Intestinal Mucin Induces More Endocytosis but Less Transcytosis of

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Intestinal mucin induces more endocytosis but less transcytosis of nanoparticles across enterocytes by triggering nanoclustering and strengthening the retrograde pathway Dan Yang, Dechun Liu, Mengmeng Qin, Binlong Chen, Siyang Song, Wenbing Dai, Hua Zhang, Xueqing Wang, Yiguang Wang, Bing He, Xing Tang, and Qiang Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19153 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 27, 2018

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ACS Applied Materials & Interfaces

Intestinal mucin induces more endocytosis but less transcytosis of nanoparticles across enterocytes by triggering nano-clustering and strengthening the retrograde pathway Dan Yang a,b,c‡, Dechun Liu

a,b,c‡

, Mengmeng Qin

b,c

, Binlong Chen

b,c

, Siyang Song a,b,c,

Wenbing Dai b,c, Hua Zhang b,c , Xueqing Wang b,c, Yiguang Wang b,c, Bing He b,c *, Xing Tang a *, Qiang Zhang a,b,c * a.

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China

b.

Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems,

School of Pharmaceutical Sciences, Peking University, Beijing 100191, China c.

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing

100191, China ‡

These authors have contributed equally to this work

*

Corresponding author

1

ACS Paragon Plus Environment

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ABSTRACT: Mucus, secreted by the goblet cells of enterocytes, constitutes the first obstacle encountered for the intestinal absorption of nanomedicines. For decades, mucus has simply been regarded as a physical barrier that hinders the permeation and absorption of drugs because of its high viscosity and reticular structure, whereas the interaction of mucus ingredients with nanomedicines is usually neglected. It is unclear whether glycoproteins, as the main components of mucus, interact with nanomedicines. We also do not know how the potential interaction affects the subsequent transportation of nanomedicines through the intestinal epithelium. In this study, mucin as the key element of mucus was investigated to characterize the interaction of nanomedicines with mucus. PEG-modified gold nanoparticles (PGNPs) were fabricated as model nanoparticles. Mucin was found to adhere to the nanoparticle surface to form a corona structure and induce the clustering of PGNPs by joining particles together, demonstrating the interaction between mucin and PGNPs. In addition, two intestinal epithelia, Caco-2 (nonmucus secretion) and HT-29 (high mucus secretion), were compared to evaluate the influence of mucin on the cellular interaction of PGNPs. Amazingly, mucin altered the trafficking characteristic of PGNPs in intestinal epithelium. Both in vitro and in vivo investigations demonstrated more nanoparticles being internalized by cells due to the mucin coverage. However, mucin induced a significant reduction in the transcytosis of PGNPs across epithelial monolayers. The mechanism exploration further revealed that the “more endocytosis but less transcytosis (MELT)” effect was mainly attributed to the strengthened retrograde pathway in which more PGNPs were transported to Golgi apparatus and exocytosed back to the apical but not the basolateral side of the epithelial 2


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monolayers. The “MELT” effect endowed mucin with duality in the nanoparticle transportation. So the rational regulation based on the “MELT” effect will provide new insight to overcome the mucus obstacle as a barrier and enhance the oral absorption rate of nanomedicines.

KEYWORDS: mucin; nano-clustering; corona; endocytosis; transcytosis; retrograde pathway

INTRODUCTION Oral administration has been widely applied clinically due to excellent patient compliance and simple operability. Over the last several decades, the successful introduction of nanotechnology has brought more oral nanomedicines into the market1, improving the bioavailability or therapy efficacy of drugs2, meanwhile, more oral nanomedicines are still in clinical trials and preclinical studies3. Notably, the mucus layer, which covers the intestinal epithelium as the first barrier, creates obstacles for the transportation of nanomedicines into blood circulation through the intestinal mucosa

4,5

.

As the key component, proteoglycans in mucus layer interact with each other to form a reticular structure, thus protecting the gastrointestinal (GI) tract against the access of exogenous substances including many types of nanomedicines6–9. Owing to the negative effects

of

mucus

layer

on

nanomedicine

transportation,

multiple

functional

nanomedicines have been fabricated to overcome the obstacles. Mucoadhesive polymers have been adopted to improve the residence time of nanoparticles in the GI tract6. For example, chitosan nanoparticles have been developed to enhance the intestinal

3



permeation of insulin significantly due to the electrostatic interaction between chitosan and the mucus layer10. Wheat germ agglutinin-functionalized nanoparticles have been designed to improve the transport amount of the carriers across intestinal epithelial cell because of the interaction between lectin and mucins in the mucus layer11. For decades, the mucus layer is always regarded as a simple physiological barrier that prevents the penetration of nanoparticles due to the compact net structure12. However, the possible interactions of mucus component with nanomedicines are almost neglected. Currently, it is unclear whether mucus can affect the properties of nanomedicines. We also do not know the detailed transport mechanism after the mucus/nanomedicine interaction despite it is so important for improving the efficiency of oral medicine. Recently, the bio-nano interaction has attracted increasing attention13–15. A large number of biomolecules including proteins in serum have been reported to adsorb onto nanoparticles to form so-called “corona layer”16, and many factors can influence the amounts and types of corona, thus affecting the original properties of the nanoparticles 17– 19

. Similar to serum, mucus is also rich in proteins, carbohydrates and lipids. This makes

us question whether mucus can interact with oral nanomedicines in a corona pattern. Compared with the abundant studies on serum corona20,21, there has been no study to investigate the possible effect of the mucus corona on oral nanomedicines at present. Notably, proteoglycans and glycoproteins, showing the most influence on corona, have a small proportion in the serum but mainly occupy the mucus. This distinction will indicate the new features in the mucus/nanomedicine interaction that differ with serum corona. Because corona proteins significantly change the physicochemical surface properties

