Transport pathways of solid lipid nanoparticles across Madin-Darby canine kidney epithelial cell monolayer.

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Transport pathways of solid lipid nanoparticles across MDCK epithelial cell monolayer Gui-Hong Chai, Fuqiang Hu, Jihong Sun, Yong-Zhong Du, Jian You, and Hong Yuan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5004674 • Publication Date (Web): 08 Sep 2014 Downloaded from http://pubs.acs.org on September 16, 2014

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Molecular Pharmaceutics

Transport pathways of solid lipid nanoparticles across MDCK epithelial cell monolayer Gui-Hong Chai a, Fu-Qiang Hu a, Jihong Sun b, Yong-Zhong Du a, Jian You a and Hong Yuan a,* a

College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, P. R. China

b

Department of Radiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, P. R. China

Corresponding author: Dr. Hong Yuan College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, P.R. China. Tel (Fax):86-571-88208439. E-mail address: [email protected] (H. Yuan)

Abstract: An understanding of drug delivery system transport across epithelial cell monolayer is very important for improving the absorption and bioavailability of the drug payload. The mechanisms of epithelial cell monolayer transport for various nanocarriers may differ significantly due to their variable components, surface properties or diameter. Solid lipid nanoparticles (SLNs), conventionally formed by lipid materials, have gained increasing attention in recent years due to their excellent biocompatibility and high oral bioavailability. However, there have been few reports about the mechanisms of SLNs transport across epithelial cell monolayer. In this study, the molecular mechanisms utilized by SLNs of approximately 100 nm in diameter crossing intestinal epithelial monolayer were carefully studied using a simulative intestinal epithelial monolayer formed by Madin-Darby canine kidney (MDCK) epithelial cells. The results 1

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demonstrated that SLNs transportation did not induce a significant change on tight junction structure. We found that the endocytosis of SLNs into the epithelial cells was energy-dependent and was significantly greater than nanoparticle exocytosis. The endocytosis of SLNs was found to be rarely mediated via macropinocytosis, as confirmed by the addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) as an inhibitory agent, and mainly depended on lipid raft/caveolae- and clathrin-mediated pathways. After SLNs was internalized into MDCK cells, lysosome was one of the main destinations for these nanoparticles. The exocytosis study indicated that the endoplasmic reticulum, Golgi complex and microtubules played important roles in the transport of SLNs out of MDCK cells. The transcytosis study indicated that only approximately 2.5 % of the total SLNs was transported from the apical side to the basolateral side. For SLNs transportation in MDCK cell monolayer, greater transport (approximately 4-fold) was observed to the apical side than to the basolateral side. Our findings may present a more comprehensive understanding on the transport of SLNs across epithelial cell monolayer.

Keywords: solid lipid nanoparticles; MDCK cell monolayer, endocytosis; exocytosis; transcytosis.

1. Introduction Biological barriers, mainly represented by epithelial tissue, are the main hindrance against the absorption of exogenous substances and protect against the invasion of pathogenic microorganisms 1. However, the barrier formed by the epithelial cell monolayer, which is extensively distributed in the gastrointestinal tract, often affects the oral bioavailability and therapeutic efficacy of drugs, especially those with poor water solubility and membrane penetration. The needs to overcome the epithelial barrier and accelerate the transport of drugs have been major challenges for pharmaceutical science for a long period of time. In recent years, the application of nanocarriers has been one of the main approaches to overcome the epithelial barrier 2. Compared with free drugs, nanocarriers can often alter the cell uptake pathways of drugs 2


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loaded in the nanoparticles and promote the transport of those drugs through the epithelial cell monolayer. In the last few years, solid lipid nanoparticles (SLNs) have been widely studied and developed as a promising nanocarriers 3. Similar to lipid nano-emulsions, liposomes and micelles, SLNs can enhance the oral bioavailability of drugs due to a high degree of dispersion 4. Conventionally, SLNs are composed of biocompatible and biodegradable materials, and have been widely used as a delivery vehicle to shuttle various biomolecules. Currently, oral delivery of SLNs is considered to be the preferred administration route due to the distribution over a larger surface area, better physical stability, greater protection of incorporated drugs from degradation, more constant plasma levels, controlled drug release, and site-specific targeting 5-7. Previous studies of the transport mechanism of SLNs have been mostly focused on tumor cell uptake pathways and partially on intracellular trafficking

8, 9

. Few studies on the transport mechanism of SLNs in

epithelial cells have been reported thus far. The transcytosis of SLNs is very complicated, involving nanoparticle internalization into cells via endocytosis from the apical side, intracellular transport, and finally exit from the basolateral side via exocytosis. Thus, a comprehensive investigation and improved understanding of the mechanisms involved in SLNs transport across the gastrointestinal epithelial monolayer are necessary to fabricate optimized SLNs with high oral bioavailability. In our previous study, we systematically investigated drug delivery using SLNs, including cell uptake 9, 10

, transport behaviors in vivo

5

and, therapeutic efficacy

11

. However, we lacked a comprehensive

understanding of the entire transport process for SLNs in MDCK cells. In this work, nano-sized SLNs was prepared, and a simulative intestinal epithelial monolayer formed by MDCK epithelial cells was employed to investigate the molecular mechanisms of SLNs transportation. Due to their similar polarity and tight junctions, MDCK epithelial cells, similar to Caco-2 cells, are often used to simulate the gastrointestinal tract 12, 13

. Herein, different pharmacological inhibitors and some novel techniques were utilized to investigate the

pathways used by SLNs during transport across epithelial cells. These pharmacological inhibitors used in our study demonstrated minimal effects on cell viability compared to the control (culture medium), a finding 3



