight, in order to not interfere with the NP quantification during the assays. The size and zeta potential of NPs were determined in a Brookhaven dynamic light scattering (DLS) instrument (Holtsville, NY, USA) to characterize the nanosystems. All formulations showed a size around 160 nm, polydispersity index below 0.2, and zeta potential around −12 mV.19 Size and surface charge are determinant characteristics for cellular uptake mechanism and transport pathways.33 Therefore, the small size and negative charge obtained can predict that these nanosystems will be able to deliver potential incorporated drugs into the brain.2,51 hCMEC/D3 Cell Culture. hCMEC/D3 cells were seeded at a concentration of 2.5 × 104 cells/cm2 and grown at 37 °C and 5% CO2 in EBM-2 medium supplemented with 1% PenStrep, 2% FBS “Gold”, 1% chemically defined lipid concentrate, 5 μg/ mL ascorbic acid, 1.4 μM hydrocortisone, 10 mM HEPES, and 1 ng/mL bFGF. Cell viability was assessed by MTT assay in the presence of SLNs and of all inhibitors and tracers used in this study, as previously described.56 Cellular Uptake of Nanoparticles. Flow Cytometry Analysis. hCMEC/D3 cells were seeded in 24-well plates (2 × 105 cells per well) and incubated with FITC-labeled functionalized and non-functionalized SLNs (10 μM) for 0, 0.5, 2, and 4 h, at 37 °C and 5% CO2. Then, cells were washed twice with PBS, detached with 0.25% trypsin−EDTA and analyzed by flow cytometry on a BD Accuri C6 (BD Biosciences, Erembodegem, Belgium). For each sample a minimum of 10 000 events were recorded. The internalization profiles reflect the delivery efficacy of the functionalized against non-functionalized SLNs. For that

Y=

Ymax ·t k+t

where Y is the uptake in real time, t is the incubation time, Ymax is the saturated cellular uptake value, and k is the time when the uptake is 50% of Ymax. Hence, a smaller k reflects a quicker cellular uptake. Confocal Laser Scanning Microscopy. For confocal laser scanning microscopy (CLSM) imaging, hCMEC/D3 cells were grown on coverslips in 24-well plates (2 × 105 cells per well) and incubated with FITC-labeled functionalized and nonfunctionalized SLNs (10 μM) for 4 h. After incubation, cells were rinsed twice with PBS and fixed with 2% PFA for 20 min at room temperature. After washing twice with PBS, membranes were stained with 5 μg mL−1 WGA-Alexa 633 for 10 min at 37 °C, followed by staining of nuclei with 300 nM DAPI for 5 min at room temperature. Coverslips were inverted over glass slides using Vectashield as mounting medium. Images were recorded on a Leica SP5 CLSM (Leica Microsystems, Wetzlar, Germany) and processed using a Leica Application Suite - LAS AF v 4.3 software. Internalization Pathways. The pathways involved in the internalization of functionalized and non-functionalized SLNs on BBB were studied by preincubating hCMEC/D3 cells in 24well plates (2 × 105 cells per well) with different inhibitors for 30 min. 0.45 M sucrose and 10 μg mL−1 chlorpromazine were used to inhibit clathrin-mediated endocytosis; 1 μg mL−1 filipin to inhibit caveolae-mediated endocytosis; 5 μg mL −1 cytochalasin D to inhibit macropinocytosis; and 1 mg mL−1 ammonium chloride is a lysosomotropic agent.57,58 4 °C temperature was applied to set the role of energy in the NPs uptake. Cells were then incubated with FITC-labeled SLNs (10 μM) for 4 h and after that were washed twice with PBS, detached with 0.25% trypsin−EDTA, and the internalization of SLNs analyzed by flow cytometry as described above. The results were compared with the cells incubated without inhibitor. Transwell Permeability Study. Permeability studies were performed on hCMEC/D3 monolayers by seeding 1 × 105 cells per polyester insert (6 wells, pore diameter of 0.4 μm, 4.67 cm2). After 7 days cells reached confluence which better mimics the BBB morphology and activity.27,28 Permeability assays were conducted by incubating functionalized and non-functionalized SLNs labeled with FITC (10 μM) on the apical side, at 37 °C and 5% CO2. Size and zeta potential parameters were investigated in both apical and basolateral sides of the monolayer. The concentration of FITC in the receptor compartment was quantified by fluorescence analysis (495/ 519 nm) and the apparent permeability coefficients (Papp) of SLNs were calculated according to Papp(cm s−1) =