4


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of nanoparticles, the bio-nano interactions are actually mediated by the “corona” rather than the pristine surface22–24. In other words, the composition and behaviors of corona will be a reflection of the interactions of nanomedicines with intestinal epithelium25,26. Although the mechanisms related to the transport of nanomedicines across the intestinal epithelial cells have been comprehensively studied for many years27–30, the effect of extracellular mucus on transport pathways is still unclear. In this study, we focus on exploring the interactions of mucus layer with nanomedicines, demonstrating the corona feature in particular. More importantly, the effects of mucus/nanoparticle interaction on the endocytosis, exocytosis and transcytosis of nanoparticles through the epithelium are emphatically analyzed. Short PEG-modified gold nanoparticles (PGNPs) were prepared as the model of nanomedicines due to the physiological stability, feasible detection by multi-technology and easy analysis of the bio-nano interaction31–33. Mucin, the dominant and representative component in mucus, was chosen for the investigation. Similar to the serum/nano interaction, mucin was found to adhere to the surface of PGNPs to form a corona layer. More interestingly, the mucin with a long strand feature further jointed surrounding PGNPs to form a nano-cluster structure, which was verified by dynamic light scattering (DLS), transmission electron microscope (TEM), fluorescence correlation spectroscopy (FCS) and multiple particle tracking (MPT). Then, the effect of mucin on the transport of PGNPs was investigated at a cellular level. Surprisingly, the nano-cluster morphology significantly altered the cellular uptake, transport mechanism and intracellular location of PGNPs compared with the monodisperse nanoparticles. The in vivo investigation further demonstrated the effects of mucus on the intestinal transportation of PGNPs. We believe that these amazing 5



findings will provide new insights for the designation of high-efficiency nanomedicine for oral administration.

RESULTS AND DISCUSSION

Preparation and characterization of PGNPs Primary gold nanoparticles (GNPs) were prepared following the method of Frens et al.34. To eliminate the negative effects of the spontaneous binding and agglomeration of GNPs in aqueous medium on subsequent mucus/nanoparticle interaction investigations, short-chain PEG (550 Da) was covalently modified on the surface of GNPs via Au-S linkage to achieve monodisperse PEG-GNPs (PGNPs). The schematic and synthetic route were shown in Figure 1a. Fourier transform infrared spectroscopy (FTIR) demonstrated the successful modification of the PEG chain. As shown in Figure 1b, when the surface of GNPs was covalently bound to mPEG-SH, the characteristic peak (2600-2700 cm-1) disappeared. Additionally, the stretching vibrations (υ(C=O), 1767 cm-1) belonging to citrate significantly weakened, indicating the replacement of the citrate groups by PEG. The Z-average diameters of pristine GNPs and PGNPs were 21.55±1.131 nm and 25.00±2.199 nm, respectively, as seen in Figure 1c and 1d. The TEM observations further demonstrated the similar size features (Figure 1e and 1f). As shown in TEM images, PEG modification also reduced the spontaneous binding of nanoparticles compared with pristine GNPs. Figure 1g illustrated that the Zeta potentials of PGNPs increased from -39.2±2.18 mV to -16.0±0.557 mV after PEG modification. Even so, the DLS and TEM investigations revealed that the dispersiveness of PGNPs in the serum-free cell culture medium after 12 h incubation was still good, and the morphology of PGNPs did not 6


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significantly change with time (Figure 1h and Figure S1). We believed that the PEG modification induced a thicker hydration shell on the particle surface compared with the pristine citrate coating, maintaining the monodispersity of the PGNPs in medium. In summary, due to the uniform size distribution of PGNPs in medium, the possibility of interference by size discrepancy and spontaneous agglomeration to mechanism investigations could be eliminated35. Mucin induced the formation of nano-clusters by bridging Mucin, as the dominant and representative component in mucus, was selected for the investigation of the mucus/PGNPs interaction. The TEM images in Figure 2a demonstrated the obvious corona feature, in which mucin coated the surface of PGNPs to form a halo-like structure (Figure 2a.(1)). Additionally, owing to the large molecular weight and intermolecular interaction, the redundant mucin linked PGNPs together as a bridge (Figure 2a. (2),(3)). The dumbbell-like structures further interplayed with each other to form bigger chain-like nano-clusters (Figure 2a. (4)). The CLSM analysis also revealed the aggregations of mucin on the nanoparticle surface via the FITC-mucin (Figure 2b). Compared with the extremely low signal of free mucin, PGNPs triggered an increase of fluorescence in the light-spot forms. Meanwhile, the aggregative feature showed significant concentration dependence (Figure 2c). Interestingly, the dynamic investigation of particle size after mucin incubation based on DLS illustrated that mucin in medium could quickly interact with PGNPs and induce the clusters (Figure 2d). When mucin increased from 800 µg/ml to 5 mg/ml, more PGNPs jointed to form larger clusters. The measurement of particle counts in clusters further demonstrated the characteristic

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(Figure 2e and 2f). Notably, the average size and proportion of the nano-clusters did not change with time, indicating that the aggregative characteristic depended on the mucin concentration but not on the incubation time. The coating and bridging of mucin on the PGNPs altered the physicochemical properties of the nanoparticles, which were verified by UV-Vis spectrum detection. As shown in Figure 2g, the absorption value of PGNPs in the range of 600 nm to 800 nm significantly increased in the presence of mucin compared with the control. Additionally, the diffusion pattern of mucin in medium after the addition of PGNPs was investigated by fluorescence correlation spectroscopy (FCS)36. After incubation with PGNPs, mucin exhibited a longer diffusion relaxation time, revealing a shorter distance of particle movement within the test time (Figure 2h). Furthermore, the diffusion rate of mucin, namely, the distance per unit time, was found to decrease as the PGNPs concentration increased (Figure 2i). In addition, the movement of mucin in the presence of PGNPs was recorded using multiple-particle tracking technology37. As demonstrated in Figure 2j and the supplementary video (S1 to S3), the moving distance of mucin decreased with the increase in nanoparticle concentrations, consistent with the finding by FCS. In summary, the dispersive features and surface properties were significantly altered by the mucin binding in a corona pattern. Mucin triggered more cellular uptake of PGNPs via endocytosis Two intestinal epithelial cell lines, Caco-2 (non- mucin secretion capability) and HT-29 (high mucin secretion capability), were cultured to evaluate and compare the influence of mucin on the cellular uptake of nanoparticles38. Caco-2 cells are always 8