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that was validated elsewhere 14. This confirmed the qualification of these inhibitors for investigation. 2. Experimental Section 2.1. Materials Monostearin was purchased from Chemical Reagent Co., Ltd. (Shanghai, China). Octadecylamine (ODA) was purchased from Fluka, USA. Chlorpromazine, sodium azide, nystatin, filipin III, cytochalasin D, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), brefeldin A, monensin, nocodazole, bafilomycin A1, Hoechst 33342 and fluorescein isothiocyanate (FITC) were obtained from Sigma Aldrich (St. Louis, MO, USA). Rhodamine-phalloidine, mouse anti-ZO-1, AlexaFluor-555 donkey anti-mouse IgG, LysoTracker® Red DND-99 dye, and tetramethylrhodamine-conjugated human Tfn were acquired from Invitrogen (Eugene, Oregon, USA). Poloxamer 188 was purchased from Shenyang Jiqi Pharmaceutical Co., Ltd. (China). MDCK cell lines were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g/L glucose) culture solution, penicillin, streptomycin, trypsin, and ethylene diamine tetraacetic acid (EDTA) were purchased from Gibco BRL (USA). All other chemical reagents were analytical grade or better. 2.2. Preparation and characterization of SLNs SLNs was prepared by a solvent diffusion method in an aqueous system reported in our previous study 15

. FITC labeled ODA (ODA-FITC) was synthesized according to our previously reported protocol

16

, and

was used as a fluorescence marker to be incorporated into the SLNs. In brief, monostearin (20 mg) and ODA-FITC (1.6 mg) were dissolved with 2 mL of ethanol under a water bath at 70 °C. Then, the ethanol solution was quickly dispersed into 20 mL of poloxamer 188 solution (0.1%, w/v) under 400 rpm stirring for 5 min at 70 °C. The freshly prepared emulsion (melted lipid droplet) was then cooled to room temperature for solidification. The particle size and surface charge of the SLNs dispersion were measured using a Zetasizer analyzer (3000HS, Malvern Instruments Ltd., UK) and particle size and Zeta potential analyzer 4


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(90Plus, Brookhaven Instruments Corporation, USA) at a nanoparticle concentration of 100 µg/mL diluted with dispersion medium. In addition, the SLNs was also analyzed by transmission electron microscopy (TEM). After being negatively stained by phosphotungstic acid, SLNs on a copper grid was examined by a transmission electron microscope (JEM1230, JEOL, Japan). The stability of the SLNs in storage was also investigated. The freshly prepared SLNs was stored at 4 °C, and particle size, as well as Zeta potential, was detected at predetermined time. 2.3. MDCK cell culture MDCK cells were grown in 75 cm2 plastic flasks containing DMEM supplemented with 10 % (v/v) fetal bovine serum, 1 % (v/v) L-glutamine, penicillin (100 U/mL) and streptomycin (100 U/mL) with the environmental conditions maintained at 37 °C in an atmosphere of 5 % CO2 with 95 % relative humidity. For the studies of endocytosis and exocytosis in MDCK cells, the cells were digested with 0.25 % (w/v) trypsin-0.02 % (w/v) EDTA solution, and seeded in 6-well sterile plates at 1×105 cells/mL. For the transcytosis studies, MDCK cells were cultured on polycarbonate filter membranes with a pore size of 0.4 µm and a surface area of 1.12 cm2 (Costar Transwell, Millipore Corp., Bedford, MA, USA). One-half milliliters of DMEM containing 3×105 cells was added into the upper compartment of each Transwell insert, and the medium volume in the basolateral side was set as 1.5 mL. During the culture for 7 days, the media in both the upper and lower compartments was changed every other day, and the integrity of the cell monolayer was verified by measuring the transepithelial electrical resistances (TEER) values using a Millicell-ERS volt-ohmmeter (Millipore Co., USA). The cell monolayers with TEER values above 180 Ω·cm2 were selected for the subsequent studies. 2.4. Transportation of SLNs in MDCK cells 2.4.1. Cellular uptake of SLNs The internalization of SLNs into MDCK cells was investigated by a flow cytometry system (FCS, FC500MCL, Beckman Coulter). MDCK cells were grown in 6-well plates for 24 h, and then incubated with 5



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FITC labeled SLNs (50 µg/mL) for varying time intervals. After incubation, the cells were rinsed with cold PBS three times. Then, the cells were collected for the FCS analysis. Moreover, to investigate whether the internalization process was energy-dependent, MDCK cells were pre-treated with low temperature (4 °C) and sodium azide (1 mg/mL) for 1 h, and then incubated with FITC labeled SLNs (50 µg/mL) for 1 h. Then, the cells were collected for detection by FCS. Table.1 Inhibitors used in this study and their functions as well as concentrations. Inhibitors Energy-dependent inhibitor Sodium azide Endocytosis inhibitors Nystatin Filipin Chlorpromazine EIPA Cytochalasin D Intracellular transport inhibitors Brefeldin A Monensin Nocodazole Bafilomycin A1

Function active transport inhibitor

lipid raft/caveolae-mediated route lipid raft/caveolae-mediated pathway inhibitor of clathrin-mediated pathway inhibitor of macropinocytosis pathway disrupt actin filaments

blocks transport from ER to Golgi complex blocks transport from Golgi complex to plasma membrane disrupts microtubules inhibitor of endosomal acidification

Final concentration 1 mg/mL

30 µM 5 µg/mL 30 µM 100 µM 5 µM

25 µg/mL 32.5 µg/mL 6 µg/mL 100 nM

2.4.2. Analysis of SLNs endocytosis pathways In the SLNs endocytosis study, MDCK cells were pre-treated with various inhibitors at the given concentrations (Table 1) for 30 min, and then incubated with FITC labeled SLNs (50 µg/mL) for 1 h. The cells were rinsed with cold PBS three times, and then collected for detection by FCS. 2.4.3. Intracellular destinations of SLNs in MDCK cells 2.4.3.1. Colocalization of SLNs with lysosomes MDCK cells were seeded in glass bottom dishes at 1×105 cells/mL for 24 h. The cells were incubated with FITC labeled SLNs (50 µg/mL) for 1 h, 2 h or 4 h. The cells were washed with PBS three times, and then stained by LysoTracker® Red DND-99 dye (50 nM) for 30 min. Next, the cells were washed with cold PBS three times, and observed by confocal laser scanning microscopy (CLSM). 6


