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Positively Charged Surface-Modified Solid Lipid Nanoparticles Promote the Intestinal Transport of Docetaxel through Multifunctional Mechanisms in Rats Li-Li Shi, Hongjuan Xie, Jia Lu, Yue Cao, Jiang-Yan Liu, XiaoXue Zhang, Hongjian Zhang, Jing-Hao Cui, and Qing-Ri Cao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00226 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 8, 2016

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Molecular Pharmaceutics

Positively Charged Surface-Modified Solid Lipid Nanoparticles Promote the Intestinal Transport of Docetaxel through Multifunctional Mechanisms in Rats

Li-Li Shia,b,1, Hongjuan Xiec,1, Jia Lua, Yue Caoa, Jiang-Yan Liua, Xiao-Xue Zhanga, Hongjian Zhanga, Jing-Hao Cuia,**, Qing-Ri Caoa,*

a

College of Pharmaceutical Sciences, Soochow University, Suzhou, People’s Republic of

China b

College of Medicine, Jiaxing University, Jiaxing, People’s Republic of China

c

Department of Pharmacy, Shanghai Changning Center Hospital, Shanghai, People’s

Republic of China Corresponding authors: *Tel.: +86-512-69564123, E-mail: [email protected] (Q.R.Cao); **

1

Tel.: +86-512- 65882077. E-mail: [email protected] (J.H.Cui)

These authors contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT Solid lipid nanoparticles (SLNs) are one of the most promising nanocarriers to increase the oral absorption of drugs with poor solubility and low permeability. However, the absorption mechanism of SLNs remains incomplete and thus requires further careful considerations. In this study, positively charged chitosan (CS)- or hydroxypropyl trimethyl ammonium chloride chitosan (HACC)-modified SLNs were designed and their absorption mechanisms were fully clarified to improve the oral absorption of docetaxel (DTX). The HACC-DTX-SLNs showed the highest cellular uptake in Caco-2 cell monolayer; the transport efficacy in the follicle-associated epithelium cell monolayer was higher than that in the Caco-2 cell monolayer. The CS or HACC-modified SLNs could reversibly regulate the trans-epithelial electrical resistance and the expressions of tight junction (TJ)-associated proteins, such as claudin-1, occludin, and zonula occludens-1. The uptake of HACC-DTX-SLNs through Peyer’s patches was higher than that of the normal tissue of the small intestine in rats. The enhanced absorption mechanisms of HACC-DTX-SLNs were mainly related to the caveolae-mediated endocytosis, M cell phagocytosis, and reversible TJ opening.

KEYWORDS: docetaxel, solid lipid nanoparticle, chitosan, hydroxypropyl trimethyl ammonium chloride chitosan, oral absorption mechanism


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1. INTRODUCTION Oral delivery of poorly water soluble drugs is a great challenge owing to their limited aqueous solubility and poor cellular permeability.1,2 Therefore, more advanced delivery systems are needed to optimize the oral delivery of these drugs. Different types of nanocarriers, such as liposomes, micelles, and solid lipid nanoparticles (SLNs), have been widely used to improve the oral bioavailability of poorly water-soluble drugs.3,4,5 Among these nanocarriers, SLNs have been extensively investigated because of their biocompatibility, large-scale production, and physicochemical stability.6,7 SLNs can improve drug transport through the intestinal epithelial layer and protect drugs against the hostile environment of gastrointestinal (GI) tracts.8,9 They also exhibit the latent advantages of direct lymphatic transport and promote the bypassing of the liver first-pass effect.10,11 However, the negative charge of SLNs is not beneficial to drug absorption because of the electrostatic repulsion between the cell membrane and SLNs; as a result, cellular uptake and oral bioavailability are reduced.12 Chitosan (CS), a cationic natural polysaccharide derived from chitin, exhibits excellent properties such as high biocompatibility, biodegradability and mucoadhesivity for biological applications.13 Especially, a quaternized CS derivative, shows excellent aqueous solubility over a wide pH range, in addition to mucoadhesive and absorption-enhancing properties at neutral pH.14 Various attempts have been performed to modify the surface of nanoparticles with CS or its derivatives, which can establish ionic interactions with negatively charged particles, to improve drug absorption.15,16 For example, CS-coated Witepsol 85E SLNs containing insulin exhibit the synergic properties of intestinal absorption. The CS coating provides high insulin permeation in Caco-2 and Caco-2/HT29 cell monolayer models.17 The potential use of CS oligosaccharide-coated nanostructured lipid carrier in ocular drug delivery has also been explored. The coated nanoparticles display an altered zeta-potential from a negative charge to a positive charge; compared with the area under the curve (AUC) of the uncoated ones, the AUC of the coated formulation significantly increases.18 CS and Pluronic®F127-modified liposomes have been engineered to investigate the differences between these mucoadhesive and mucus-penetrating systems in the oral absorption of cyclosporine A, a poorly soluble drug.3 However, the absorption mechanism of these nanocarriers remains incomplete and thus requires further careful considerations.