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chosen as a common cell line to investigate the transport and absorptive mechanisms of nanomedicines. HT-29 cells, as mucus-secreting cells of the gut, can secrete mucus to the enteric cavity from the apical exocytosis, and they had been widely applied in the study of intestinal immune response to bacterial infection and microorganism invasion. Meanwhile, the adhesion and retardant for nanomedicines had also been studied in recent years39,40. Here, the alcian blue staining, which was specifically labeled with acidic polysaccharides, confirmed the secretion of mucin from the HT-29 cells first (Figure 3a). Throughout the cellular studies, both mono-dispersive PGNPs and nano-clusters (PGNPs@mucin) were fabricated and compared with each other. First, the cytotoxicity analysis demonstrated that the nanoparticle incubation had no effect on the cell viability (Figure S2). Then, the cellular uptake was tested on Caco-2 cells at different temperatures. As shown in Figure 3b, the intracellular gold content at 37 °C was significantly greater than that at 4 °C both in the PGNPs@mucin and PGNPs groups, and these were also confirmed by CLSM analysis (Figure 3c) based on a testing method that benefit by the great light scattering characteristic of inorganic nanomaterials32. The intracellular uptakes of PGNPs and PGNPs@mucin were found to occur mainly through energy-dependent endocytosis. In addition, a small fraction of PGNPs and PGNPs@mucin were still detected to be internalized by cells via an energy-independent process. Besides, the intracellular contents of both nanoparticles were proportional to the concentration and incubation time (Figure 3d and 3e). Interestingly, Figure 3f illustrated that the cellular uptakes of PGNPs@mucin were significantly greater than that by PGNPs in both cell lines. The ratio difference in the cell lines were 2.6-fold (Caco-2) and 3.1-fold (HT-29), indicating the significant effects of mucin on nanoparticle internalizations. The CLSM

9



analysis in Figure 3 g showed that the intracellular spotlights of PGNPs@mucin were larger than that of PGNPs. It revealed that the intracellular PGNPs@mucin still existed in nano-cluster forms, indicating that mucin also entered the cells and constrained the nanoparticles from dispersion in cells. The effects of mucin were also verified in the HT-29 cells with natural mucin secretion. As shown in Figure 3h, when cells were treated with NAC to specifically eliminate the extracellular mucin, the cellular uptake of mono-dispersive PGNPs decreased significantly. Generally, it was clearly indicated here that in both cells the mucin corona coating and resultant nano-clusters dramatically facilitated the endocytosis of nanoparticles. Mucin altered the cellular interaction characteristic and endocytosis mechanism of nanoparticles Before nanoparticles were internalized by cells, they must first adhere to the surface of cells. Hence, the bio-adhesion feature was investigated in the following. Through detecting the signal changes of the forward scatter (FSC) and side scatter (SSC) of cells after incubated with PGNPs and PGNPs@mucin separately based on flow cytometry technology, greater intensities of FSC and SSC were found for the PGNPs@mucin group (Figure 4a), indicating the greater bio-adhesion characteristic of PGNPs@mucin versus PGNPs41. Additionally, the interaction of nanoparticles with cell membrane was investigated by the FRAP technique42. As illustrated in Figure 4b and Figure S3, mucin-coated nanoparticles (PGNPs@mucin) significantly lowered the cell membrane fluidity more than mono-dispersive PGNPs alone. Thus, the great inducibility of mucin manifested for the membrane interaction of nanoparticles.

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The effect of mucin on the endocytosis mechanism of nanoparticles was studied using pharmacological inhibitors. It was first verified that pharmacological inhibitors caused little change in cell viability compared with the control group (Figure S4). As illustrated in Figure 4c and Figure 4d, MβCD, a cholesterol depletion agent43,44, significantly reduced the intracellular gold content of both nanoparticles. The endocytosis percentage of PGNPs and PGNPs@mucin decreased by 66.3 % and 56.3 %, respectively, in the presence of MβCD. It was demonstrated that cholesterol played an important role in the endocytosis of both nanoparticles. The intracellular gold content of PGNPs and PGNPs@mucin was reduced to 0.41±0.13 µg (decreasing by 58.1 %) and 2.26±0.17 µg (decreasing by 11.0 %), respectively, when pre-incubated with CPZ. CPZ is a specific inhibitor of clathrin-mediated endocytosis (CME)45. The corresponding CLSM analysis in Figure 4e and 4f also reflected the consistent inhibition effects. Meanwhile, the internalization of PGNPs was inhibited by hyperosmotic sucrose, a non-specific inhibitor of CME by the K+ depletion effect46 (Figure 4e and 4f), further proving the importance of CME in the endocytosis of nanoparticles. Interestingly, CME was mainly involved in the cellular uptake of PGNPs while having negligible effect on the internalization of PGNPs@mucin. EIPA, as an inhibitor of the macropinocytosis pathway via the sodium-proton exchange47, significantly reduced the intracellular gold content of PGNPs@mucin (decreasing by 48 %). However, the endocytosis of PGNPs was hardly influenced by EIPA (only decreasing by 15.3 %). The CLSM analysis also showed a similar result (Figure 4e and 4f). It was indicated that the endocytosis of PGNPs@mucin was mainly via the macropinocytosis pathway. The difference in endocytosis might be related to the clustering of nanoparticles induced by mucin because particle sizes larger