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2.4.3.2. Colocalization of SLNs with endosomes mediated via clathrin MDCK cells were seeded in glass bottom dishes at 1×105 cells/mL for 24 h. The cells were incubated with tetramethylrhodamine-conjugated human Tfn (40 µg/mL) for 30 min and then with FITC labeled SLNs (50 µg/mL) for 15 min, 30 min or 60 min, and were observed by CLSM. 2.4.4. Analysis of SLNs exocytosis pathways For the exocytosis study, MDCK cells were incubated with FITC labeled SLNs (50 µg/mL) at 37 °C for 1 h. After aspiration of the nanoparticle dispersion, the cells were rinsed by cold PBS three times and then incubated in fresh medium containing various inhibitors (intracellular transport inhibitors in Table 1) for another 2 h. The cells were rinsed with cold PBS three times, and the nanoparticles remaining within the cells were determined by FCS. 2.5. Transportation of SLNs in MDCK cell monolayer 2.5.1. Verification of the transportation of SLNs across MDCK cell monolayer MDCK cell monolayers grown on porous Transwell insert membranes were incubated with FITC labeled SLNs (100 µg/mL) in HBSS solution at 37 °C for 4 h. Then, the polycarbonate membranes covered by the cell monolayers were carefully separated from the Transwell insert, washed with cold PBS, fixed with 4 % paraformaldehyde, and stained with rhodamine-phalloidine (165 nM) and Hoechst 33342 (2 µg/mL) to label the F-actin and cell nuclei, respectively. Finally, the membranes were placed on glass slides, sealed and observed by CLSM on an x-y-z scanning mode. As a separate experiment, the transport medium in the apical and basolateral sides after 4 h transportation of SLNs (transport in cell culture medium, DMEM) was collected and sampled for TEM observation. 2.5.2. Quantitative detection of the cumulative and total transport of SLNs MDCK cell monolayers cultured on Transwell insert membranes were incubated with FITC labeled SLNs (100 µg/mL) in HBSS. At predetermined time, the medium in the basolateral compartment was completely aspirated and replaced by fresh HBSS. The amount of SLNs in the basolateral medium was 7



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measured using a fluorescence spectrophotometer (F-2500, HITACHI Co., Japan). The cumulative transport was calculated as the sum of SLNs quantified at each time point. At the end of the experiment (4 h), the amount of SLNs in the apical compartment was measured using a fluorescence spectrophotometer. The total transport was calculated as the initial amount of SLNs minus the amount of SLNs at 4 h in the apical compartment. During the whole experiment, the TEER values were monitored to determine whether the transport of the SLNs was dependent on paracellular transport through the cell monolayer or on transport across the entire cell membrane by the transcytosis pathway. 2.5.3. The exocytosis of SLNs from MDCK cell monolayer To evaluate the amount of SLNs exocytosed from the cell monolayer via the apical and basolateral sides, MDCK cells were cultured on polycarbonate membranes for 7 days to form a tight monolayer, and then incubated with FITC labeled SLNs (100 µg/mL) for 2 h. The cell monolayers were rinsed with cold HBSS three times, and then covered by another polycarbonate membrane to keep the external environment identical for both the apical and basolateral sides. The media in apical and basolateral compartments was replaced by fresh HBSS. The cell monolayers were incubated for another 3 h. Then, the amount of SLNs in the apical and basolateral compartments was quantified using a fluorescence spectrophotometer. 2.5.4. Immunofluorescence staining of MDCK cell monolayers MDCK cell monolayers on polycarbonate membrane were incubated with FITC labeled SLNs (100 µg/mL) for 4 h. Then, the membrane was cut, washed with cold PBS three times, treated with cold triton X-100 (0.2 % w/v, pH 7.1, 100 mM KCL, 3 mM MgCL2, 1 mM CaCL2, 200 mM sucrose, and 10 mM HEPES) for 2 min in an ice bath and fixed with 4 % paraformaldehyde in PBS for 15 min. The fixed cell monolayer was further washed three times with PBS, treated with Triton X-100 (0.05 %) in PBS for 5 min, and washed with PBS two times. The cell monolayers were incubated with BLOTTO (5 % instant nonfat dry milk in PBS) solution for 30 min, and then with mouse anti-ZO-1 (1:50 in BLOTTO solution) for 1.5 h at room temperature. After three washes with the BLOTTO solution, the cell monolayers were incubated with 8


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AlexaFluor-555 donkey anti-mouse IgG (1:500 in BLOTTO solution) for 1 h. Thereafter, the cell monolayers were washed with BLOTTO and PBS again, mounted with glycerol/PBS (1:1, v:v) and finally visualized by CLSM. 2.5.5. Investigation of the molecular mechanism of SLNs transport across MDCK cell monolayer MDCK cell monolayers were pre-treated with various inhibitors (Table 1) in HBSS for 30 min, then incubated with FITC labeled SLNs (100 µg/mL) in HBSS containing the inhibitors (Table 1) for 4 h. After incubation, the total amount of SLNs in the basolateral compartment was detected using a fluorescence spectrophotometer. As a separate experiment, MDCK cell monolayers were pre-treated with FITC labeled SLNs (100 µg/mL) in HBSS for 2 h. The monolayers were washed with HBSS three times, and the media in the apical and basolateral compartments was replaced by fresh HBSS containing various inhibitors (intracellular transport inhibitors in Table 1). The monolayer was incubated for another 3 h, after which the total amount of SLNs in the apical and basolateral compartments was detected using a fluorescence spectrophotometer. 2.6. Statistics All data (ANOVA) are presented as the mean value ± SD from at least three independent experiments. A p-value less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Preparation and characterization of SLNs As reported in our previous study, approximately 97.9 % of added ODA-FITC was incorporated into the SLNs

