Transferrin Functionization Elevates Transcytosis of Nanogranules

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Transferrin Functionization Elevates Transcytosis of Nanogranules across Epithelium by Triggering PolarityAssociated Transport Flow and Positive Cellular Feedback Loop Dan Yang, Dechun Liu, Hailiang Deng, Jian Zhang, Mengmeng Qin, Lan Yuan, Xiajuan Zou, Bin Shao, Huiping Li, Wenbing Dai, Hua Zhang, Xueqing Wang, Bing He, Xing Tang, and Qiang Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07231 • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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Transferrin Functionization Elevates Transcytosis of Nanogranules across Epithelium by Triggering Polarity-Associated Transport Flow and Positive Cellular Feedback Loop Dan Yang a,b,c‡, Dechun Liu a,c‡, Hailiang Deng a,c, Jian Zhang a,c, Mengmeng Qin a,c, Lan Yuan d, Xiajuan Zou d, Bin Shao e, Huiping Li e, Wenbing Dai a,c, Hua Zhang a,c , Xueqing Wang a,c, Bing He a,c *, Xing Tang b *, Qiang Zhang a,b,c * a.

Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery

Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China b.

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016,

China c.

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

100191, China d. Centre

e.

of Medical and Health Analysis, Peking University, Beijing 100191, China.

Department of Medical Oncology, Key Laboratory of Carcinogenesis and

1

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Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, 100142, China. ‡ These authors have contributed equally to this work * Corresponding author ABSTRACT Overcoming the epithelial barriers to enhance drug transport is a focused topic for gastrointestinal, intratracheal, intranasal, vaginal and intrauterine delivery. Nanomedicines with targeting functionization promote such a process owing to specific ligand-receptor interaction. However, compared to the cell uptake of targeting nano-therapies, currently few studies concentrate on their transcytosis including endocytosis for ‘in’ and exocytosis for ‘out’. In fact, the cellular regulatory mechanism for these pathways, as well as the principle of ligand’s effect on the transcytosis, are almost ignored. Here, we fabricated transferrin (Tf) functionalized nanogranules (Tf-NG) as the nanomedicine model, and confirmed the difference in polar distributions of Tf receptors (TfR) between two epithelium models (bipolarity for Caco-2 and unipolarity for MDCK cells). Compared to the non-specific reference, Tf-conjugation boosted the endocytosis by different pathways in two cell models, and transformed the intracellular route of Tf-NG in both cells differently, affecting exocytosis, recycling, degradation but not secretion pathway. Only bipolar cells could establish a complete transport flow from ‘in’ to ‘out’, leading to the enhanced 2


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transcytosis of Tf-NG. Importantly, epithelia could make responses to Tf-NG transcytosis. Based on the quantitative proteomics, the intracellular trafficking of Tf-NG altered the protein expression profiles, in which the endocytosis- and transcytosis-related proteins were specifically upregulated. Particularly, only bipolar cells could positively feed back to such trafficking via accelerating the subsequent Tf-NG transcytosis. Here, all the cell transport of Tf-NG was polarity-associated. In summary, Tf-modification elevated the transcytosis of Tf-NG across epithelium by triggering polarity-associated transport flow and positive cell feedback loop. These findings provided an insight to the targeting nano-delivery for efficient transport through epithelial barriers. KEYWORDS: transcytosis, transferrin functionalized nanogranules, polarity, cell response, proteomics, nano systematic biology Biological barriers (BB), existing in multiple parts of body, protect from the invasion of different exogenous pathogens, but usually become the primary obstacle for drug transport.1 As one of the main components of BB, epithelium regulates the balance between internal milieu and external environment by controlling different cargos in and out.2 Epithelial cells are largely distributed in gastrointestinal tract, lung, kidney, nose, vagina, uterus, and so forth. Because of the compact monolayer structure, they restrict the transport or absorption of multiple drugs, especially the insoluble medicines and biomacromolecules.3 Overcoming the biological obstacle to 3


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enhance the drug transport efficiency is a focused topic for various nonparenteral drug deliveries. Nanomedicines are widely applied for improving drug efficacies owing to obvious advantages in conquering the epithelial barriers.4,5 Distinct from the conventional formulations, nanopreparations usually transport across the epithelial monolayer in vesicle-mediated pathways.6 Ligand modification or active targeting exhibits enormous potentials in boosting the transcytosis of nanomedicines across epithelial cells.7,8 For instance, Vitamin B12 was used to modify nanoparticles which achieved a transcytosis 5.6 folds high than the ligand-free control.9 Our previous work demonstrated the positive effect of active targeting on promoting the transcellular trafficking of nanoparticle through Caco-2 cells.10 Nevertheless, compared to the enthusiasms in targeting nano-therapy that only concern endocytosis, few studies currently focus on the transcytosis of targeting nanomedicines. In mechanism, ligand modification elevates cellular uptake of nanomedicines by the receptor-mediated endocytosis, but the situation in transcytosis is more complicated.8 In fact, the transcytosis of nanomedicines is composed of endocytosis for ‘in’ and exocytosis for ‘out’. So far, such double regulatory mechanism of cells for functionized nanomedicines is unclear. Moreover, not all targeting ligands that increased the cellular uptake could accelerate the transcytosis. This might be attributed to the different features and functions of receptors,11 but the detailed reason and mechanism is unknown. 4


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Polarity of receptor, as a specific characteristic, makes epithelium distinct from other types of cells in histomorphology. Polar receptors asymmetrically distribute in apical and basolateral sides that are separated by tight junctions. These receptors are reported to have directivity during the transport and recycling processes.12,13 It means that if a certain polar receptor is targeted by specific ligand-modified nanomedicines, the receptor feature may affect the trafficking of nanomedicines. However, this impact is usually neglected in the nano-therapy studies, since all of which only concern the endocytosis in targeting cells. In contrast, when nanomedicines are transported through epithelium from ‘in’ to ‘out’, the polarity will become a non-negligible factor. Currently, it is unclear how the polarity of receptors regulates the transcytosis of nanomedicines. During the trafficking of active targeting nanomedicines, the ligand-receptor interaction causes the conformational change of receptors, then transmitting the external signal to intracellular space.14 To maintain the cellular homeostasis, the aroused cells respond to signal by altering the genetic and protein expression profiles.15,16 When nanomedicines are delivered for therapy, this responsiveness is often neglected, because the loaded drugs induce greater cell response than carrier materials. However, in terms of the transcytosis, the treated epithelial cell monolayer is not the therapeutic target for drugs. So the cellular response to nanomaterials could come out and influence the subsequent transportation.17,18 Nevertheless, most existing studies just focus on the trafficking pathway of nanomedicines in cells. The 5