11



than 500 nm have been reported to enter cells mainly via macropinocytosis48. In addition, the cholesterol-sequestering agent filipin, which was treated as a specific inhibitor of caveolin-mediated endocytosis46, also inhibited the internalization of PGNPs while having no effect on the endocytosis of PGNPs@mucin (Figure 4e and 4f). In summary, PGNPs@mucin was mainly internalized by Caco-2 cells via the macropinocytosis pathway while PGNPs were mainly internalized through CME and caveolin-mediated endocytosis. In other words, mucin significantly altered the endocytosis mechanism of nanoparticles. Mucin induced less transcytosis but more exocytosis of nanoparticles through the epithelial monolayer To explore the transport characteristics of nanoparticles through intestinal epithelia, cells were cultured in a transwell plate to establish the cell monolayer model. First, the TEM images in Figure 5a demonstrated that nanoparticles were transported across the cell monolayer to the basolateral side. The trans-epithelial electric resistance (TEER) and Papp detection further showed that the integrity of the epithelia was not affected by the trans-cellular process of the nanoparticles (Figure 5b and Figure S5). The TEM analysis of the tight junctions also verified the same result (Figure 5c). It revealed that transcytosis but not paracytosis was the main trans-cellular pathway. By determining the transcytosis amount of nanoparticles based on the Au content, the transcytosis of PGNPs@mucin was found to be significantly less than that of PGNPs (Figure 5d). Combined with the comparison of endocytosis between the two nanoparticles (Figure 3d), the different effects of mucin on the transcytosis and endocytosis of PGNPs were elucidated. Thus, we

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hypothesized that the intracellular PGNPs@mucin might be recycled again to the extracellular side (apical side) via apical exocytosis. To verify this hypothesis, the apical exocytosis study was performed to clarify the possible recycling of PGNPs@mucin to the extracellular apical side. Consequently, Figure 5e showed that the efflux amount of PGNPs@mucin was significantly greater than that of PGNPs. The comparison based on relative amounts further revealed less transcytosis but more exocytosis for PGNPs@mucin (transcytosis: 8.7±3.4 %; apical exocytosis: 89.8±0.7 %) compared with mono-dispersive PGNPs (transcytosis: 31.3±12.4 %; apical exocytosis: 62.5±5.8 %) (Figure 5f), and this was also confirmed by Figure 5g. As a result, the lower transcytosis of PGNPs@mucin was attributed to the greater exocytosis induced by mucin. In summary, these findings confirmed the “more endocytosis but less transcytosis (MELT)” effect for the influence of mucin on the transportation of nanoparticles. Mucin caused the “MELT” effect by reinforcing the retrograde pathway of nanoparticles in the epithelia To explore the detailed mechanism of the “MELT” effect induced by mucin, the exocytosis mechanisms of PGNPs and PGNPs@mucin were investigated and compared using pharmacological inhibitors related to efflux. During the exocytosis process, intracellular cargo are usually transported to the endoplasmic reticulum (ER) and Golgi apparatus and finally delivered out of cells through a secretion pattern. This pathway is also termed as a retrograde route49,50. Reportedly, brefeldin A inhibits the transport from the Golgi apparatus to the ER51, while monensin blocks transport from the Golgi apparatus to the plasma membrane52. As shown in Figure 6a, the pre-incubation of

13



brefeldin A and monensin significantly increased the intracellular retention of nanoparticles during the exocytosis investigation. Notably, both pharmacological inhibitors showed greater influence on the efflux of PGNPs@mucin than PGNP. The intracellular amount of PGNPs@mucin increased to 2.11±0.22 µg in the presence of monensin compared with 0.28±0.02 µg in the control group. After pre-incubation with brefeldin A, the intracellular PGNPs@mucin reached 2.03±0.14 µg. These findings demonstrated that mucin strengthened the exocytosis of PGNPs through the retrograde pathway53. For further exploration, the effect of mucin on the intracellular location of nanoparticles was also investigated. As detected by TEM analysis (Figure 6b), the intracellular nanoparticles were mainly located in endosomes and lysosomes when mono-dispersive PGNPs were incubated with cells37. However, most PGNPs@mucin existed in endosomes and the Golgi apparatus and still maintained the nano-cluster morphology. Additionally, the co-localization characteristics of both nanoparticles with different organelles were analyzed by CLSM. To quantitatively compare the degree of co-localization, the Pearson correlation coefficient (PCC) and the Mander’s overlap coefficient (MOC) were used. PCC and MOC represent the deviation from the mean and absolute intensities, respectively54. Besides, because PGNPs and PGNPs@mucin showed a punctate distribution in the cells, the M1 value derived from the MOC was also used. M1 represents the co-localization of nanoparticles in the proportion of total nanoparticles. When the nanoparticles were incubated with Caco-2 cells (Figure 6c and 6d), the PGNPs were more inclined to enter into lysosomes. Quantitatively, the co-localization coefficients of PGNPs with lysosomes in Caco-2 cells were greater than that of 14


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PGNPs@mucin (Figure 6d). However, the co-localization parameters of PGNPs@mucin with recycling endosome and Golgi apparatus in the same cells were higher than that of the PGNPs (Figure 6e to 6h). The location study was also studied in HT-29 cells that with natural mucin secretion capacity (Figure S6). The co-localization trend was consistent with the results from the Caco-2 cells investigation. Reportedly, mucin is only specifically secreted to the enteric cavity. Thus, the intracellular mucin could only be transported to the apical side of enterocytes. Hence, these findings demonstrated that mucin facilitated the recycling of nanoparticles back to the apical space, which was consistent with the retrograde pathway described above. In conclusion, it provided a reasonable account of the “MELT” effect induced by mucin. Mucin affected the bio-adhesion and absorption of nanoparticles in intestinal segments differently To confirm the effect of mucin on nanoparticle transportation in vivo, different intestinal segments were ligated and incubated with PGNPs. The amounts of nanoparticles that adhered to and absorbed by enterocytes were detected and compared in the presence and absence of a mucus layer using the environmental scanning electron microscope (ESEM) technique6,55. As illustrated in Figure 7a to 7c, the amounts of PGNPs adhering to the surface of natural intestine (mucin +) were clearly higher than the mucus removed from the gut (mucin -), especially at the duodenum. Notably, the corresponding EDS analysis revealed that the quantities of adhered nanoparticles in both intestinal environments (mucin + and -) gradually decreased from the duodenum and jejunum to the ileum. The CLSM detection on the different intestinal sections further

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demonstrated a similar result (Figure 7d to 7f). The spotlights distributed within enterocytes indicated the intestinal absorption of nanoparticles. In addition, the uptake amounts of PGNPs in natural intestine (mucin +) were evidently greater than that in the mucin negative group (mucin -). Similarly, the signals also decreased gradually from the duodenum and jejunum to the ileum. In summary, mucin facilitated the bio-adhesion and absorption of nanoparticles in intestinal segments differently. In other words, mucin caused more retention of nanoparticles in the enterocytes by inducing the formation of nano-clusters, whereas it could not prompt complete trans-cellular absorption.