5, 16

. Moreover, under sink conditions, less than 7 % of ODA-FITC leaked from the loaded SLNs

after 24 h of incubation in different medium, i.e. plasma, phosphate buffered saline (PBS, pH 6.8) containing 0.3 wt% sodium dodecyl sulfate (SDS), cell culture medium (DMEM) and transport medium (HBSS). Therefore, there was negligible ODA-FITC leakage from the SLNs in our experiment, indicating that ODA-FITC could be used as an effective fluorescence marker for the SLNs. The characterization of the 9



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FITC labeled SLNs was detected by dynamic light scattering (DLS). The number diameter was 88.3 nm, and the polydispersity index was 0.128. The Zeta potential of the FITC labeled SLNs was -28.78 mV. The transmission electron microscope (TEM) results (Fig. 1A) indicated a spherical morphology and that the particle size was also less than 100 nm, with uniform distribution, which is in agreement with the DLS analysis. The uniform size distribution of SLNs was an advantage in eliminating the potential influence of particle size on their cellular internalization 17, 18. The stability of the FITC labeled SLNs is illustrated in Fig. 1B, and the particle size and Zeta potential over 7 days were not altered significantly. Thus, the quality of the SLNs could be ensured over the experimental period.

Fig. 1 Characterization of SLNs. (A) TEM image of SLNs (bar=0.1 µm). (B) The stability of SLNs monitored by particle size distribution and Zeta potential over one week. 3.2. Transportation of SLNs in MDCK cells 3.2.1. Cellular uptake of SLNs Fig.2 A1 shows the results of the cellular uptake of SLNs at 37 °C after different time intervals (0.5 h, 1 h, 2 h and 4 h). The endocytosis of SLNs increased remarkably with greater incubation time. In addition, the MDCK cells were treated with low temperature (4 °C) and sodium azide to investigate whether the internalization of the SLNs was energy-dependent, as active transport would be inhibited by these two treatment processes

19

. Fig. 2A2 presents the cellular uptake of the SLNs under these two treatment

processes, and both of these treatments dramatically decreased the endocytosis of the SLNs. Therefore, the 10


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internalization of SLNs can be concluded to be both time and energy dependent.

Fig. 2 The endocytosis and exocytosis of SLNs in MDCK epithelial cells. (A1) The effects of incubation time on SLNs internalization. (A2) The effects of sodium azide and low temperature (4 °C) on the endocytosis of SLNs. (B) The effects of different inhibitors (endocytosis inhibitors in Table 1) on the endocytosis of SLNs. (C) The effects of different inhibitors (intracellular transport inhibitors in Table 1) on the endocytosis and exocytosis of SLNs. The italic numbers (X-Mean values from FCS results) in the brackets represent the fluorescence intensity in MDCK cells, and each data represents the mean value of three tests. 3.2.2. Endocytosis pathways utilized by SLNs The endocytosis of nanomedicines into cells have been reported to involve several pathways, including 11



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lipid raft/caveolae- and clathrin-mediated pathways, as well as macropinocytosis

20

. The endocytosis of

many extracellular macromolecules is reported to be mediated by lipid rafts or caveolae via specific or non-specific interactions. Filipin, which is a cholesterol binding agent, is known to function as an inhibitor of lipid raft/caveolae-mediated endocytosis. In addition, nystatin, which is a cholesterol sequestering agent, triggers the loss of lipid rafts and caveolae in cells and inhibits the internalization of nanoparticles. The results presented in Fig. 2B1 indicated that filipin and nystatin were able to significantly reduce the endocytosis of SLNs. Lipid rafts or caveolae have been reported to distribute to different extents in various cell membranes 21. In MDCK cells, the caveolae has been verified to distribute on the cell membrane in a polarized manner

22, 23

, with the cellular apical membrane devoid of caveolae but containing lipid rafts.

Therefore, the endocytosis of the SLNs by MDCK cells from the apical side can be concluded to be mediated by lipid rafts, and the endocytosis of SLNs is more reliant upon cholesterol. Clathrin-mediated endocytosis is a classical internalization pathway for macromolecules

24, 25

.

Extracellular substances are incorporated into clathrin-coated pits, which pinch off from plasma membrane, and are then internalized by the cell along with these pits. Chlorpromazine, a cationic drug that inhibits clathrin assembly at the plasma membrane 26, was used to clarify whether the endocytosis of SLNs was via a clathrin-mediated pathway. The results (Fig. 2B1) demonstrated that the endocytosis of SLNs was remarkably reduced with the addition of chlorpromazine. Therefore, the endocytosis of SLNs was confirmed to be mediated by clathrin-coated vesicles. Macropinocytosis is a bulk internalization process involving a dynamin-independent pathway that differs from the lipid raft/caveolae- and clathrin-mediated pathways

27

. Extracellular macromolecules are

internalized via plasma membrane ruffling that forms macropinosomes, and the size of macropinosomes is often larger than 1 mm. Many substances, including bacteria, viruses and apoptotic bodies can trigger macropinocytosis by different types of cells. EIPA, a Na+/H+ ion channel blocking agent derived from amiloride, inhibits the macropinocytosis mediated pathway 28. In the present study, no impact of EIPA on the 12