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Several theories have been proposed to explain the mechanisms by which the oral bioavailability of drugs can be improved by using nanocarriers.19 For instance, studies have supported the idea that the absorbed lipid in enterocytes can be assembled into intestinal lipoproteins in the endoplasmic reticulum and the Golgi body; the absorbed lipid is then selectively taken up by the intestinal lymphatic system after the lipid is exocytosed from the enterocytes.20 Nanoparticles can be modified with specific materials, such as polyethylene glycol, Pluronic® F127, and penetrating peptides; once modified, these nanoparticles can improve the oral bioavailability of drugs by promoting cross static water level and cell membrane permeability.21,22 In addition, some nanoparticles can transiently regulate the expression of tight junction (TJ)-associated proteins, thereby opening the TJs, which promote paracellular transport.23However, these studies have not yet fully clarified the absorption and transport mechanism of CS- or its derivative-modified SLNs. On the basis of previous studies, we used hydroxypropyl trimethyl ammonium chloride chitosan (HACC) exhibiting pH-independent solubility and CS showing pH-dependent solubility to modify glyceryl monostearate-based SLN loading DTX. The cellular uptake and transport capacity of SLNs were fully investigated in Caco-2 and follicle-associated epithelium (FAE) cell models through flow cytometry, fluorescence microscopy, confocal laser scanning microscopy (CLSM), and high-performance liquid chromatography (HPLC). In addition, the effects of temperature and various inhibitors on the endocytosis of SLNs in Caco-2 cells were investigated. The disruption of TJs by SLNs in Caco-2 cells was evaluated by measuring the trans-epithelial electrical resistance (TEER) and analyzing the expression of TJ-associated proteins, such as claudin-1, occludin and ZO-1 by western blotting. The site specific absorption in the GI tract was also visualized using a small animal imaging system. 2. MATERIALS AND METHODS 2.1. Materials. Docetaxel (DTX) was purchased from Xi’an Natural Field Bio-technique Co., Ltd. (Xi’an, China). Gycerol monostearate was purchased from Tianjin Kemiou Chemical Reagent Co. (Tianjin, China). Soybean lecithin was purchased from Shanghai Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Tween 80 was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Chitosan (CS, Mw=50,000-60,000 Da) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).


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Hydroxypropyl trimethyl ammonium chloride chitosan (HACC) was purchased from Lushen bioengineering Co., Ltd. (Nantong, China). Coumarin 6 (C6) was purchased from sigma (Pittsburgh, USA). Rabbit anti-Occludin and Rabbit anti-ZO-1 were purchased from Invitrogen Corporation (California, USA). Anti-Claudin-1 was purchased from abcam (Cambridge, USA). GAPDH was purchased from beyotime Biotechnology (Nantong, China). DyLightTM 680-Labeled Antibody TO Rabbit IgG (H+L) (Gaithersburg, USA). DyLightTM 800-Labeled Antibody TO Mouse IgG (H+L) (Gaithersburg, USA). All other chemicals were of reagent grade and were used without further purification. All other chemicals were of reagent grade and were used without further purification. Caco-2 cells and Human Burkitt’s lymphoma Raji B cells were purchased from culture collection of the Chinese Academy of Sciences (Shanghai, China). 2.2. Preparation of DTX-SLNs. DTX-SLNs were prepared by emulsion solvent evaporation method. In brief, DTX (3 mg) and soybean lecithin (20 mg) were dissolved in 2 mL of dichloromethane. Glyceryl monostearate (60 mg) was melted at 60 °C. The uniform oil phase was achieved by mixing the dichloromethane solution and the melted liquid. The aqueous phase was prepared by dissolving Tween 80 (40 mg) in 4 mL of double distilled water and heated to the same temperature as that of the lipid phase. Hot aqueous solution was quickly added to the lipid phase, and the fine emulsion was obtained after the coarse emulsion was sonicated at 200 W for 60 s. Dichloromethane was evaporated under constant stirring at room temperature to obtain DTX-SLNs. The coumarin-6 (C6)-loaded SLNs (C6-SLNs) were also prepared by dissolving 1 µg of C6 instead of DTX in dichloromethane. The surface-modified SLNs were obtained by dispersing DTX-SLNs in 0.1% (w/v) CS or HACC solution, followed by further stirring for 3 h. The formulation compositions of the various SLNs are listed in Table S1. 2.3. Measurement of particle size and zeta potential. The size and surface charge of various SLNs were measured by a dynamic light scattering instrument (HPP 5001, Malvern, UK). The samples were diluted in distilled water before measurement. 2.4. Determination of entrapment efficiency (EE) and drug loading (DL) capacity. The free drug was separated from SLNs by ultracentrifugation at130,000 × g for 45 min (4 °C) to determine the EE and DL capacity. The amount of DTX in the supernatant was determined