CONCLUSIONS In this study, we uncovered a new effect of mucin on the oral absorption of nanoparticles. Unlike the post-cognition on mucus that only functioned as a physical barrier to hinder the infiltration of nanomedicine, mucin as the dominant component of mucus interacts with nanoparticles and alters their transport characteristics. As shown in Figure 8, mucin adsorbed onto the PGNPs surface to form a corona and induced nano-clustering by jointing particles together. The double effects caused more endocytosis but less transcytosis of nanoparticles through the epithelial monolayer. Notably, mucin was also internalized by cells along with PGNP due to the formation of a corona. The intrinsic transport feature of mucin altered the intracellular locations of the nanoparticles. Compared with the mono-dispersive PGNPs, more PGNPs@mucin were transported through retrograde pathway (PM-Golgi/ER-PM (plasma membrane)), thus accelerating the exocytosis of nanoparticles and rationally explaining the “MELT” effect caused by mucin. We believe these findings on mucin/nanoparticle interaction endow a

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new perspective for improving the oral absorption of nanomedicines (Figure 8). Despite the trans-cellular capability of nanoparticles being significantly inhibited by mucin, the enhanced cellular uptake might increase the local release of drugs from nanomedicines in enterocytes, providing a potential strategy for enhancing drug absorption. In conclusion, precisely regulating and balancing the effects of mucin will contribute to the design of more novel nanomedicines to overcome the obstacle of mucus as a barrier and enhance the oral absorption rate of loaded drugs.

MATERIALS AND METHODS

Materials and cells Chloroauric acid (HAuCl4·3H2O) was purchased from Sahn chemical technology (Shanghai) Co. LTD. Sodium citrate was supplied from National medicine group chemical reagent Beijing Co. LTD. Thiol modified methoxyl polyethylene glycol (mPEG-SH, Creative PEGWorks, the molecular weight of 550 Da) was purchased from Shanghai ZZBIO Co. LTD. LysoTracker probe was obtained from Invitrogen (Thermo Fisher Scientific, USA). Rabbit anti-Rab11 antibody and Texas Red-conjugated goat anti-rabbit IgG were achieved from Abcam (Cambridge, MA, USA). Golgi-Tracker probe, Hoechst 33258, Brefeldin A, Monensin and Cell Counting Kit-8 (CCK-8) were all purchased from Beyotime (Haimen, Jiangsu, China). Fluorescein isothiocyanate (FITC), chlorpromazine

(CPZ),

methyl-β-cyclodextran

(MβCD),

fillipin,

5-(N-ethyl-N-isopropyl)-amiloride (EIPA)， sucrose and porcine stomach mucin were obtained from Sigma (St. Louis, MO, USA). Penicillin, streptomycin, trypsin containing

17



0.02 % EDTA, RIPA lysis buffer, phosphate buffer solution (PBS), cell culture medium was purchased from Mcgene Co. (Beijing, China). All chemical reagents used in this experiment were analytical grade. Caco-2 and HT29 cell lines were obtained from National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China). Caco-2 cells were grown in a culture flask containing MEM medium supplemented with 10 % (v/v) fetal bovine serum, 1 % (v/v) non-essential amimo-acid, 100 UI/mL penicillin and 100 µg/mL streptomycin and 1 % (v/v) sodium pyruvate at a humidified atmosphere containing 5 % CO2 at 37 °C. HT29 cells were cultured using DMEM-Ham's F12 medium containing 10 % (v/v) fetal bovine serum, 1% (v/v) non-essential amimo-acid, 1 % (v/v) L-glutamine and 100 UI/mL penicillin and 100 µg/mL streptomycin. Both cells were passaged to a new culture flask digested with 0.25 % trypsin/0.02 % EDTA after the proliferation of 4-5 days. Synthesis and Characterization of PEG modified gold nanoparticles (PGNPs) Original gold nanoparticles (GNPs) were prepared by citrate reduction. The detailed process was as following: to 25 mL of deionized water, 1 mL of 50 mM HAuCl4·3H2O was added, and the mixture was heated to boiling under reflux, then, 1 mL 150 mM sodium citrate was quickly added and the reaction mixture was left stirring for another 20 min at the state of boiling and then cooled to room temperature under stirring to obtain GNPs. Then a certain amount of mPEG-SH as the ratio of 50:1 of Au/ligand was slowly dropped into GNPs solution under continuously stirring and this reaction was performed for 6 h. Then, excess mPEG-SH was removed through centrifugation at 13000 rpm for three times and the PEG modified gold nanoparticles (PGNPs) were obtained. 18


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The particle size and surface charge of nanoparticles were detected by the dynamic light scattering (DLS) analyzer (Malvern, Zetasizer Nano ZS) at 25 °C. Then, nanoparticles were also observed by transmission electron microscope (TEM, JEM-1200EX, JEOL) without negatively staining. Fourier Transform Infrared spectroscopy (FTIR) analysis was conducted to verify mPEG-SH covalent attachment to the surface of GNPs. Dry KBr was mixed with appropriate amount of samples (GNPs, PGNPs, mPEG-SH). Then, samples were pressed into pellets after sufficient milling. The stability of PGNPs were carried out as following: PGNPs were dispersed into deionized water, PBS and serum-free culture medium, respectively. Then, the particle size was measured by DLS and TEM after they were placed into a shaking table at 37 °C for 12 h or 7days. The interaction between PGNPs and mucin PGNPs were incubated with different concentrations of mucin in a shaking table at 37 °C for 2 h with a speed of 100 rpm. Then, unbound mucin was removed by centrifugation at 13000 rpm for 20 min, and the supernatant was discarded. The precipitates were re-suspended with an equal volume of PBS, and a complex of PGNPs and mucin (PGNPs@mucin) was obtained. DLS and TEM analysis. The particle size and dispersion morphology of PGNPs@mucin as well as PGNPs were detected by DLS and TEM (with/without negative staining).