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complete mechanism is still lacking on how these cells respond to the nanomaterial transportation. Here, we committed to clarify the cell regulatory mechanism to the transcytosis of ligand modified nanomedicines across epithelium as well as the cell response during the transcytosis by exploring the effect of receptor polarity, which is believed to be a part of nano systematic biology. Given the intracellular stability and detectability, the canonical gold nanogranules (GNG) were fabricated as the models of nanomedicines, and then modified with transferrin (Tf) as the well-established targeting ligand (Tf-NG).11,19 Bovine serum albumin modified nanogranules (BSA-NG) were prepared as the non-specific reference. Caco-2 and MDCK cells as the most frequently used epithelium were chosen as cell models thanks to their naturally difference in transferrin receptor polarity or distribution.10,20 The whole study was focused on the comparison between Tf-NG and BSA-NG in two cell models. Besides other technologies, confocal laser scanning microscopy (CLSM) based on laser reflection (LR) was applied to investigate the trafficking pathway of Tf-NG, after the polarity evaluation of Tf receptor (TfR). Quantitative proteomics, a powerful high-throughput technology, was utilized to study the cellular response in terms of protein expression, followed by bioinformatical analysis based on big data. RESULTS AND DISCUSSION Engineering and characterizing of Tf-NG 6


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Firstly, primal rough gold nanogranules (GNG) were synthesized by classical citrate reduction method.21 GNG were further modified by SH-PEG to enhance the dispersibility and stability. Transferrin (Tf), as the active targeting ligand, was conjugated to GNG surface via amide reaction with PEG-COOH, thus obtaining the Tf modified nanogranules (Tf-NG). BSA-NG were prepared via the same technique and set as reference throughout the study, which could eliminate the potential negative effect of non-specific interaction between transferrin and cells in study (Figure S3). Figure S4 illustrates the integrated fabrication flow for different nanogranules. X-ray photoelectron spectroscopy (XPS) demonstrated the conjugation of PEG with GNG. As shown in Figure S5a, the peaks of Au (4f) at 87.68 eV and S (2p) at 162.48 eV illustrated the formation of Au-S bond. Besides, the excessive SH-PEG was also detected in reaction solution based on the characteristic peak of free thiol group at 164.5 eV, indicating the complete covering of PEG on GNG surface.22 Gel electrophoresis was utilized in study to confirm the protein modifications. Figure S5b exhibited the obvious relevant bands in Tf-NG and BSA-NG groups, revealing the successful ligand conjugation. The computations of ligand densities showed that almost four protein molecules were bound on each particle (Table S1). The modification was further verified by transmission electron microscopy (TEM), and a distinct corona-like structure (Figure S5c, arrows) was observed in periphery of nanogranules after linking of protein with PEG-NG. Compared to the significant aggregation for pristine gold nanogranules, PEG coating and ligand modification 7


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endowed GNG with better dispersity (Figure S5d). Also, the dispersitiy of NG was associated with the test methods and mediums. For instance, the PEG-NG, in PBS but not in distilled water, partially formed clustering during the static TEM observation (Figure S5e), and there was no such impact during the dynamic measurement like dynamic light scattering (DLS) technology (Table S2). Further protein-modification on PEG-NG achieved better dispersity in static and ionic environment. The surface hydration layer on NG might be responsible to such difference. The dispersive feature of nanogranules in aqueous medium was further investigated by UV/Vis spectrophotometer and DLS, respectively (Figure S6). Figure S6a showed that Tf-NG and BSA-NG had similar maximal absorption peak with GNG at the wavelength of 520 nm, indicating that the ligand modification did not induce nanogranule aggregation.23 All monodisperse nanogranules showed a burgundy appearance (Figure S6b). The statistical analysis revealed the similar mean diameters (near 30 nm) between Tf-NG and BSA-NG, greater than that of GNG, detected by DLS (Figure S6c, Table S2). Notably, due to the elimination of hydrated layer, the average particle sizes of TEM measurement were obviously smaller than that of DLS test. Meanwhile, as shown in Figure S6d, nanogranules demonstrated a long-term stability in aqueous medium. Particle size, size uniformity and dispersity in medium are all significant factors for cellular transport of nanomedicines, and the alteration of any one may impact their cellular trafficking.24 To avoid their potential interferences on the transport of active 8


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targeting nanomedicines, their diameter distribution and dispersive characteristic were regulated to keep consistency between Tf-NG and BSA-NG, thus guaranteeing the robustness of subsequent comparisons. To make sure that the number of particles and proteins modified is comparable in this study between Tf-NG and BSA-NG, we calculated and compared the particle numbers based on the mass concentrations of NG (Table S3), as well as the number of protein molecules on the particle surface (Table S1). It was confirmed that the number of nanogranules was consistent between two NG groups, and about four protein molecules were conjugated on each particle in both NG groups. Verification of bipolar and unipolar cell models based on TfR distribution Both Caco-2 and MDCK cells are canonical epithelial models for trans-cellular studies of nanomedicines. Here, CLSM was used to compare the different expressions and distributions of TfR in two cell lines. Caco-2 and MDCK cells were cultured in transwell porous membrane to form cell monolayers and finish polar differentiations. Especially, occludin, a constituent of cellular tight junction, was labeled to indicate the boundary between apical and basolateral sides in confocal imaging study. As shown in Figure 1a and 1b, the horizontal plane graphs (X-Y mode) illustrated that TfR significantly expressed in both cells. However, the longitudinal section images (X-Z mode) exhibited their difference in distributions of TfR. Compared to the co-expression of TfR in both apical and basolateral sides of Caco-2 9