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by a waters 2996 HPLC system with Inertsil ODS-SP column (5 µm, 4.6 mm × 150 mm) under the following conditions: mobile phase, acetonitrile-water (50:50, v/v); flow rate, 1.0 mL/min; and detection wavelength, 230 nm. The total drug amount in the drug-loaded SLNs was determined by dissolving SLNs in methanol to release the encapsulated DTX. EE was defined as the ratio between the encapsulated drug in SLNs and the total DTX. DL capacity was calculated as the ratio between the encapsulated drug in SLNs and the polymer. The EE and DL were calculated according to following equations. EE =（W Total DTX – W DTX in the supernatant）/ W Total DTX × 100 % DL =（W Total DTX – W DTX in the supernatant）/ W Polymer × 100 % 2.5. TEM. The morphology of various SLNs was observed through a transmission electron microscope (TecnaiG220, FEI, USA). The samples were placed on copper grids with films, dried under infrared lamp and then viewed under the TEM. 2.6. Cell culture. Caco-2 cells were cultured in DMEM with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) non-essential amino-acids at 37 °C under an atmosphere of 5% CO2 and 90% relative humidity. Raji B cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% (v/v) non-essential amino-acids at 37 °C in a 5% CO2/95% air atmosphere. To obtain the Caco-2 monolayer, Caco-2 cells were seeded at a density of 2.5 × 105 cells/well on Millicell® Hanging Cell Culture Inserts (12 mm insert diameter, 1 µm pore size; Millipore, USA) and cultured over 15 days. The medium was replaced every second day for the first week and every day in the second week. To verify the integrity of the Caco-2 monolayer, the morphology of Caco-2 cells was characterized by inverted microscope. The ratio of alkaline phosphatase between the apical and basal compartments and the TEER was measured. The FAE model was obtained by co-culturing Caco-2 cells and Raji B cells. Briefly, Caco-2 cells were seeded at a density of 2.5 × 105 cells/well on Millicell® Hanging Cell Culture Inserts. The medium was replaced every other day, until days 13–15, when Raji B cells were added to the basolateral compartment for conversion of Caco-2 cells into M cells at a density of 1 ×106 cells/well. The FAE model was verified by measuring the TEER and the expression of UEA-1. 2.7. Cellular uptake in Caco-2 cells. The cellular uptake of various SLNs was quantified


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by HPLC and flow cytometry, and further visualized by fluorescence microscopy and CLSM. For the HPLC study, Caco-2 cells were seeded at a density of 2.5 × 105 cells/well on a 6-well tissue culture plate and cultured for 4 days prior to the study. The cells were incubated with DTX, DTX-SLNs, CS-DTX-SLNs, and HACC-DTX-SLNs, respectively. After incubation, the medium was removed and washed with cold PBS three times. The cells were then lysed by adding a RAPI buffer, scraped from the plate, and the cell suspension was sonicated under 400 w for 90 s to facilitatelysis. Cell suspension was centrifuged at 10,000 ×g for 10 min. Afterward, the supernatant was stored to measure the DTX concentration adsorbed by the cells and the total protein by HPLC and BCA Protein Quantitation Kit, respectively. The uptake was calculated as follows: Uptake = CDTX/Cprotein C6-SLNs were used for the for the flow cytometry study. Cells were seeded in a 6-well tissue culture plate at a density of 2.5 × 105 cells/well for four days prior to study. The cells were incubated with C6, C6-SLNs, CS-C6-SLNs, and HACC-C6-SLNs, respectively. After the cells were incubated for 2 h, these cells were washed thrice with cold PBS, trypsinized from the plates, and centrifuged at 1,500 × g. The cell pellets were washed with PBS, resuspended in PBS, and analyzed using a BD FACS Calibur flow cytometer and BD CellQuest software. Cell fluorescence was quantified by measuring the fluorescence of C6 at 488 nm (FL1); approximately 10,000 events were collected for each sample. The FlowJo data analysis software package (TreeStar, USA) was used to analyze the data. In fluorescent microscopy, the cells were cultured in a six-well tissue culture plate at a density of 1.0 × 105 cells/well for four days before the study was performed. The cells were incubated with C6, C6-SLNs, CS-C6-SLNs, and HACC-C6-SLNs for 2 h and then washed with cold PBS. The cell nucleus was stained with DAPI, and the sample was observed using an inverted fluorescent microscope (Olympus, Japan) under excitation at 488-nm wavelength and UV channels. In confocal microscopy, the cells were cultured on cover slips at a density of 1.0 × 105 cells/well for four days before the study was performed. After the cells were incubated with C6, C6-SLNs, CS-C6-SLNs, and HACC-C6-SLNs for 2 h, the cells were washed with cold PBS and fixed with 4% paraformaldehyde for 30 min. The cell nucleus was stained with