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Ultraviolet-visible (UV) spectroscopy assay. The adsorption spectra of PGNPs@mucin and PGNPs were measured via a UV spectrophotometer. The scanning wavelength range was set from 300 to 1200 nm with a scanning interval of 1 nm. Fluorescence Correlation Spectroscopy (FCS) study. Fluorescein isothiocyanate (FITC)-labeled mucin was prepared initially. In brief, a certain amount of FITC was dissolved in deionized water, and the pH was adjusted to 9.0 with 1 M NaOH. Then, mucin was added into the FITC solution at a molar ratio of 1:2, and the reaction was performed in a shaking table for 24 h. Then, excess FITC was removed by dialysis, and FITC-labeled mucin (FITC-mucin) was freeze-dried for storage. Different concentrations of PGNPs were first incubated with FITC-mucin in a shaking table at 37 °C for 2 h at a speed of 100 rpm. After subsequent centrifugation and washing 3 times, the samples were dropped onto a slide, covered with a coverslip, and detected by confocal laser scanning microscope (CLSM, Leica, TCS, SP5) at room temperature. The FITC-mucin solution was set as the control group. Then, a FCS experiment was conducted to record the fluctuation of the fluorescence intensity. Furthermore, a two component function was adopted to fit the measured autocorrelation function as shown in formula 1-1.

Gτ =

1 〈N〉

1

1 + ττ 1 + a ττ

1

−1

Where ＜N＞ represented the average number of fluorophores in the focal volume and τ represented the characteristic residence time of particles. In addition, the diffusion rate of Brownian motion of particles could be calculated by the corresponding software.

20


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Multiple particle tracking (MPT) assay. Different concentrations of PGNPs were also first incubated with FITC-mucin as mentioned above. After subsequent centrifugation and washing 3 times, the samples were added to an 8-well chamber slide system, and movies were captured at 30 frames/s for 10 s by Spinning-disk confocal microscopes (Nikon, Japan). Additionally, the x and y positional data over time was extracted from the movies and analyzed using Volocity Demo software to record the change of track. Cytotoxicity analysis of nanoparticles Caco-2 or HT-29 cells were cultured in 96-well plates. After the cell confluence reached 80 %, the cells were incubated with various of setting concentration nanoparticles at 37 °C for 24 h, the serum-free medium without nanoparticles were taken as control. After incubation, cell viability were analyzed with CCK-8 kits. Cellular uptake of nanoparticles in both cell lines For Caco-2 cells, PGNPs or PGNPs@mucin were dispersed into a serum-free MEM medium and incubated with cells. For HT29 cells, the mucus layer was removed with 10 mM N-Acetyl-cysteine (NAC) at 37 °C for 1 h as the negative group (mucin(-)) prior to the experiment, and then PGNPs dispersed into a serum-free DMEM-F12 medium were allowed to interact with the cells treated with/without NAC (mucin(-) or mucin(+)). Quantitative analysis. First, 2 ×105/ mL cells were seeded onto a 12-well sterile plate until a confluence between 70 % and 80 % prior to exposure to the nanoparticle suspension, and then they were ready for use. After treatment with 600 µg/mL

21



nanoparticles at 37 °C for 12 h, the cells were washed with cold PBS 3 times and digested. After collection, the cells were centrifuged and washed 3 times to remove the nanoparticles adhering to the cell surface. Then, the cells were treated with a RIPA lysis buffer at 4 °C with gentle shaking. The aqua regia was added to dissolve the gold nanoparticles overnight, and the samples were diluted to 0.5 mL for ICP-MS analysis (inductively coupled plasma mass spectrometry, Elan DRCⅡ, Perkin-Elmer Sciex). Notably, the internalization refers to a series of tests on nanoparticle concentration and interaction time were performed to screen the appropriate operating conditions first. Qualitative analysis. First, 5 ×104/ mL cells were seeded onto a sterile glass bottom dish until the confluence to 70 %. Caco-2 cells were incubated with 600 µg/mL PGNPs or PGNPs@mucin (PBS was added to the control group) at 37 °C for 12 h, respectively. Then, the cells were washed with cold PBS 3 times, fixed by 3.7 % paraformaldehyde at room temperature for 20 min, stained by Hoechst 33258 to mark the cell nuclei and finally sealed with glycine/PBS (v/v=1:1). Lastly, the cell samples were detected by CLSM (Leica, TCS, SP8) with a 633 nm laser for nanoparticles. For HT-29 cells, the cells (mucin(-) or mucin(+)) were incubated with 600 µg/mL PGNPs at 37 °C for 12 h and then treated as Caco-2 cells mentioned above. The influence of nanoparticles on the plasma membrane (PM) Cell membrane adhesion. Flow cytometry (FCM, FACS Calibur, BD) was used to estimate the adhesive characteristics of the nanoparticles on the cell membrane. Caco-2 cells were incubated with 600 µg/mL PGNPs or PGNPs@mucin for 12 h, rinsed with cold PBS 3 times, detached by trypsinization and re-suspended with PBS. Through 22