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cell monolayer, TfR only distributed in the basolateral membrane of MDCK cells, which was also confirmed previously.12,20 Western blot analysis showed that the TfR expression in Caco-2 cells was almost 1.3 folds that in MDCK cells (Figure 1c). It revealed that two cells were naturally different in terms of the polar distribution of TfR. Based on that, we established and identified the bipolar (Caco-2) and unipolar (MDCK) cell models in transwell culture plate (Figure 1d). It was noteworthy to mention that we mainly analyzed the different impacts of Tf-modification on the trafficking of nanogranules in two models, thus expounding their transport mechanisms, as illustrated in Figure 1d. By the way, it is a pity that Caco-2 and MDCK cells come from different species, owing to the current limitation in finding cell models with different polar distribution of TfR and the same species origin. Tf-functionalization promoted cellular internalizations of nanogranules in both models CLSM

and

inductively coupled plasma mass spectrometry

(ICP-MS)

were

utilized to evaluate the effect of active targeting modification on the internalizations of nanogranules. First, cytotoxic detections illustrated that both Caco-2 and MDCK cells could tolerate the nanogranules incubation with high concentrations even at 1000 μg/mL (Figure S7). It revealed that the following mechanism investigations were not influenced by the potential toxic effect of gold nanogranules because we chose the lower incubative concentration (500 μg/mL, 2.53 mM). Besides, laser reflection (LR) 10


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technology, as an effective label-free strategy, was introduced in CLSM to detect intracellular nanogranules. Because of the great light scattering characteristic, most inorganic nanomaterials, including gold, silicon, etc., could be directly detected by capturing the reflected light, without functional modifications such as fluorescence labeling (Figure S8).25,26 To qualify BSA-NG as the negative control or to rule out the role of possible albumin receptors in test cells,27 we pre-incubated cells with human serum albumin (HSA, the ligand of albumin receptor) or human transferrin (Tf, the non-albumin control ) and then analyzed the internalization of BSA-FITC. As a result, there was no significant difference between HSA and Tf pre-incubation groups, no matter in Caco-2 or MDCK cells (Figure S9), indicating that albumin receptor had a weak influence on BSA-NG which could be used as a negative control in our study. Figure 1e and 1f showed the cellular uptake comparisons between Tf-NG and BSA-NG in bipolar and unipolar cells, respectively. Interestingly, Tf-modification simultaneously caused more nanogranules internalizations in both models, although no TfR was found to express in the apical side of unipolar cells. By collecting the cell suspensions after nanogranule incubation and eliminating the surface bound particles (Figure S10), the intracellular nanogranules were also quantified based on the Au detection by ICP-MS. Compared to BSA-NG or PEG-NG, the obtained results further demonstrated the increased uptakes of Tf-NG in both cell models (Figure 1g and 1h, 11


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Figure S11). In other bipolar cell models (tumor cell HT-29 and primary cell HUVEC) based on the TfR expression, Tf-NG showed similar promotion in cell uptakes (Figure S12). These findings revealed that the facilitation of ligand functionalization to endocytosis might be independent on the receptor distribution. In other words, the surface Tf-TfR interaction might not be the key for the internalization of Tf-NG in unipolar cells. So, it was hypothesized that the polarity-associated intracellular ligand-receptor interaction might take a critical role in the transport process of nanogranules across epithelium.28,29　 Protein corona is another factor to regulate the active internalization of targeting nanomedicines.30 We also detected its existence in the cellular uptake investigations (Figure S13). To guarantee that the accelerated uptake of Tf-NG was not affected by it, we identified the amount and composition of protein corona by incubating different NG with cell lysates. Based on the SDS-PAGE analysis and quantitative proteomics (Figure S14), the minor difference in protein corona was found among three NG groups which shared all high-content proteins. It was also indicated that the presence of protein corona basically did not affect the observation and targeting ability of Tf-NG (Figure 1e to 1h). Tf-conjugation regulated the endocytosis of nanogranules in two models with different mechanisms To explore the effects of Tf-modification on the endocytosis mechanisms in two 12


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models, nanogranules were firstly incubated with two cells at low temperature. As shown in Figure 2a and 2b, 4 °C incubation significantly reduced the cellular uptakes of Tf-NG and BSA-NG in both cells. The addition of different ATP inhibitors also brought down the internalizations of nanogranules (Figure S15). These findings all indicated an energy-dependent endocytosis. The pharmacological inhibition analysis directly demonstrated the vesicle-mediated mechanism (Figure 2c and 2d), in which two typical inhibitors, nocodazole (inducing microtubule depolymerization) and cytochalasin D (disturbing actin aggregation) decreased the internalizations of nanogranules in two cells.31 TEM observations also manifested the similar results (Figure 2e). The magnified micrographs showed that dozens of Tf-NG were internalized in cells and located in endosomes. During the endocytosis process, Tf-modification exhibited different regulatory mechanisms in two models (Figure 2c and 2d). In terms of MDCK cells, the corporate inhibitions of chlorpromazine (CPZ), filipin and EIPA indicated that Tf-NG were internalized through multiple pathways consist of clathrin-mediated endocytosis (CME), caveolin-dependent endocytosis (CDE) and macropinocytosis (Figure 2c).32,33 In contrast, only CME and macropinocytosis functioned in Caco-2 cells (Figure 2d). More importantly, the competitive inhibition analysis illustrated that Tf complement significantly reduced the uptake of Tf-NG by Caco-2 cells compared to BSA-NG group (61%, Figure 2f), while it had less effect on the Tf-NG internalization in MDCK cells (28%, without significant, Figure 2g). Notably, the addition of free transferrin 13