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DAPI, and the sample was observed using a Z-stack pattern via a Leica TCS SP2 CLSM (Leica, Germany) under excitation at 488-nm wavelength and UV channels. 2.8. Endocytosis mechanism in Caco-2 monolayer. The endocytosis of HACC-C6-SLNs was investigated using different types of inhibitors, such as chlorpromazine, filipin, M-β-CD, and low temperature (4 °C). Caco-2 cells were pre-incubated with these inhibitors for 30 min and then co-cultured with HACC-C6-SLNs for 2 h. In addition, the endocytosis at 4 °C was evaluated. In flow cytometry, the untreated cells were used as control. The cells were incubated, washed, trypsinized from the plates, and centrifuged under the same conditions described in a previous section. Afterward, the cell pellets were washed with PBS, resuspended in PBS, and analyzed using a BD FACSCalibur flow cytometer under excitation at 488-nm wavelength and BD CellQuest software. In confocal microscopy, the cells were fixed and the nuclei were stained at the end of incubation. The sample was observed using a Leica TCS SP2 CLSM (Leica, Germany) under excitation at 488-nm wavelength and UV channels. 2.9. Cellular uptake in FAE monolayer. The FAE monolayer model with TEER value above 200 Ω/cm2 was used to evaluate cellular uptake and to investigate the effect of M cells on the SLN uptake. Before the experiment was performed, the monolayer was washed thrice with Hank’s balanced salt solution (HBSS). After the cells were incubated with HACC-C6-SLNs for 2 h, the cells were washed with cold PBS and fixed with paraformaldehyde for 30 min. The cells were further washed with PBS, cultured with UEA-1 antibody at 4 °C overnight, and cultured with rhodamine-IgG at 37 °C for 1 h. The cell nucleus was stained with DAPI, and the sample was observed using a Leica TCS SP2 CLSM (Leica, Germany). The Caco-2 monolayer was used as the control group. 2.10. Transport across Caco-2 and FAE monolayers. After a 15-day differentiation, eligible Caco-2 and FAE monolayers were chosen for the transport study. DTX or DTX-SLNs were suspended in HBSS to a final concentration of 40 µg/mL of DTX. Approximately 0.5% DMSO was added when necessary to dissolve DTX. Prior to the study, the monolayer was washed with pre-warmed HBSS for three times. At the start of the experiment, the drug solutions were added to the upper side of the Millicell® Hanging Cell Culture Inserts, and HBSS without drug was added to the basolateral side. Then, the cells were incubated with


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these drug solutions at 37 °C for 4 h. At the end of the time point, 100 µL of basolateral medium was collected and analyzed by HPLC. The transport efficiency was evaluated by the percentage of cumulatively transported DTX in the basolateral medium versus the total DTX added to the apical medium. 2.11. Dynamic change in TEER. Caco-2 cells were cultured on Millicell® Hanging Cell Culture Inserts (12 mm insert diameter, 1 µm pore size; Millipore, USA) at a density of 2.5 × 105 cells/well and cultured over 15 days. Eligible Caco-2 monolayer with TEER value above 500 Ω/cm2 was used to conduct the study. The SLNs suspended in HBSS was applied to the apical chamber, and TEER was measured at different time intervals for 4 h. After incubation, the test suspension was removed and the monolayer was washed with PBS for two times. TEER was monitored for another 18 h after adding a fresh medium. The change in TEER for the tightness of cell monolayers was measured with a Millicell electrical resistance system (Millipore Corp., Bedford, MA, USA) connected to a pair of chopstick electrodes. The monolayer that was not cultured with SLN suspension was used as control. Data are expressed as a percentage of the initial values. 2.12. Expression of TJ-associated proteins. In brief, the Caco-2 cells were cultured on a six-well tissue culture plate and cultured for 15 days. The cells were co-cultured with SLNs suspension for 4 h and then washed with PBS thrice after the suspension was removed. Afterward, the cells were cultured with fresh medium in different interval times. The monolayer was gently washed with PBS twice and the cells were lysed with a lysis buffer to extract the total protein. The samples were subsequently lysed through sonication at 400 w for 60 s and then centrifuged at 10,000 ×g for 5 min. The supernatant was collected for the subsequent Western blot analysis. The total protein concentration of each sample was calculated by BCA method and adjusted to a suitable concentration. The suspension was boiled for 10 min and then immediately cooled to room temperature. An equal amount of protein samples were separated on SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was incubated in diluted primary antibodies at 4 °C overnight after blocking in 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST). The membrane was washed thrice with TBST, incubated with secondary antibodies, and