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observations of the change in forward scatter (FSC) and side scatter (SSC), the morphologies of the cells were analyzed compared with the group without nanoparticles. Fluorescence recovery after bleaching (FRAP) technology. Caco-2 cells were cultured in a glass bottom dish until a confluence above 70 %. Prior to the cellular uptake experiment, the cell membrane was stained with DiI at 37 °C for 1 h, and the cells were incubated with PGNPs or PGNPs@mucin for 12 h. After that, they were washed with cold PBS three times and imaged via CLSM before photobleaching. Afterword, a region of interest (ROI) on the labeled cell membrane was selected, and the intensity of the exciting laser was regulated to the maximum to photobleach this area for 3 s; then, the recovery of fluorescence in the ROI was recorded during the following 90 s. At the same, the group without nanoparticles was set as the control. Endocytosis mechanism investigation Energy dependence study. The ready cells and nanoparticles were pre-treated at 4 °C and 37 °C for 0.5 h, respectively. Then, the cell medium was removed and replaced with corresponding nanoparticles (PGNPs or PGNPs@mucin) and continuously incubated for 12 h at 4 °C or 37 °C, respectively. After incubation, the cells were treated as described in the Qualitative or Quantitative analysis. Endocytosis pathway analysis with pharmacological inhibitors. The endocytosis pathway of PGNPs and PGNPs@mucin were conducted by the addition of common pharmacological inhibitors as described in Table S1 and investigated simultaneously by ICP-MS and CLSM. Prior to the addition of nanoparticle dispersions, Caco-2 cells were

23



pre-incubated with a certain concentration of inhibitors for 30 min at 37 °C. Then, 600 µg/mL PGNPs or PGNPs@mucin re-suspended with a serum-free MEM medium containing the same pre-treated concentrations of inhibitors were incubated with cells for another 12 h. After incubation, the cells were treated as described in the Qualitative or Quantitative analysis. Trans-cellular transport across intestinal cell monolayer analysis First, 4×105/mL Caco-2 cells were seeded onto the transwell polycarbonate membrane (Corning, 12 well, 3 µm) and allowed to culture for three weeks. The culture medium was changed every day. When the transepithelial electrical resistance (TEER) was higher than 500 Ω/cm2, the cell monolayer was used. Transcytosis analysis. First, 600 µg/mL (0.5 mL) PGNPs or PGNPs@mucin were added to the apical side, and 1.5 mL of blank medium was used for the basolateral chambers. The cells were incubated at 37 °C for 12 h. The TEER value was measured before and after incubation. Then, all 1.5 mL basolateral solutions of each well were completely collected and measured by ICP-MS. The polycarbonate membrane was carefully cut with a knife and placed into a new 12-well plate; then, the cells on the membrane were digested and detected as in the Quantitative analysis. TEM observation. The Caco-2 cell monolayer was incubated with PGNPs or PGNPs@mucin. After incubation for 12 h, the polycarbonate membrane covered with compact cells were carefully cut with a knife and placed into a centrifuge tube. Then, they were was fixed with 2.5 % glutaraldehyde, post-fixed in osmium tetroxide (1 %, w/v)

24


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for 1 h and dehydrated by washing in 30, 50, 70, 90 and 100 % acetone in sequence before the cells were embedded in epoxy resin. Ultrathin sections were cut with a diamond knife and further stained with uranyl acetate and lead citrate; then, the samples were analyzed via TEM. Apparent permeability coefficients (Papp) measurement. FITC-labeled dextran (FD4, molecular weight: 4 KDa) was added into the apical side of the cell monolayer, which had been co-incubated with nanoparticles for 12 h. Then, 200 µL of basolateral medium was withdrawn at specific time points. The paracellular amount of FD4 at different times was detected by a fluorescence spectrophotometer, and the group without nanoparticles was set as the negative control. Then, Papp values at determined times were calculated as seen in formula 1-2.

P =

∆Q 1 × 1 − 2 ∆t AC

Where Q represented the amount of fluorescent molecule across the cell membrane; A represented membrane area and C0 was the initial FD4 concentration. Then, the permeability of FD4 and Papp data in the experiment group were compared with control group to evaluate the integrity of cell monolayer. Exocytosis analysis Caco-2 cells were cultured in 12-well plates until the proliferation reached above 70 %. Prior to the exocytosis experiment, the Caco-2 cells were incubated with 600 µg/mL PGNPs or PGNPs@mucin at 37 °C for 12 h. Then, the nanoparticle suspension

25



was replaced with a serum containing MEM medium and allowed to incubate for 6 h at 37 °C. Finally, the intracellular gold contents were detected as described in the Quantitative analysis via ICP-MS. Intracellular transport pathway study of nanoparticles Intracellular location of nanoparticles observed via TEM. Caco-2 cells were cultured in 6-well plates, and when the confluence reached 80 %, cells were used. 600 µg/mL PGNPs or PGNPs@mucin were added and co-incubated at 37 °C for 12 h. Then, the cells were digested and collected, fixed with 2.5 % glutaraldehyde at 4 °C for 12 h, and dehydrated, following the same protocol as described in the TEM observation. Co-localization analysis. Caco-2 cells were incubated with PGNPs or PGNPs@mucin at 37 °C for 12 h, which were then replaced by fresh a serum-free MEM medium containing Lysotracker Probe (75 nM) or Golgi-tracker (150 µg/mL) to incubate for determined times according to the protocol described in the website. Then, the samples were detected by CLSM. For HT-29 cells, cells (mucin(-) or mucin(+)) were interacted with 600 µg/mL PGNPs at 37 °C for 12 h, and then the same process as Caco-2 cells was followed. For recycling endosome, the cells were washed with cold PBS after the treatment with nanoparticles and then fixed with 3.7 % paraformaldehyde, permeabilized by TPBS and blocked with a 5 % bovine serum albumin (BSA) solution. Then, they were incubated with rabbit anti-Rab11 antibody at 4 °C overnight and treated with AlexaFlour®647 labeled goat anti-rabbit IgG for another 2 h at 37 °C. After cell nuclei staining, the samples were detected via CLSM. In addition, co-localization-related parameters including Pearson’s correlation coefficient (PCC), Mander’s overlap 26