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increased the cellular uptakes of both nanogranules in MDCK cells (Figure 2g). We deemed that this might be caused by the potential adsorption of free Tf on particle surface. As a result, the competitive inhibition effect of Tf was attenuated. Additionally, it gave nanogranules, especially BSA-NG, with the active targeting capacity due to the physisorption of Tf. So, similar as the uptake result in Figure 1h, the intracellular Tf-TfR interaction might make more retention of nanogranules in unipolar cells, thus increasing the cellular uptakes. Generally, it was indicated here that Tf-modification influenced the endocytosis of nanogranules differently. With the bipolar TfR expression in Caco-2 cells, the surface Tf-TfR crosstalk was involved in the regulation of nanogranule endocytosis behaviors. However, when nanogranules transported in unipolar cells, the impetus might mainly come from the intracellular but not the extracellular space.34 Tf-modification altered the intracellular transport pathways of nanogranules in two models To explore the intracellular effects of Tf-modification, the transport pathways of nanogranules in different routes (‘in’ and ‘out’) were analyzed in two models. First, apical exocytosis analyses in Figure 3a and 3b illustrated that more Tf-NG were detained in cells compared to BSA-NG during exocytosis in both cell models, and the between-group difference were more significant along with the exocytosis time. So, it demonstrated that Tf still affected the nanogranules transport even Tf-NG was 14


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internalized into cells. Because the amount of cellular uptake depends on both endocytosis and exocytosis, the reduction of latter will indirectly increase the uptake level of nanogranules in both cell models. Additionally, it was found in Figure 3a and 3b that the retention ratios (Tf-NG/BSA-NG) in two cells were similar, so the polar distribution of receptors hardly affected the apical exocytosis of active targeting nanomedicines from epithelium. Combining with internalization study in Figure 1, it was indicated that the intracellular ligand-receptor interaction played more significant role in cellular uptake and apical exocytosis process. From another point of view, this finding might offer a strategy to improve the intracellular delivery of nanomedicines for cellular targeting therapies. Additionally, it was worth noting that the one-directional exocytosis model here was designed for the study on intracellular ligand-receptor interaction, but not a reflection of real transport situation which should be two-directional. The intracellular nanogranules transport are usually divided into three subclasses including secretion, recycling and degradation pathways.35,36 To characterize the intracellular Tf-TfR interaction and the effects of Tf-modification on nanogranules transport pathways in two cell models, each subclass was analyzed via CLSM based on LR technology. The co-localization features of nanogranules in different organelles were evaluated by measuring the Mander’s overlap coefficients (M values).37,38 First, secretion pathway refers to the transport from endosome to endoplasmic reticulum (ER) and Golgi complex, and finally to extracellular space.21,39 15


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Figure 3c to 3j showed that a small part of intracellular Tf-NG located in ER and Golgi complex when they were internalized. The quantitative comparisons of co-localization parameters40 further revealed that the Tf-modification did not significantly change the locations of nanogranules in ER and Golgi complex compared to BSA-NG. In addition, the pharmacological inhibition analysis was utilized to evaluate the effect of ER and Golgi complex on the transportation of nanogranules. As shown in Figure S16, two secretion-related inhibitors, brefeldin A and monensin, significantly decreased the apical exocytosis of Tf-NG and BSA-NG in two cell models.21,26 It revealed the involvement of ER and Golgi complex in the trafficking of fractional nanogranules (Figure 3k). However, in this pathway, Tf-NG did not manifest more advantages than BSA-NG in both cell models, indicating that the specific ligand-receptor interaction did not work in ER/Golgi route or secretion pathway. The degradation pathway of nanogranules refers to their transport in cells from early endosomes (EE) to multi-vesicular bodies (MVB)/late endosomes (LE) and finally to lysosomes, while the recycling pathway means that from EE to recycling endosome (RE) and continue to extracellular space.41 The confocal images of EE (Figure 4a to 4d) and the corresponding co-localization study exhibited the locations of Tf-NG in EE. Similar as the situation in unipolar cells, Tf-NG did not show more co-localization with EE compared to BSA-NG in bipolar cells, although TfR significantly expressed in the apical side. Besides, Figure 4e to 4h revealed the significant differences on the MVB/LE locations of Tf-NG in two cells. Compared to 16


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BSA-NG, Tf-modification triggered more nanogranules transport to MVB/LE in unipolar cells. However, the observation in bipolar cells was just reversed. So, the MVB/LE, but not EE, was the key regulatory sites for the intracellular trafficking of the modified nanogranules. Additionally, Tf-NG manifested a co-localization M value with RE near 3.02 folds higher compared to BSA-NG in Caco-2 cells (Figure 4i and 4j), but showed no significant difference with BSA-NG in MDCK cells (Figure 4k and 4l). These findings revealed the different emphases on the trafficking pathways of Tf-NG in unipolar and bipolar cells. In this regard, Tf-modification caused more nanogranules transport via degradation pathway in unipolar cells, but more through recycling route in bipolar cells, compared to BSA-NG (Figure 4q). Last but not the least, the lysosome locations of Tf-NG were detected based on the fluorescence labeling of specific marker LAMP1 via CLSM.42 As shown in Figure 4m and 4n, less Tf-NG transported to lysosomes in bipolar cells compared to BSA-NG. It was consistent with the aforementioned findings that more Tf-NG located in RE through recycling pathway, which led to the attenuation of other routes, including lysosomes.43 In MDCK cell model, although the degradation pathway (MVB/LE/lysosomes) dominated in the trafficking of Tf-NG (Figure 4e to 4h), Tf-NG showed lower co-localization values than BSA-NG (Figure 4o and 4p). So, it seemed that the regulation effect of Tf-TfR interaction on nanogranules trafficking in MDCK cells was mainly confined in MVB/LE, but not in lysosomes. In other words, despite the lack of apical expression of TfR in unipolar cells, the intracellular Tf-TfR 17


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interactions made more Tf-NG detained in MVB/LE, thus reducing the exocytosis and increasing the cellular uptake indirectly. In summary, Tf-modification changed the intracellular transport characteristics of nanogranules in bipolar and unipolar cells with different patterns. Although the secretion, recycling and degradation pathways all functioned in both models, the different polar TfR resulted in distinct transport priorities in two cells. Depending on the specific ligand-receptor interactions, Tf-NG mainly transported to RE in bipolar model, but turned to MVB/LE in unipolar cells. It was indicated that the intracellular transport of active targeting nanogranules in epithelium was polarity-associated. Incidentally, the co-localization analysis here was based on tests at single time point, so it hardly indicated the dynamic processes of NG. Tf-functionalization led to a complete transport flow for nanogranules across bipolar monolayer Distinct from simple endocytosis, the transcytosis of nanogranules through epithelium was involved with both endocytosis from one side and exocytosis from other side.44 Here, to explore the effect of Tf-modification on the transcytosis of active targeting nanogranules, Caco-2 and MDCK cells were cultured in transwell porous membrane to construct the monolayer models (Figure 5a to 5c). Considering the different distributions of TfR in apical and basolateral membranes, MDCK cell monolayers were further cultured with upright and inverted patterns respectively as 18