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visualized with the odyssey color development kit (9201-00, LI-COR, USA). Densitometric analysis of specific bands was performed using the Image J software (National Institute of Health, Bethesda, MD, USA). 2.13. Transport images in GI tract. To demonstrate the transportation of HACC-C6-SLNs through the GI tract, male Sprague-Dawley rats (180–200 g, Slac, Shanghai, China) were used to conduct the study. Prior to the study, rats were fasted for 24 h with access to water ad libitum. Two milliliters of C6-HACC-SLNs were administered orally to the rats; the concentration of C6 was 5 µg/mL. At different time intervals (1, 2, 6 h), the rats were anesthetized with chloral hydrate (5%, m/v) through intraperitoneal injection and then killed by cervical dislocation. The digestive tract of the rats was isolated, rearranged onto a disk, and visualized by small animal imaging system (Caliper IVIS Lumina II, Xenogen, USA). The

optimal excitation and emission wavelength were 465 nm and 509 nm, respectively. The time at which the rats were not orally administered with SLNs was set as 0 h. 2.14. Transport capacity through the Peyer’s patch. Peyer’s patch and the small intestine near the patch were removed after the SLNs were orally administered for 2 h to compare the transport capacity of Peyer’s patch and common small intestine. These segments were washed with PBS and then prepared into freezing microtome sections. The sections were subsequently fixed with 4% paraformaldehyde and washed with PBS. The nuclear was stained with DAPI, and the image was recorded by CLSM under excitation at 488-nm wavelength and UV channels. 3. RESULTS AND DISCUSSION 3.1.

Preparation and characterization of SLNs. Nanocarriers have been extensively

investigated for the oral absorption of anticancer drugs with poor solubility and low permeability. The neutral or negatively charged surface of nanoparticles can prevent the interaction of nanoparticles with the cell membrane; as a result, cellular uptake is inhibited.24,25,26 In our current study, we modified the glyceryl monostearate-based SLNs with positively charged CS or HACC because these SLNs can strongly adhere to cellular surfaces and can promote intestinal paracellular drug transport. Figure 1 shows the particle size, zeta potential, EE, and DL capacity of the three different SLNs. The particle size of CS/or HACC-modified SLNs were larger than that of unmodified SLNs, indicating that the CS/or


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HACC was adsorbed onto the drug-loaded SLNs by non-covalent interactions (Figure 1A). After modifying with CS/or HACC, the zeta potential of SLNs changed from −20.24±0.49 to 8.83±0.12 or 23.10 ±0.20 mV (Figure 1A). The surface charge of the glyceryl monostearate-based SLNs was converted from negative to positive after the SLNs were modified with CS or HACC. The EEs of the three SLNs were more than 80%, while the DL capacities were around 3% (Figure 1B). Figure 1C shows that the DTX-SLN surface becomes rough after modifying with CS/ or HACC, which further proved that SLNs have been modified by CS/ or HACC. 3.2.

Establishment of Caco-2 and FAE monolayer models. The morphology observed

from inverted microscope showed that the Caco-2 monolayer have favorable morphology and clear border after incubation for 15 days (Figure S1A). The cell growth curve showed that the absorbance value on the seventh day was similar to that on the fifth day, indicating that the differentiation of cells was saturated on the fifth day and the formation of polarity structure occurred mainly after culturing for 5 days (Figure S1B). The activity ratio of ALP was 3.64 (Figure S1C) and the TEER was above 500 Ω/cm2 (Figure S1D) on the fifteenth day, indicating that the monolayer can be used for transport experiments after culturing for 15 days. After co-cultivation of Caco-2 cells and human Raji B lymphocytes, the TEER decreased by approximately 50%, which is approximately 300 Ω/cm2 (Figure S2A). UEA-1 is a specific protein expressed on the M cell surface. As shown in Figure S2B, a red fluorescent signal indicates the M cell formation. 3.3.