Q A×C×t

where Q represents the total amount of permeated FITC (μg), A is the surface area of the filter (cm2), C is the initial FITC concentration (μg cm−3), and t is the experiment time (s). Confocal Laser Scanning Microscopy. Images of hCMEC/ D3 monolayers grown on transwell inserts were obtained after 15 and 60 min of permeability assay. The monolayers were rinsed twice with PBS and fixed with 2% PFA for 20 min at room temperature. Membranes and nuclei were then stained as 1001


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Center for Biomaterials and Regenerative Therapies (b.IMAGE) and Mariá Gómez Lázaro for technical assistance in confocal microscopy. The authors also thank Dr. Babette Weksler from Weill Cornell Medical College (New York, USA), Dr. Ignacio A. Romero from The Open University (Milton Keynes, UK) and Dr. Pierre-Olivier Couraud from INSERM (Paris, France) for technical assistance and support with the hCMEC/D3 cell culture. SACL thanks Operaçaõ NORTE-01-0145-FEDER-000011 for her Investigator contract.

described before. Monolayers on polyester membranes were carefully cut off from the inserts and placed between a glass slide and coverslip, using Vectashield as mounting medium. The preparations were visualized in a Leica SP5 CLSM (Leica Microsystems, Wetzlar, Germany) and images were acquired at xy planes with a step length of 0.5 μm in z-axis from 0 to 12 μm. The images were then processed and the intracellular fluorescence quantified at different depths of cell monolayer, using a Leica Application Suite - LAS AF v 4.3 software. Transmission Electron Microscopy. After 4 h of BBB permeability assay, monolayers were rinsed twice with PBS, fixed with 2.5% glutaraldehyde and 2% PFA in cacodylate buffer 0.1 M (pH 7.4), and post fixed in 2% osmium tetroxide in the same buffer. The monolayers were then dehydrated in ethanol, carefully cut off from the inserts and embedded in Epon resin. Ultrathin sections of 40−60 nm thickness were prepared on a RMC Ultramicrotome (PowerTome, USA), using diamond knives (DDK, Wilmington, DE, USA). These sections were mounted on 200 mesh copper or nickel grids, stained with uranyl acetate and lead citrate for 15 min each, and examined under a JEOL JEM 1400 TEM (Tokyo, Japan). The images were digitally acquired by a CCD digital camera Orious 1100W (Tokyo, Japan) at the HEMS/IBMC of the University of Porto. Transendothelial Pathways. In order to study the preferential transendothelial pathway of functionalized and non-functionalized SLNs across the BBB barrier, lucifer yellow (20 μM) and propranolol (10 μM) were used as paracellular and transcellular tracers, respectively.43 Tracers were incubated with each type of NPs on the apical side of the hCMEC/D3 monolayer for 1 h, at 37 °C and 5% CO2. Finally, the tracer concentration in the basolateral compartment was quantified by fluorescence analysis (428/540 nm for lucifer yellow and 290/ 330 nm for propranolol). The permeability of each tracer in the presence of NPs was then compared to that in the absence of NPs, for the same studied conditions. Statistical Analysis. Data was expressed as mean ± SD and all experiments were independently repeated at least three times. Statistical analysis was assessed using one-way ANOVA followed by Bonferroni, Tukey, and Dunnett post hoc tests calculated using SPSS software (v 20.0; IBM, Armonk, NY, USA). p values

Apo E-Functionalization of Solid Lipid Nanoparticles Enhances Brain

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