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endocytosis of SLNs was observed (Fig. 2B2), indicating that the internalization of the nanoparticles is rarely mediated by the macropinocytosis pathway. Particles exceeding 150 nm in size have been reported to be able to be internalized by cells through the macropinocytosis pathway 29, but the SLNs in this study was less than 100 nm. Cytochalasin D, a cell permeable toxin, can disrupt actin filaments 30. In the present study, the addition of cytochalasin D exerted gentle suppression of SLNs endocytosis (Fig. 2B2), implying that the endocytosis of SLNs involved actin to some extent. Some reports have elucidated that the process of nanoparticle internalization and the transport of nanoparticles are reliant on the role of the actin cytoskeleton in the endocytic machinery 20, 31. Thus, the internalization of SLNs can be concluded to also depend on actin. 3.2.3. Intracellular destinations of SLNs in MDCK cells 3.2.3.1. Colocalization of SLNs with lysosomes In the present study, the colocalization of SLNs with lysosomes was investigated. Fig. 3 shows the colocalization of SLNs with lysosomes after MDCK cells were incubated with FITC labeled SLNs (50 µg/mL) for varying time periods. Over time, namely from 1 h to 4 h, the area A (ODA-FITC labeled SLNs) overlap B (lysosomes) increased from 18.15 % to 32.75 %, and this result indicated that increasing amount of SLNs was transported into lysosomes. In contrast, the area B (lysosomes) overlap A (ODA-FITC labeled SLNs) increased from 83.26 % to 97.83 %, meaning that more lysosomes were occupied by SLNs. The results clearly indicate that the SLNs traveled into lysosomes gradually over time, but the proportion of transported nanoparticles was approximately 30 % after 4 h incubation. Therefore, the internalized SLNs was found to also be transported to lysosomes in this study. During the endocytosis process, some of internalized SLNs was delivered from apical early endosomes (AEE) to late endosomes (LE) or multivesicular bodies (MVB), and ultimately entered into the lysosomes. However, most of the SLNs remained in other organelles or in the cytoplasm, and some of these nanoparticles would likely be transported to the basolateral side in MDCK cells. 13



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Fig. 3 Colocalization micrographs and scatterplots of SLNs with lysosomes. The green color represented SLNs, and the red color represented lysosomes. In the scatterplots graph, the closer scatterplots to diagonal the more obvious colocalization of SLNs and lysosomes was obtained. Area A (ODA-FITC labeled SLNs) overlap B (lysosomes) indicated the proportion of SLNs that was internalized into lysosomes, and area B overlap A represented the proportion of lysosomes that was occupied by SLNs. Scale bar: 20 µm. 3.2.3.2. Colocalization of SLNs with endosomes mediated via clathrin Transferrin (Tfn) related endocytosis is a well-known clathrin-mediated pathway

32

. After endocytosis

via clathrin-coated pits, the initial delivery sites of Tfn are the sorting endosomes (or early endosomes), and then the molecules rapidly exit from the sorting endosomes and are either returned directly to the plasma membrane or are transported to the recycling endosomes (RE). From the RE, essentially all of the transferrin recycles to the cell surface. The colocalization of SLNs and these endosomes was investigated in the present study. Fig. 4 illustrates the colocalization between the SLNs and these endosomes, and the area B (clathrin related endosomes) overlap A (ODA-FITC labeled SLNs) increased approximately from 75 % to 95 % at 15 min and 60 min, respectively, which indicated that an increasing amount of these endosomes were occupied by SLNs. However, the area A (ODA-FITC labeled SLNs) overlap B (clathrin related endosomes) was almost unchanged, and the values were approximately 20 %. These results indicated that over longer time 14


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intervals, increasing numbers of SLNs entered into clathrin related endosomes, but the proportion of the SLNs that was internalized via clathrin-coated pits was a constant value (approximately 20 %).

Fig. 4 Colocalization of SLNs with endosomes formed by clathrin coated pits. The green color represented SLNs, and the red color indicated tetramethylrhodamine-conjugated human Tfn labeled endosomes. Area A (ODA-FITC labeled SLNs) overlap B (clathrin related endosomes) indicated the proportion of SLNs that was internalized into clathrin coated pits. Area B overlap A represented the proportion of tetramethylrhodamine-conjugated human Tfn contained vesicles, namely clathrin coated pits, which was occupied by SLNs. Scale bar: 20 µm. 3.2.4. Investigation of SLNs exocytosis pathways Exocytosis is a transport process antidromic to endocytosis, by which a cell discharges internalized cargo out of the cell 33. To simplify the exocytosis process, the exocytosis pathways evaluated in this study only included the discharge of SLNs from the apical side. After SLNs was internalized into MDCK cells, specific organelles and structures will participate in its transport, and finally resulting in the exocytosis of the nanoparticles out of the cells. To investigate the contribution of the Golgi complex, endoplasmic reticulum (ER), microtubules and endosomal acidification process to the transport of SLNs, brefeldin A, monensin, nocodazole and bafilomycin A1 were added as inhibitory agents to elucidate the intracellular 15



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process of SLNs. Furthermore, the effects of these inhibitory agents on the endocytosis of SLNs were also investigated. The Golgi complex has been reported to serve as an important station for both the secretory pathway from the ER to the plasma membrane (PM) and endocytic recycling

34

. Brefeldin A (BFA), a fungal

metabolite, blocks forward transport between the ER and Golgi complex

35

. Monensin (MON), another

inhibitor often used for the disruption of the Golgi complex, has been shown to effectively block the transportation of macromolecules from the Golgi complex to the PM

36

. As shown in Fig. 2C1, these two

inhibitors exhibited no effects on the endocytosis of SLNs, indicating that the ER and Golgi complex did not regulate the endocytosis of SLNs. However, for the exocytosis study, the remained SLNs in the MDCK cells increased with the adding of brefeldin A and monensin (Fig. 2C2), which indicates that these two inhibitors decreased the exocytosis of SLNs from the MDCK cells. Therefore, the exocytosis of SLNs might involve in these two pathways; namely, both the ER and Golgi complex were important regulatory organelles for the exocytic transportation of SLNs. Nocodazole is an antibiotic agent that disrupts microtubules, resulting in a kinetic effect on the delivery of proteins to both surfaces