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Figure 5b and 5c illustrated. Nanogranules were all added into the upper chambers of transwell culture plate, thus establishing two distinct transcytosis models (A-B and B-A) for the unipolar monolayers. By comparing the different effects of Tf-modification on the transcytosis in three models, the polar association in the whole process could be certified. Notably, there existed the difference of cellular culture sites between A-B and B-A models for unipolar cells. Cells placed on the transwell membrane in A-B model, but located beneath membrane in B-A model. To eliminate the interference that came from the ‘site effect’, we also constructed the inverted bipolar monolayer model in transwell plate for comparison (Figure S17a). Based on the trans-epithelial electronic resistance (TEER) detection and Papp analysis based on the FITC-Dextran (FD4) permeation, we first verified the integrities of cell monolayers (Figure S18).45,46 TEM images in Figure 5d and 5e showed that nanogranules entered cell monolayers from the apical side and fluxed out across basolateral membrane, directly demonstrating the transcytosis mechanism from ‘in’ to ‘out’. Figure 5f to 5h illustrates the quantitative results of nanogranule transcytosis in three monolayer models. The trans-cellular and cellular uptake amounts were simultaneously measured by ICP-MS. In bipolar monolayer model, Tf modification resulted in the increase of transcytosis near 4.5 folds that of BSA-NG (Figure 5f). The similar promotion was also found in the inverted bipolar monolayer model (2.3 folds, Figure S17b and S19). However, no significant difference between Tf-NG and BSA-NG was detected in the transcytosis through unipolar monolayer, no matter in 19


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A-B or B-A pattern (Figure 5g and 5h). Besides, the monolayer uptakes of Tf-NG in three models were also different. As shown in Figure 5f, during the transport across bipolar monolayer, no dissimilarity in cellular uptakes was found between Tf-NG and BSA-NG. But in unipolar monolayers, Tf-NG manifested more cellular uptake than BSA-NG in A-B model (Figure 5g) and less internalization in B-A model (Figure 5h). When adding the PEG-NG as another reference for transcytosis comparison, Tf-NG also showed the highest trans-cellular efficiency in bipolar model, but had no significant difference from the control in unipolar cell monolayer (Figure S20). These findings demonstrated that the transport of the functionalized nanogranules across epitheliums was polarity-associated. Next, the data from transcytosis and endocytosis investigations were combined to clarify the detailed regulation mechanisms of polar TfR in different cell models. First, the endocytosis analysis of Tf-NG in bipolar cells manifested more cellular uptakes and RE locations than BSA-NG (Figure 5i). Nevertheless, during the transcytosis in Caco-2 cells, the monolayer uptakes of two nanogranules showed no difference. While, more Tf-NG were found to transport cross bipolar monolayer to basolateral side. So, it seemed that the cellular uptake promoted by Tf-modification was offset by the increased transcytosis. In other words, Tf-modification caused more nanogranules flowed out of cell monolayer during the trans-cellular process. Besides, the RE was the key site that regulated the intracellular interaction of Tf-NG with TfR. As shown in Figure 5i, most Tf-NG were internalized by cells via specific Tf-TfR 20


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interaction and located in RE. Owing to the TfR expression in two sides, intracellular Tf-NG could transport along with the track of TfR, in which nanogranules migrated from the apical RE (ARE) to basolateral RE (BRE) and finally out of cells.6,13 In short, Tf-modification induced a complete transport flow from ‘in’ to ‘out’ for nanogranules through bipolar cell monolayer. Finally, the cellular and trans-cellular transports of Tf-NG in unipolar cell model also exhibited obvious dissimilarities (Figure 5j). Tf-NG showed more cellular uptakes than BSA-NG but no difference in transcytosis. Namely, the increased endocytosis of nanogranules did not contribute to their transcytosis. Distinct from the situation in bipolar cells, the enhanced cellular uptake in unipolar cell was closely related to the MVB/LE location of Tf-NG (Figure 5j and 5k). As the key site of degradation pathway, MVB/LE is difficult to link up with the exocytosis of nanogranules.47 It revealed that the role of Tf-modification in transcytosis was cut off in MVB/LE, thus losing the capacity for establishing the full transport flow. The cellular transport experiment in the inverted cell monolayer (B-A model) also showed the unavailability of Tf-modification for the transcytosis, although Tf-NG could bind with the surface TfR in this model. It suggested that the bipolar feature of receptors was the prerequisite for improving the transcytosis efficiency of active targeting nanogranules. Via the transport flow along the intracellular track of TfR, more active targeting nanogranules could break through the epithelial barrier via the ligand-receptor interaction.

21


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Interestingly, the transwell porous membrane cell monolayer may not be a perfect model though it is used for years. The nanoparticles during transcytosis from apical to basolateral side, may get entrapped between cell monolayer and the transwell membrane due to the limited pores on the membrane. From there, they may enter cells, transport inside cells, or traffic to the apical and basolateral side again. Besides, the 12 h observation here is only for the mechanism study at the cell level, such as the transport equilibrium, cellular response and feedback to the nanoparticle trafficking, and it might not be a direction reflection of physiological situation. Next, the in vivo tests were performed. We firstly explored the stability of two NG groups in the gastrointestinal condition. Here, we regulated the pH values in medium to simulate the different gastrointestinal environments for the stability investigation. As illustrated in Figure S21, Tf-NG and BSA-NG were stable and dispersed well in different pH environment (pH 6.0, pH 6.8 and pH 8.0) up to 8 h. Although, both NG groups tended to aggregate in pH 1.2, but the neutralization of pH enable them to re-disperse in medium. Totally, the stability of Tf-NG and BSA-NG in the gastrointestinal condition was comparable. Then, by orally administrating rats with different nanogranules, Tf-NG exhibited the improved absorption capacity almost 1.5 folds that of BSA-NG based on their area under the curve (AUC) (Figure S22).8,48 Notably, based on the conversion of particle number (Table S4), near 14.93 million particles might enter blood 12 h after oral administration of Tf-NG, further confirming the high efficiency and advantage of active targeting nanogranules. 22