Cellular uptake in Caco-2 monolayer. In order to verify whether the absorption of

DTX was improved after encapsulated in SLNs, the cellular uptake study was conducted in Caco-2 cells model. Figure 2A shows that the cellular uptake of DTX was time dependent and HACC-DTX-SLNs can significantly improve the cellular uptake of the drug. Flow cytometry (Figure 2B) showed a result similar to that of HPLC. The fluorescent microscopy images shown in Figure 2C further confirmed the data from HPLC and flow cytometry. When the cells were cultured with the C6 solution, green fluorescence was barely detected in the Caco-2 cells. However, significantly enhanced fluorescence intensity was detected after C6 was incorporated in the SLNs. The fluorescence intensity was further enhanced after the SLN



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surface was modified with CS or HACC. Figure 3 shows the cellular uptake by the Z-stack model of CLSM. Green fluorescence was hardly observed in the transverse (X–Y) and longitudinal sections (X–Z or Y–Z) when cultured with C6 solution (Figure 3A). After culturing with C6-SLNs, the green fluorescence increased in the transverse section (X–Y) and longitudinal section (X-Z or Y-Z), indicating that SLNs can improve cellular uptake (Figure 3B). Moreover, further enhancement in longitudinal section was observed after culturing with CS/or HACC-C6-SLNs; the green fluorescence in HACC-C6-SLNs was stronger than that in CS-C6-SLNs (Figure 3C and 3D). 3.4.

Endocytosis in Caco-2 monolayer. Endocytosis is a major absorption

mechanism for drugs to transport across the cellular membranes. We extensively studied the oral absorption mechanisms of CS or HACC-modified SLNs at cellular and animal levels because these SLNs exhibit excellent cellular uptake behaviors. Various oral absorption mechanisms, such as transcellular route, paracellular route, and M-cell uptake route for SLNs, are employed.11,27,28,29 Endocytosis is an important mechanism by which drugs are transported across cells.30,31 Various inhibitors, such as chlorpromazine, filipin, and m-β-CD, were used to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively, to clarify the endocytosis mechanism of HACC-C6-SLNs in Caco-2 cells. Compared with the cellular uptake of the control group, the cellular uptake of HACC-C6-SLNs significantly decreased to 23±1.9% at 4 °C (Figure 4A), indicating that the uptake of SLNs was energy-dependent. The cellular uptake of HACC-C6-SLNs decreased to 62±2.0% in the presence of filipin; by contrast, the cellular uptake of HACC-C6-SLNs was slightly inhibited after the cells were pre-incubated with chlorpromazine or m-β-CD. The effects of temperature and inhibitors on cellular uptake of the nanoparticles were also imaged by CLSM. As shown in Figure 4B, the fluorescence intensity of HACC-C6-SLNs was much lower than that at room temperature. As shown in Figure 4C, the cellular uptake of HACC-C6-SLNs in the presence of filipin was the lowest compared with the control. Caco-2 cells pre-incubated with m-β-CD and chlorpromazine also reduced cellular uptake. This result suggests that HACC-C6-SLNs were mainly absorbed through caveolae-mediated endocytosis, but were partly absorbed through clathrin-mediated endocytosis and macropinocytosis. 3.5.

Cellular uptake in FAE monolayer. M cells locate on the FAE of the Peyer’s


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patch follicle and play an important role in the phagocytosis of exotic particles.32,33 So we

studied the cellular uptake of HACC-SLNs in FAE monolayer to investigate the function of M cells for enhancing drug absorption. The green fluorescence interspersed around the blue fluorescence-like star dots in the FAE monolayer, and this pattern differed from that in the Caco-2 monolayer (Figure 5A). This finding confirms that the uptake behavior of M cells is different from that of Caco-2 cells; M cells likely absorb particles through phagocytosis. 3.6.

Transport across Caco-2 and FAE monolayers. Oral bioavailability of drugs is

closely relative with the transport efficiency through a cell monolayer at the absorption site. Transport studies in Caco-2 and FAE monolayers were conducted to evaluate the potential of SLNs as suitable DTX carrier. Figure 5B shows the transport efficiency of each SLN formulation. In the Caco-2 monolayer, only few DTXs can transport across the cells, indicating that the permeability efficiency of DTX-SLNs was higher than that of free DTX. After modifying with CS/HACC, further increase in permeability efficiency was observed. Moreover, the permeability rate of HACC-DTX-SLNs (3.98±0.48%) was 1.99-fold higher than that of DTX-SLNs (2.00±0.04%) and 1.24-fold higher than that of CS-DTX-SLNs (3.21±0.35%). In the FAE model, a significant increase was observed in the permeability of all three SLNs compared with the Caco-2 model. The permeability of HACC-DTX-SLNs (4.67±1.82%) was 1.58-fold higher than that of DTX-SLNs (2.97±0.03%), but similar to that of CS-DTX-SLNs (4.71±0.24%) in the FAE monolayer. This reveals that the three SLNs displayed a greater transport ability in the FAE model than in the Caco-2 cells; after the SLNs were modified, the transport efficiency of HACC-DTX-SLNs was significantly higher than that of DTX-SLNs. 3.7.