37

. Some vesicles have been reported to be transported directionally along

microtubules (MTs) using vesicle-associated motors. The results presented in Fig. 2C1 indicate that nocodazole exerted few effects on endocytosis but dramatically affected the exocytosis of the SLNs (Fig. 2C2). The endocytosis of nanoparticles into MDCK cells has been reported to be relatively easier than the exocytosis process

14

. As demonstrated in our study, although these inhibitors (intracellular transport

inhibitors in Table 1) could exert significant effects on the exocytosis of the SLNs, most of the SLNs remained within the MDCK cells. These results further indicated that endocytosis is a relative facile process compared to exocytosis. Therefore, the SLNs exocytosis process may be more dependent on microtubules compared with SLNs endocytosis. These findings further confirmed that microtubules play an important role on the transport of SLNs containing vesicles. 16


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Lysosomes are the prime destination for internalized macromolecules

38

, and following acidification,

early endosomes mature into late endosomes and finally to lysosomes. Bafilomycin A1, which is known to be an inhibitor of endosomal acidification 39, was added to investigate the effects of the acidification process on the endocytosis and exocytosis of SLNs. As illustrated in Fig. 2C1, the inhibition of endosomal acidification increased the fluorescence intensity in MDCK cells, but exhibited no effects on the exocytosis of SLNs (Fig. 2C2). Because the lysosomal pathway is a degradation route for most macromolecules, the inhibition of acidification may prevent SLNs from being transported to lysosomes, resulting in the avoidance of degradation. Thus, the net result was to increase the amount of SLNs remaining in the MDCK cells rather than increasing endocytosis. For exocytosis, the results indicated that the acidification process exerted no effects on the exocytosis of SLNs. The transport of SLNs in MDCK cells was complex, and involved different structures and organelles. Inhibition of these intracellular processes exerted negligible effects on the endocytosis of SLNs, which meant that the endocytosis is a facile procedure and independent on intracellular process. However, inhibition of these intracellular processes can significantly decrease the exocytosis of SLNs. Our results indicated that the endoplasmic reticulum, Golgi complex and microtubules played important roles in the transport of SLNs out of MDCK cells. 3.3. Transportation of SLNs in MDCK cell monolayer 3.3.1. The evaluation of SLNs transport across MDCK cell monolayer After incubation with the FITC labeled SLNs (100 µg/mL) for 4 h, the insert membranes with MDCK cell monolayers were cut off and prepared for CLSM observation via an x-y-z mode. To illustrate the results vividly, the data were processed by Imaris software into a three-dimensional micrograph. As displayed in Fig. 5A, the MDCK cell monolayers incubated without FITC labeled SLNs only exhibited stained cell nuclei and F-actin. However, with the same parameters as those in Fig. 5A, Fig. 5B clearly shows that the SLNs (green) was transported throughout the cell monolayer. Fig. 5C further presents the slices of the 17



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CLSM results that represent representative images from the apical, middle and basolateral focal planes of the MDCK cell monolayer. In the basolateral focal plane, the nuclei (blue) and F-actin (red) were almost imperceptible, indicating that the focal plane had reached the bottom of the cell monolayer, but the SLNs (green) could be clearly observed. These results suggested that the SLNs was transported across the MDCK cell monolayer. Furthermore, the TEM results of SLNs in the apical side and basolateral side after 4 h transportation in cell culture medium (DMEM) demonstrated that the SLNs after transcytosis was intact (data not shown). The reason for changing the transport medium (HBSS replaced by DMEM) was that there was a large amount of crystals (a variety of salts in HBSS) precipitated on the copper grid when used HBSS as transport medium. This further verified the integrity of SLNs during the transport across MDCK cell monolayer.

Fig. 5 Verifying the transport of SLNs across MDCK cell monolayer. (A)(B)Three-dimensional micrograph of MDCK cell monolayer grown on porous membrane after incubation without FITC labeled SLNs or with FITC labeled SLNs (100 µg/mL) for 4 h at 37 °C, respectively. (C) Slices of confocal fluorescence micrograph series achieved via an x-y-z mode, which typically represent apical, middle and basolateral focal 18


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plane of MDCK cell monolayer. Cells were stained with rhodamine-phalloidin to label F-actin (red) and Hoechst 33342 to mark cell nuclei (blue). All data were processed by Imaris software, and the scale bar marked in pictures was 20 µm. 3.3.2. Quantitative detection of the cumulative and total transport of SLNs The amount of SLNs transported across the MDCK cell monolayer was detected, and the cumulative transcytosis of SLNs is shown in Fig. 6A1. The accumulated transcytosis of SLNs increased significantly over time, and the transcytosis rate between 2 h and 3 h was the fastest. The final transcytosis amount of SLNs at 4 h was approximately 1.25 µg, which was approximately 2.5 % of the amount (100 µg/mL, 0.5 mL) added into the apical compartment at the beginning. For the determination of total SLNs transport, Fig. 6A2 indicates that approximately 10 % of the total amount of SLNs added into the apical compartment was transported into and across (transcytosis) the MDCK cell monolayer. Therefore, the transcytosis of the SLNs was rather difficult compared to internalization into the MDCK cell monolayer.