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In addition to the PK study, we further performed several animal investigations to highlight our finding. As illustrated in the CLSM images (Figure S23 to S25), more laser reflection signals (red fluorescence) were detected in duodenum, jejunum and ileum after the Tf-NG incubation. Additionally, the Tf-modification triggered more NG transport across epithelium to the basolateral cells, even into the central lacteal (arrows, Figure S23 to S25). Besides, the ultrathin sections were used to detect the transcytosis feature of NG under TEM. Compared to BSA-NG, more Tf-NG passed through the intervals of microvilli and entered epithelial cells (red boxes, Figure S26 to S28). This enhanced uptake was more obvious in jejunum and ileum. Additionally, more Tf-NG transferred through basolateral membrane into lacteal. Generally, all tests demonstrated that Tf-NG could facilitate the intestinal transport of NG in animal level, which was in accordance with the cellular studies. Tf-conjugation induced greater transport-related cell responses in both models In order to comprehensively evaluate the cell responses caused by the continuous endocytosis of functionalized nanogranules in molecular level, the cellular expression profiles after cellular uptake were established based on the label-free quantification (LFQ) proteomics detected by LC-MS/MS.49–51 More than 2000 proteins were identified (2044 for bipolar cells and 2131 for unipolar cells) (Table S5 and S6). Two independent measurements were performed for each sample. As the result, 481 proteins were simultaneously identified after Tf-NG incubation with Caco-2 cells, and 23


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1336 proteins were found in MDCK cells (Figure S29).The correlation analysis among the shared proteins in duplicated tests showed coefficients all exceed 0.8 (Figure S29), indicating the feasibility and reproduction of detection. By comparing the LFQ intensities of identified proteins between nanogranules and control group, the proteins with ratios beyond 1.4 (test/control) were regarded as up-regulation and were selected for the subsequent studies. Figure 6a and 6b showed the Venn diagrams of up-regulated proteins induced by Tf-NG and BSA-NG in bipolar and unipolar cells, respectively. Notably, the number of the shared proteins in Caco-2 cells was lower than that in MDCK cells (44 in Figure 6a vs 89 in Figure 6b). However, the proteins that specifically induced by Tf-NG obviously increased in bipolar cells (474 in Figure 6a vs 180 in Figure 6b). The proportion of shared and specific proteins in each group further verified the discrepancies between Tf-NG and BSA-NG in two models (Figure 6c). It was clear that, compared to the non-specific cellular interaction of BSA-NG, Tf-NG induced the up-regulation of more specific proteins in two cells, especially in bipolar cell model, further revealing the influence of Tf-modification on the endocytosis of nanogranules. The up-regulated proteins in both models were further classified and analyzed via gene ontology (GO) strategy based on molecular function (MF), cellular component (CC) and biological process (BP).16,26 In most specific categories, Tf-NG exhibited similarities with BSA-NG group (Figure S30 and S31). However, 24


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Tf-modification caused the expressions of receptor and location-related proteins with different degrees (Figure 6d). More proteins were involved in receptor activity (GO: 0004872) and localization (GO: 0051179) classes during the trafficking of Tf-NG in bipolar cells. GO analysis also exhibited the differences in endocytosis pathways of Tf-NG between Caco-2 and MDCK cells. As shown in Figure 6e, when Tf-NG were internalized by unipolar cells, nearly half of proteins were identified for catalytic activity (GO: 0003824), while this value obviously reduced in bipolar model. It was further illustrated that hydrolases (GO: 0016787), which mainly located in lysosomes,52 predominated in the subclasses of catalytic activity. So these studies manifested that, in unipolar cells, more Tf-NG transported through degradation (BSE/LE) pathway, which was consistent with the co-localization observation. Overrepresentation test (ORT) was performed to clarify the functional differences of proteins induced by Tf-NG and BSA-NG in two cell models (Figure S32 to Figure S35). The lower p-value represented greater enrichment for the identified protein function.53,54 As shown in Figure 6f and 6g, in bipolar cells, Tf-NG exhibited significant enrichments of proteins in transport (GO: 0006810), localization (GO: 0051179), organization (GO: 0006996, GO: 0071822) and receptor activity classes (GO: 0038023, GO: 0099600). But these category of proteins were not found during the endocytosis of BSA-NG. So, it was confirmed that, during the endocytosis of nanogranules, Tf-modification up-regulated specific proteins which were closely associated with their cell uptake. Additionally, Tf-NG had lower p-values than 25


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BSA-NG in CC classes, especially in vesicle (GO: 0031982, GO: 0070062) and organelle (GO: 0043226, GO: 0044422). It manifested that cells could respond to the endocytosis by strengthening the vesicle-related transport process. The heat map in Figure 6h directly illustrated the overall up-regulations of vesicle-related proteins after Tf-NG internalization, including cytoskeleton and involved organelles, further demonstrating the promotion of Tf-modification to the vesicular trafficking. So it could be reasonably predicted that this promotion would feedback to the nanogranules transport.55,56 Tf-modification

triggered

positive

cellular

feedback

to

nanogranules

transcytosis through bipolar monolayer Same as the cell response study on endocytosis, the LFQ proteomics strategy was further utilized to measure the protein expression profiles in transcytosis of nanogranules. In order to establish the relevance of cellular response between endocytosis and transcytosis, we used the same incubation time in transcytosis as in endocytosis test (24 h). As a result, Tf-NG showed higher transcytosis efficiency in Caco-2 cells, but more retention in MDCK cells (Figure S36). In terms of the cellular response, near 2000 proteins were identified in two models after the trans-cellular process of nanogranules (1458 for bipolar and 1958 for unipolar models, Table S7 and S8). The Venn diagrams of up-regulatory proteins in Figure 7a and 7b illustrated the specific effects of Tf-modification on the transcytosis of nanogranules (279 for 26