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coefficient (MOC) and M1 coefficients were obtained from Image-Pro Plus software. In vivo experiment Male Wistar rats were divided into three groups (control, nanoparticles with/without mucus), fasted overnight before the experiments and anesthetized by an intraperitoneal injection of 4 % (w/v) chloral hydrate (1 mL/100 g). A midline incision was performed in the belly, and the upper side of the duodenum was ligated with a cotton thread. For the nanoparticles without mucus group, rat small intestine was first pre-treated with NAC for 1 h. After the whole intestinal segments were further ligated and divided into the duodenum, jejunum and ileum, 1.2 mg/mL PGNPs (the same volume of physiological saline was used for the control group) was injected into the corresponding parts, which were incubated for 2 h. During the experiment, the rats operated upon were alive. After the end of the experiment, the rats were executed. All of the animal experiments conformed with the Principles of Laboratory Animal Care. Environmental scanning electron microscopy (ESEM) assay. A 1 cm segment of duodenum, jejunum and ileum was taken from the rat small intestine after incubation, washed with physiological saline (0.9 % w/v, NaCl solution) several times, fixed with 2.5 % glutaraldehyde, and raised with a cacodylate buffer and dehydrated in an acetone series except for sputter-coating with a thin film of gold. To avoid background interference, the PGNPs were imaged using backscattered electron mode. The accelerating voltage and ESEM Vacuum were set as 10 kV and 0.1-1.5 Torr, respectively. ESEM with Energy-Dispersive Spectrometry (EDS) analysis was used to determine the position of PGNPs on the surface of rat small intestine in the presence/absence of the 27



Page 28 of 47

mucus layer. CLSM analysis. After 2 h of incubation, the intestinal segments including the duodenum, jejunum and ileum were collected and washed three times with physiological saline. Then, they were cut into 1 cm segments and opened along with the mesentery. All samples were fixed with 4 % paraformaldehyde at 4 °C for 12 h, and the following steps followed the protocol for frozen sections. The sample of frozen sections was further stained with acridine orange at 37 °C for 10 min and detected by CLSM (LEICA, TCS, SP8). Statistical analysis All data shown as the mean value ± SD were collected through at least three independent experiments. Two-tailed Student’s t-test or one-way analyses of variance (ANOVA) were conducted. The significant difference was defined according to a p-value less than 0.05.

ASSOCIATED CONTENT Supporting Information Supplementary

Information

is

available:

The

detailed

information

of

pharmacological inhibitors used in the pathway study, Stability of nanoparticles detected by TEM, Cytotoxicity of nanoparticles, bio-membrane fluidity detected by CLSM, Cytotoxicity of pharmacological inhibitors, Papp value measurement, Co-localization

28


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information in HT-29 cell line, supplementary video: movement track of mucin incubated with nanoparticles. AUTHOR INFORMATION

Corresponding Authors

* E-mail: [email protected] (Qiang Zhang）

[email protected] (Xing Tang)

[email protected] (Bing He)

Author Contributions ‡

These authors have contributed equally to this work

Q.Z., X.T. and B.H. designed the research and wrote the manuscript. D.Y. and D.C.L. performed and analyzed most experiments. M.M.Q assisted with the preparation of gold nanoparticles.

B.L.C.

assisted

with

cell

culture.

S.Y.S

assisted

with

the

immunofluorescence experiments. W.B.D., H.Z., X.Q.W. and Y.G.W. assisted with data analysis. All authors discussed the results and commented on the manuscript.

Notes

The authors disclose no conflicts.

29



Page 30 of 47

ACKNOWLEDGMENTS

Supported by the National Basic Research Program of China (2015CB932100), the National Natural Science Foundation of China (81690264), and the Innovation Team of the Ministry of Education (BMU20110263).

The authors thank Lan Yuan for CLSM study

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Figure 1. Preparation and characterization of PGNPs. (a) The schematic and synthetic route of PGNPs. (b) Verification of PEG successfully covalent-linked on the surface of bare GNPs using FTIR. Size distributions of GNPs and PGNPs detected by DLS (c) normal distribution map and (d) average particle size, parallel operation for three times. The dispersed states of (e) GNPs and (f) PGNPs in PBS observed by TEM. (g) Zeta potentials of GNPs and PGNPs detected by DLS, n=3. (h) The stability of PGNPs dispersed in different medium for 12 h (sf-MEM: serum-free culture medium). 179x154mm (300 x 300 DPI)



Figure 2. Nano-clusters appeared when PGNPs incubated with mucin. (a) The effect of mucin on PGNPs detected by TEM with negative staining, sketch map indicated mucin induced the formation of nano-clusters by bridging, scale bar: 50 nm. (b) CLSM analysis of FITC-mucin after incubated with PGNPs, the intensity of fluorescence signal were reduced following a unified standard to observe the phenomenon of clusters. Scale bar: 25 µm. (c) TEM observation of PGNPs incubated with different concentration of mucin, scale bar: 500 nm. (d) The variation of Z-average diameter of PGNPs incubated with mucin at different time detected by DLS. (e) The measurement of average nano-clusters counts in unit area from TEM images. (f) The dynamic measurement of particle size (left Y) and average monodisperse particle counts in per nano-cluster (right Y) from TEM images. (g) UV scanning spectrum of PGNPs and PGNPs@mucin. (h) Normalized autocorrelation curves of PGNPs@mucin exhibited longer correlation time with increasing concentration of PGNPs. (i) The diffusion rate of mucin and PGNPs@mucin detected by FCS. (j) The trajectories of the Brownian movement of mucin incubated with different concentrations of PGNPs.


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209x280mm (300 x 300 DPI)



Figure 3. Mucin triggered more cellular uptake of PGNPs via endocytosis. (a) Staining of mucus secreted by HT29 cells with alcian blue. In the left image, mucus was removed by NAC before staining with alcian blue. The internalization analysis of nanoparticles at different temperatures in Caco-2 cells detected by (b) ICP-MS and (c) CLSM, ###p

Intestinal Mucin Induces More Endocytosis but Less Transcytosis of

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