Fig. 6 Evaluation of the transport of SLNs across MDCK cell monolayer. (A1) Transcytosis of SLNs across MDCK cell monolayer by monitoring the cumulative amount of SLNs in the basolateral chamber as the 19



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function of time (n=6). (A2) The amount of SLNs in the apical and basolateral chambers after 4 h transportation (n=6). (A3) The amount of SLNs in the apical and basolateral chambers after re-incubation with fresh transport medium for another 3 h. (B) Evaluation of the integrity of tight junctions (TJ) after 4 h transportation of SLNs, where B1 represents the control group (without FITC labeled SLNs), and B2 was the group that was added FITC labeled SLNs (100 µg/mL). Scale bar: 10 µm. 3.3.3. The exocytosis of SLNs from the MDCK cell monolayer After SLNs was internalized into the MDCK cell monolayer from the apical side via endocytosis, the nanoparticles were able to be exocytosed from both sides. To evaluate the amount of SLNs transported from within the MDCK cell monolayer to the apical and basolateral sides, a novel sandwich-like structure (MDCK cell monolayer between two polycarbonate membranes) was established. As shown in Fig. 6A3, the amount of SLNs in the apical chamber was 4-fold higher than that in basolateral chamber after another 3 h incubation. This finding indicated that the SLNs internalized into the MDCK cell monolayer was more inclined to be transported to the apical side than to the basolateral side. These results further indicated that the transport of SLNs across MDCK cell monolayer is rather difficult compared with endocytosis. 3.3.4. Effects of SLNs on the integrity of MDCK cell monolayer Transepithelial electrical resistance is widely used to evaluate the integrity of epithelial cell monolayers. The TEER values of the MDCK cell monolayers were monitored during 4 h of SLNs transportation, and no significant changes were observed (data not shown). This finding indicated that the transport of SLNs did not trigger an increase in MDCK cell monolayer permeability. Furthermore, the influence of the SLNs on the tight junctions of the MDCK cell monolayers was detected by CLSM. As illustrated in Fig. 6B, the integrity of the cellular tight junctions was not changed following SLNs transport, and this result suggests that the influence of SLNs on tight junctions may be ignored. As is well known, the toxicity of nanoparticles is the main restriction to their application. In the present study, the SLNs exhibited almost no effect on the integrity of the epithelial cell monolayer, ensuring the safety of the SLNs as an oral drug delivery system. 20


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3.3.5. Investigation of the molecular mechanism through which SLNs cross the MDCK cell monolayer The effects of different inhibitors (Table 1) on the transport of SLNs through MDCK cell monolayer was investigated, and all inhibitors used in the present study exerted little influence on the TEER values of the MDCK cell monolayer (data not shown). These findings suggest that the inhibitors utilized in this study did not affect the integrity of the MDCK cell monolayer, helping to ensure the credibility of the results. The various inhibitors were used under two conditions to study the transportation of SLNs across MDCK cell monolayer. One condition was the whole transport condition, which included endocytosis from apical side, exocytosis from the apical side and transcytosis. The second transport condition excluded endocytosis and only evaluated exocytosis from the apical side and basolateral side.

Fig. 7 Effects of different inhibitors on the transport of SLNs across MDCK cell monolayer. (A) The effects of different inhibitors on the transport of SLNs across MDCK cell monolayer in the whole transport condition, where A1 was the results of endocytosis inhibitors, and A2 was the results of intracellular transport inhibitors. (B) The effects of intracellular transport inhibitors on the transport of SLNs across MDCK cell monolayer in the second transport condition, where B1 was the results of SLNs exocytosis to 21



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the apical side, and B2 was the results of SLNs exocytosis to the basolateral side. For the whole transport condition, sodium azide, which inhibits energy-dependent transport, was able to significantly decrease the transcytosis of SLNs (Fig. 7A1). This result indicated that the transport of SLNs is energy dependent. Filipin and nystatin significantly decreased the transcytosis of SLNs (Fig. 7A1), and these results indicated that the transport of SLNs is through a lipid raft/caveolae-mediated pathway. For the clathrin-mediated pathway, chlorpromazine also significantly decreased the transcytosis of the SLNs (Fig. 7A1), and the results indicated that SLNs transport for the whole transport condition was also via a clathrin-mediated pathway. As to the macropinocytosis pathway, EIPA exhibited minimal effects on the transcytosis of SLNs for the whole transport condition (Fig. 7A1). This finding further suggests that macropinocytosis contributes negligibly to the transport of SLNs. Cytochalasin D significantly decreased the transcytosis of SLNs for the whole transport condition (Fig. 7A1), suggesting that actin may participate in the overall transport of SLNs. Fig. 7A2 shows the effects of intracellular transport inhibitors on the transcytosis of SLNs for the whole transport condition. The transcytosis of SLNs was significantly increased with the addition of brefeldin A and monensin. Based on the exocytosis study in the MDCK cells, the addition of brefeldin A and monensin would be expected to decrease the transcytosis of SLNs, because they inhibited the exocytosis of the SLNs. However, these agents increased the transcytosis of SLNs for the whole transport condition. The reason for this result may be that other existing pathways mediated the transcytosis of the SLNs. The inhibition of the ER/Golgi and Golgi/PM pathways may lead to more SLNs containing vesicles remaining in the plasma and may facilitate the direct transcytosis of the nanoparticles across the MDCK cell monolayer. Vesicles budding from common recycling endosomes (CRE) have been reported to be able to transport directly to the basolateral recycling endosomes (BRE), finally fusing with the basolateral plasma membrane 40

. Therefore, there may be direct pathways that transport SLNs to the basolateral side, and the inhibition of

ER and Golgi mediated pathways could improve the activity of these direct pathways. These findings give 22


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us an enlightment to fabricate SLNs with less transportation via ER/Golgi and Golgi/PM pathways may increase transcytosis of the nanoparticles, and finally increase the oral bioavailability of the drug payload. Our results demonstrated that the addition of bafilomycin A1 increased the transcytosis of the SLNs, and the reason for this finding may due to the inhibition of endosomal acidification, resulting in more SLNs remaining in the early endosomes and, lead to greater transport to the basolateral side rather than to lysosomes for degradation. However, nocodazole dramatically decreased the transcytosis of the SLNs, which was similar to the results of the exocytosis study in MDCK cells. This finding further confirmed that microtubules play an important role on the transcytosis of the SLNs. After the SLNs was internalized into the MDCK cell monolayer via endocytosis from the apical side, the nanoparticles can be exocytosed from the apical and basolateral sides. To investigate the exocytosis of SLNs from the MDCK cell monolayer, the second transport condition was employed to study the effects of different intracellular transport inhibitors on the exocytosis of SLNs from the apical and basolateral sides. The effects of brefeldin A, monensin, nocodazole and bafilomycin A1 on the exocytosis of SLNs from the apical and basolateral sides of the MDCK cell monolayer are depicted in Fig. 7B1 and Fig. 7B2, respectively. With the addition of brefeldin A and monensin, the exocytosis of SLNs from both the apical and basolateral sides was dramatically decreased. This result was consistent with the previous exocytosis experiment in MDCK cells, and further indicated that the ER/Golgi and Golgi/PM pathways played important roles in the transport of SLNs out of the MDCK cell monolayer. The same conclusions were also obtained from the nocodazole and bafilomycin A1 inhibition studies. These results further suggested that microtubules played important roles in the movement of the SLNs, whereas endosomal acidification had few effects on the transport of the SLNs.