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Tf-NG vs 45 for BSA-NG in Caco-2 cell monolayer). The GO analysis manifested the similar results with endocytosis study in functional proportion detections (Figure 7c and 7d, Figure S37 and S38). Differently, it was shown in ORT analyses that Tf-modification played diverse roles in the cellular responses to transcytosis in two models (Figure S39 to Figure S42). When nanogranules were transported through bipolar monolayer, Tf-modification caused obvious up-regulations of proteins in multiple categories, including vesicle (GO: 0031982), binding (GO: 0005488) and organelle (GO: 0043226). In contrast, these up-regulatory effects were attenuated in the unipolar model compared to BSA-NG group. As shown in Figure 7e, the transcytosis of Tf-NG through bipolar monolayer increased the protein expressions in cytoskeleton, organelle and protein transport classes, all of which were related to the vesicle-mediated trafficking.57 However, the inducibility of Tf-NG to these proteins was significantly reduced in the transcytosis across unipolar monolayer. As shown in supporting information, the western blotting analysis demonstrated that the incubation of Tf-NG obviously up-regulated the expression of clathrin in bipolar cells, but had no effect in unipolar model (Figure S43). It revealed that the bipolar cells responded to the transcytosis of nanogranules by accelerating the vesicle trafficking, but unipolar cells did not. In conclusion, the cell response to the transcytosis of active targeting nanogranules was polarity-associated. Besides, by comparing the cellular responses between endocytosis and transcytosis for Tf-NG group, it was found that their protein expressions were different, 27


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even though they belonged to the same functional category. As illustrated in Figure 7f, part of proteins shared in both cellular responses, and others functioned exclusively for endocytosis or transcytosis of Tf-NG. So, it seems that cellular regulatory mechanisms are different between the two transport processes of active targeting nanomedicines.51 Although the cell responses to transcytosis were clarified, we still did not know if they could feedback to the nanogranules transport. To address that, we designed a nanogranules re-incubation experiment as shown in Figure 7g. After the transcytosis of Tf-NG or BSA-NG, two cell monolayer models were re-incubated with a type of fluorescent nanogranules (Coumarin 6 loaded micelle, C6-M). The feedback effect was evaluated by detecting the trans-cellular amount of C6-M in the basolateral side. Interestingly, the pre-incubation of Tf-NG significantly increased the transcytosis of C6-M compared to the BSA-NG group in bipolar model (Figure 7h). Nevertheless, no difference between Tf-NG and BSA-NG pre-incubations was detected during the tran-cellular process of C6-M across unipolar monolayer (Figure 7i). These findings directly indicated that Tf-modification increased the nanogranules transcytosis by triggering a positive cellular feedback loop, in which the up-regulated proteins further promoted and accelerated the vesicle-related trafficking, thus continuously improved the transcytosis efficiency of the later administered nanogranules.58 Importantly, this positive feedback was also polarity-associated.

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CONCLUSION In this study, we found a closed-loop regulatory pathway which uncovered the trancytosis mechanism of active targeting nanomedicines through epithelium. As shown in Figure 8a, Tf modified nanogranules entered epithelial cells through endocytosis based on the surface binding with TfR in bipolar cells. By means of the intracellular Tf-TfR interaction, nanogranules transported from EE to RE, and finally out of cell monolayer from the basolateral side, thus establishing a complete transport flow. In unipolar cells, Tf-NG were internalized into cells and then transported from EE to MVB/LE, while stagnating there due to the lack of an incomplete transport flow. In addition, the transportation also affected the cells by altering the expression profile. To respond and adapt to the nanogranules transport, cell themselves increased the expressions of vesicle-related proteins, which in turn promoted the subsequent transcytosis in bipolar cell monolayers (Figure 8b). This closed-loop regulatory mechanism clarified the effect of ligand modification on the transcytosis of active targeting nanomedicines. Notably, polarity was the key factor during the whole transportation. Only bipolar epithelial cells could construct a complete transport flow and induce the positive feedback. By the way, it was interesting to find that ligand modification was more significant than the composition of core material in their cell transport, because the NG with micelle core exhibited very similar features with the gold nanogranules (Figure S44 to S46). Totally, this study provided a preliminary foundation for the nanomedicine design aimed at efficient trans-cellular delivery. 29


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Meanwhile, unipolar epithelial cells were found not be able to promote the transcytosis for active targeting nanomedicines cross epithelial barrier, due to the break-off in cell transport flow and the failure in triggering positive cell feedback. However, it was clear that the surface binding of active targeting nanomedicines with receptors was not an indispensable factor for the epithelial uptake. The contralateral expression of receptors could also increase the uptakes on account of the retention effect caused by intracellular ligand-receptor interaction. We believed that this might offer a strategy to the targeting nano-therapy for the epithelioid diseases.

MATERIALS AND METHODS Materials and cells. Chloroauric acid (HAuCl4·3H2O) was obtained from Sahn chemical technology (Shanghai, China). Sodium citrate was purchased from National medicine group chemical reagent (Beijing, China). Carboxyl or methoxyl modified thiolation polyethylene glycol (COOH-PEG-SH, mPEG-SH, Creative PEGWorks, Mw = 550 Da) were supplied by ZZBIO (Shanghai, China). Holo-Transferrin, filipin, 5-(N-ethyl-N-isopropyl)-amiloride

(EIPA),

chlorpromazine

(CPZ),

nocodazole,

cytochalasin D and N-hydroxysuccinimide (NHS) was got from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA), hoechst 33258, Cell Counting Kit-8 (CCK-8), brefeldin A, monensin, bafilomycin A, blebbistatin, oligonmycin A and Golgi-tracker were obtained from Beyotime (Haimen, China). Phosphate buffer 30


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solution (PBS), 4% paraformaldehyde, Triton X-100 and all chemical reagents for SDS-PAGE were purchased from Solarbio (Beijing, China). Rabbit anti-TfR (ab84036), anti-occludin (ab216327), anti-Rab5 (ab13253), anti-Rab7 (ab187868), anti-Rab11 (ab3612), anti-LAMP1 (ab24170), Goat anti-Rabbit IgG H&L (Alexa Fluor® 488, ab150077), Goat anti-mouse IgG H&L (Alexa Fluor® 647, ab150115) and Donkey anti-Rabbit IgG H&L (Alexa Fluor® 647, ab150075) were obtained from Abcam (Cambridge, MA, USA). Mouse anti-TfR (136800) were purchased from Invitrogen. Rhodamine-phalloidine was purchased from YEASEN (Shanghai, China). 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) were purchased from Thermo Fisher Scientific (New York, USA).