Dynamic change in TEER. Paracellular transport is a useful route for small

water-soluble drugs but is less useful for macromolecular fat-soluble substances because of a narrow TJ between cells.34 In this study, CS and HACC were used as TJ modulators to promote the paracellular transport of DTX. The TEER value can reflect the tightness of TJs; hence, the dynamic changes in TEER after adding and removing SLN suspension in Caco-2 cells were evaluated. As shown in Figure 6, a significant decrease in TEER was observed after treatment with DTX-SLNs, CS-DTX-SLNs, and HACC-DTX-SLNs, which was



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44.58±9.33%, 21.38±2.05% and 23.68±2.22% of control at 4 h, respectively. This illustrates that the TEER was lower after the positively charged SLNs were applied than after the negatively charged SLNs were employed. After the removal of SLN suspension, a gradual recovery in TEER was observed, suggesting that the regulating effect of SLNs in TEER was reversible. However, DTX-SLNs, CS-DTX-SLNs, and HACC-DTX-SLNs showed relatively low recovery of 74.28±28.21%, 76.04±12.23% and 83.90±13.40% at 18 h, respectively. As a result, drugs released from SLNs pass through gaps between cells. Moreover, the concentration of CS and HACC was the same, leading to similar dynamic change in TEER between CS-DTX-SLNs and HACC-DTX-SLNs.28,35 3.8.

Expression of TJ associated proteins. Drugs that gradually released from SLNs

can be absorbed through the membrane pores if the TJs were opened by CS. TJs is composed of a complex combination of transmembrane integral proteins.36,37 To observe the correlation between TJs and the transport of DTX-SLNs, the expression of TJ-related proteins after culturing with SLN suspension was determined. Figure 7 shows the quantitative results of the TJ-related proteins (claudin-1, occludin and ZO-1) in Caco-2 cells incubated with various SLNs. The suspension of CS-SLNs (64.03±7.25%) or HACC-SLNs (52.22±5.76%) resulted in significant decrease in claudin-1 compared with the control group (100%), whereas no significant decrease was observed for the unmodified SLN suspension. Occludin expression was significantly decreased when cultured with HACC-SLNs (63.31±4.93%), whereas no significant change was observed for SLNs or CS-SLNs. Unlike claudin-1 and occludin, ZO-1 expression was affected by SLNs, CS-SLNs and HACC-SLNs and decreased to 78.98±5.35%, 13.96±1.67% and 10.63±3.03% when compared with the control group, respectively. Especially, the negative regulation of CS/or HACC-SLNs was more obvious than SLNs. Figure S3 shows the recovery of the TJ-related proteins when the SLN suspension was removed from the Caco-2 cells. The expressions of claudin-1, occludin and ZO-1 increased during cell recovery. Interestingly, the removal of HACC-SLNs resulted in a significant increase in claudin-1 and occludin compared with control group. By contrast, ZO-1 did not show the phenomenon. We found that SLNs could reduce the expression of the TJ-related proteins, and this downregulation could be recovered in the absence of SLNs. The paracellular route can be promoted by opening the TJs, and CS and its derivatives can adhere


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to the epithelial surface and transiently open the TJs. 3.9.

Transport images of HACC-C6-SLNs in GI tract. The oral absorption

mechanism of HACC-modified SLNs was also clarified in vivo by using rats as animal models. The transportation of HACC-C6-SLNs through the GI tract of rats was visualized by Caliper IVIS-Lumina II imaging system (Xenogen, USA). The optimal excitation and emission wavelength were 465 nm and 509 nm, respectively (Figure S4). As shown in Figure 8A and 8B, HACC-C6-SLNs were distributed throughout the small intestine at 1 h after oral administration; most of the SLNs were observed in the stomach. After 2 h, most of the SLNs were retained in the stomach, and the amount of SLNs that entered the ileum was similar to that at 1 h. However, after 6 h, only weak fluorescence can be detected in the gut. Comparing the different segments of the small intestine, higher fluorescence intensity was found in the ileum, indicating that the maximum amount of orally administered HACC-SLNs is absorbed through the ileum. Figure 8C shows the fluorescence intensities in the liver, spleen, heart, and kidney after oral administration. The intensities in the kidney gradually increased with the extension of time, suggesting that the SLNs were eliminated from the body through the excretory system. The HACC-SLNs were rapidly distributed throughout the small intestine after these SLNs were orally administered. The largest amount of the orally administered HACC-SLNs was absorbed through the ileum 3.10. Transport ability of HACC-C6-SLNs through Peyer’s patch. Peyer's patch plays an important role in the absorption of particles though the distribution of peyer's patch in the small intestine is not abandant. Figure 9A shows the images of the Peyer’s patch incorporating small intestine and normal small intestine isolated from rats after oral administration of HACC-C6-SLNs. The fluorescence intensity in the Peyer’s patch was higher than that in the normal small intestine, indicating that the transport of HACC-C6-SLNs through the Peyer’s patch was prior to the small intestine. Figure 9B shows the quantitative results of the fluorescence intensity, which further validated the results from Figure 9A. As shown in Figure 9C, distribution of SLNs in the Peyer’s patch compared with the normal small intestine was also observed by CLSM. Green fluorescence was detected in the Peyer’s patch and small intestine. Moreover, the intensity of the fluorescence in the Peyer’s patch was higher than that in the small intestine, which may be due to the extensive phagocytosis of M