23



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Fig. 8 Schematic diagram of transport pathways of SLNs across MDCK cell monolayer. The SLNs was internalized via lipid raft/caveolaes and clathrin-coated pits. The budded SLNs containing vesicles fused with apical early endosomes (AEE). From the AEE, there may be three pathways for SLNs traveling, such as directly transport to plasma membrane, transport to multivescular bodies (MVB)/late endosomes (LE) and finally transport to lysosomes, and transport to apical recycling endosomes (ARE). Lysosomes pathway is a degradation route, and nanoparticles entered into lysosomes could not be easily discharged. Following degradation, some nanoparticles residuum may further transport to the apical or basolateral sides. SLNs entered into ARE may ether be transported to common recycling endosomes (CRE), or transport to the plasma membrane. From CRE, SLNs was transported to Golgi complex, endoplasmic reticulum (ER), or directly to basolateral recycling endosomes (BRE) for transcytosis. After SLNs entered into ER and Golgi complex, the following travel pathways may like the excretion of proteins or chylomicrons. The transport 24


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between different organelles may bidirectional processes, e.g. ARE and CRE. All these pathways were directly (represented by black solid arrows) or indirectly (indicated by black dotted arrows) proven in the present study. 4. Conclusion Oral delivery is an attractive route to deliver therapeutics via nanoparticles due to its ease of administration and patient compliance. Solid lipid nanoparticles, which are promising nanocarriers for oral drug delivery, have gained growing attention in recent years. However, the mechanisms of SLNs transporting across the epithelial cell monolayer have rarely been reported. In the present study, MDCK cells were employed to investigate the molecular mechanisms of SLNs trafficking across the epithelial cell monolayer. The results indicate that SLNs transportation exhibited no effects on tight junctions, and that the transport of SLNs was rarely mediated via macropinocytosis, as confirmed by inhibition experiments. Moreover, the endocytosis of SLNs was energy-dependent and a relatively facile procedure compared to exocytosis. The endocytosis of the SLNs was demonstrated to be mainly dependent on lipid raft/caveolaeand clathrin-mediated pathways, and the inhibition of these pathways decreased the endocytosis of the SLNs on the apical side. For the transcytosis of SLNs, the results demonstrated that only approximately 2.5 % of the total SLNs was transported across the MDCK cell monolayer, and that the exocytosis of the SLNs from the apical side was more facile than that from the basolateral side. The lysosomes were the main destinations for the SLNs, and the inhibition of endosomal acidification increased the transcytosis of the SLNs. The endoplasmic reticulum and Golgi complex may be two important regulatory organelles for SLNs transportation. Microtubules also played an important role in the transport of SLNs. A schematic diagram of transport pathways for SLNs traversing MDCK cell monolayer is presented in Fig. 8. The transport of SLNs across MDCK cell monolayer is complex, and involves different structures and organelles. Our findings may present a more comprehensive understanding of the transport of SLNs across epithelial cell monolayers, and may provide further instructions on designing promising SLNs systems with high transportation efficiency 25



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and excellent bioavailability. Acknowledgments We appreciate the financial support from the National Nature Science Foundation of China under Contract No. 81373349, Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents, Qianjiang Talent Program of Zhejiang Province, China (2012R10027), Scientific Research Foundation of the Health Bureau of Zhejiang Province in China (WKJ2012-2-030) and SRF for ROCS, SEM.

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Ferritin polarization and iron transport across monolayer epithelial barriers in mammals.

MS.

Enhanced oral bioavailability of insulin-loaded solid lipid nanoparticles: pharmacokinetic bioavailability of insulin-loaded solid lipid nanoparticles in diabetic rats.

Lipid transport and epithelial barrier integrity.

Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways.

Epithelial transport during septic acute kidney injury.

Epithelial transport of noscapine across cell monolayer and influence of absorption enhancers on in vitro permeation and bioavailability: implications for intestinal absorption.

Liquid-solid and solid-solid phase transition of monolayer water: high-density rhombic monolayer ice.

Caco-2 cell line: a system for studying intestinal iron transport across epithelial cell monolayers.

Gastrointestinal HCO3- transport and epithelial protection in the gut: new techniques, transport pathways and regulatory pathways.

Nutrient transport pathways across the epithelium of the placenta.

Kinetics of macrotetrolide-induced ion transport across lipid bilayer membranes.

Dynamic covalent transport of amino acids across lipid bilayers.

Anion transport across varying lipid membranes--the effect of lipophilicity.

Growth of Madin-Darby canine kidney epithelial cell (MDCK) line in hormone-supplemented, serum-free medium.

Preparation and evaluation of novel octylmethoxycinnamate-loaded solid lipid nanoparticles.

Preparation, characterization, and optimization of primaquine-loaded solid lipid nanoparticles.

Formulation and Physicochemical Characterization of Lycopene-Loaded Solid Lipid Nanoparticles.

Pharmacokinetic and anti-inflammatory effects of sanguinarine solid lipid nanoparticles.

Enhanced photocytotoxicity of curcumin delivered by solid lipid nanoparticles.