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cells located in the Peyer’s patch. The HACC-SLNs were more likely transported through Peyer’s patches than through the small intestine. This observation indicated that M cells play an important role in the SLN transport. 4. CONCLUSIONS In summary, the cellular uptake and transport of DTX-SLNs, especially HACC-modified SLNs, in Caco-2 cells can be significantly improved. The HACC-DTX-SLNs were mainly absorbed through caveolae-mediated endocytosis and partly through clathrin-mediated endocytosis and pinocytosis. DTX was transported across the FAE monolayers to a higher extent than across the Caco-2 monolayers because of the phagocytic ability of the M cells. Moreover, the CS- or HACC-modified SLNs could reversibly regulate the TEER. Our in vivo studies further revealed that HACC-DTX-SLNs could improve the oral absorption of DTX through a significant uptake in Peyer’s patches. Therefore, the oral absorption mechanisms of the positively charged HACC-DTX-SLNs were mainly related to caveolae-mediated endocytosis, M cell phagocytosis, and reversible TJ opening. ACKNOWLEDGMENTS This work was partially supported by the National Natural Science Foundation of China (81373333, 81311140267), the project sponsored by the Applied Basic Research Program, Suzhou Science and Technology Bureau (SYS201204), and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. REFERENCES (1) Soudry-Kochavi, L.; Naraykin, N.; Nassar, T.; Benita, S. Improved oral absorption of exenatide using an original nanoencapsulation and microencapsulation approach. J. Control. Release 2015, 217, 202–210. (2) Wang, J.; Li, L.; Du, Y.; Sun, J.; Han, X.; Luo, C.; Ai, X.; Zhang, Q.; Wang, Y.; Fu, Q.; Yang, Z.; He, Z. Improved oral absorption of doxorubicin by amphiphilic copolymer of lysine-linked ditocopherol polyethylene glycol 2000 succinate: In vitro characterization and in vivo evaluation. Mol. Pharm. 2015, 12 (2), 463–473. (3) Chen, D.; Xia, D.; Zhu, Q.; Yu, H.; Zhu, C.; Gan, Y. Comparative study of Pluronic® F127-modified liposomes and chitosan-modified liposomes for mucus penetration and oral


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List of Figures Figure 1. Physical properties of various SLNs. (A) Particle size and zeta potential. (B) EE and DL capacity. (C) TEM. Data are expressed as mean ±SD (n = 3). Figure 2. (A) Cellular uptake of DTX, DTX-SLNs, CS-DTX-SLNs, and HACC-DTX-SLNs in Caco-2 cells through HPLC. (B) Cellular uptake of C6, C6-SLNs, CS-C6-SLNs, and HACC-C6-SLNs through flow cytometry. (C) Cellular uptake of C6, C6-SLNs, CS-C6-SLNs, and HACC-C6-SLNs after incubation for 2 h through fluorescent microscopy. Data are expressed as mean ± SD (n = 3). Figure 3. CLSM images of the Caco-2 cells with a Z-stack pattern after incubation with (A) C6, (B) C6-SLNs, (C) CS-C6-SLNs, and (D) HACC-C6-SLNs for 2 h. Scale bars = 50 µm. Figure 4. Effects of the inhibitors on the uptake of HACC-C6-SLNs in Caco-2 cells. (A) Intracellular fluorescence intensity of HACC-C6-SLNs in Caco-2 cells preincubated with chlorpromazine, filipin, and M-β-CD for 2 h at 37 °C. (B) CLSM images of Caco-2 cells after treatment with HACC-C6-SLNs at 4 °C. (C) CLSM images of HACC-C6-SLNs in Caco-2 cells preincubated with various inhibitors. Scale bars for images are 20 µm. *p

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