ER-tracker were supplied from

Invitrogen (Carlsbad, CA, USA). All other chemical reagents were analytical grade. Caco-2, MDCK, HT-29 and HUVEC cell lines were obtained from National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China). Fetal bovine serum (FBS) was got from Gibco (Grand Island, NY, USA). DMEM (4.5 g/L glucose), DMEM-F12, F12K, penicillin-streptomycin, trypsin containing 0.02% EDTA, heparin sodium salt, endothelial cell growth supplement and sodium pyruvate were all supplied by Macgene Biotechnology Ltd. (Beijing, China). MEM cell culture medium was purchase from Corning (Cambridge, MA, USA). Preparations of Tf and BSA modified gold nanogranules (Tf-NG, BSA-NG). Original gold nanogranules were synthesized by the citrate reduction method. Briefly, 31


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100 mL HAuCl4 solution (2 mL, 50 mM) was heated to reflux with stirring and 2 mL (150 mM) sodium citrate was added rapidly. When the red vine color was observed, the solution continued to keep boiling for another 20 min and then left to stir until return to room temperature. In order to obtain stable and functional gold nanogranules, mixed thiolated short chain PEG (Mw= 550 Da, COOH-PEG-SH: mPEG-SH = 4:1) were applied with a molar ratio of 50:1(Au: PEG) and reacted 8 h at room temperature. After purification by high-speed centrifugation and resuspended into MES (0.1 mol/L), 100 mL PEGylated gold nanogranules were activated with 20 mg NHS and 15 mg EDC for 20 min, Then PBS was used to replace MES. 12.5 mg Tf or BSA were added to the activated gold nanogranules and keep sufficient reaction for 12 h. The desired nanogranules were collected with centrifuging at 13000 rpm for 20 min of three times to remove the excess protein. Characterizations of the Tf-NG and BSA-NG. Transmission electron microscopy (TEM, JEM-1200EX, JEOL) and dynamic light scattering (DLS, Malvern, Zetasizer Nano ZS) were applied in measuring the morphology, size and zeta-potentials of nanogranules. The stability of gold nanogranules in the different dispersed medium was validated by TEM, DLS and UV-vis spectrophotometry as our previous work.21 Besides, Tf/BSA-modified nanogranules were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad, Hercules, CA, USA) as the published literature.26

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Tf Receptor expression comparison between two cell models. MDCK and Caco-2 monolayers were selected as models and cultured for the transferrin receptor (TfR) expression study. MDCK cells were cultured on a polycarbonate membrane (Corning, transwell, 12 well, 0.4 μm) and seeded at 1×105/well. Then, 0.5 mL or 1.5 mL DMEM was respectively added into the upper and basolateral side and the medium was changed every two days. The cells were allowed to grow for 4-5 days until transepithelial electric resistance (TEER) reached 200 Ω/cm2. Caco-2 cells were seeded at 2×105/well on the transwell polycarbonate membrane (Corning, transwell, 12 well, 3 μm) and continued to culture for three weeks (medium were changed every two days during the first two weeks and every day for the last week ) until TEER was higher than 500 Ω/cm2. When cell monolayers were ready, the polycarbonate membrane covered with cells were cut along the edge carefully. Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After being treated with the blocking solution (PBS containing 0.1% Triton X-100 and 5% BSA) for 1 h at 37 °C, cells were incubated with anti-TfR (1:200) and anti-occludin (1:200) for 16 h at 4 °C (PBS was taken as the negative control) and then with secondary antibodies (1:300) for 2 h at 37 °C. Finally, Hoechst 33258 was added and then incubated for 20 min at room temperature to label the cell nuclei. Samples were observed by CLSM (LEICA TCS SP8, Germany). The western blot (WB) technology was also used to compare the TfR expressions in two cells. The cells were firstly lysed by RIPA (Beyotime, Haimen, 33


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China)). After mild shaking at 4 °C for 30 min, the obtained lysis were centrifuged (13000 rpm, 20 min, 4 °C). 15 μg supernatant protein were collected and loaded in 4%-20% gradient precast gels for SDS-PAGE (120 mV, 60 min). And then protein were transferred into the polyviinglidene difluoride (PVDF, Merckmillipore, Canada) membrane (200 mA, 60 min，4 °C) and incubated with anti-TfR (1:1000, 16 h, 4 °C), and HRP-labeled Goat anti-rabbit IgG (H+L) (Beyotime, Haimen, China) (1:1000, 1 h, 37 °C) in sequence. The results were detected after adding the developer (Beyotime, Haimen, China) at last and detected by Gel Illuminator (ChemiDoc XRS, Bio-Rad, Hercules, CA, USA). The establishments of polar cell models. MDCK cell line was grown in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at a humidified atmosphere containing 5% CO2 at 37 °C. Caco-2 cell line was cultured in DMEM media supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% sodium pyruvate and 1% non-essential amino-acid at a humidified atmosphere containing 5% CO2 at 37 °C. Both cell lines were detached from the culture flask with 0.25% trypsin/0.02% EDTA. For the cellular uptakes and endocytosis investigations, both cells were cultured in 12-well plate at 1×105/well or 6-well plate at 2×105/well. After the cell culture for at least two days, the cells with the convergence rate up to 90% were utilized for subsequent experiments. In order to construct the cell monolayers for the transcytosis experiments, MDCK 34


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cells and Caco-2 cells were cultured on a polycarbonate membrane (Corning, transwell, 12 well, 3 μm) as described above for the A-B models. To establish the reversed cell monolayer (B-A) models based on the polar distribution of transferrin receptors. MDCK and Caco-2 cells were selected to develop an inverted-transwell monolayer. Specifically, the inserted wells of 12-well (transwell, 3 μm) were placed upside down atop a special glass-sterile device (as Figure S1), medium was added till just reaching the polycarbonate membrane and cells were seed at 2×105/well (MDCK cells) or 4×105/well (Caco-2 cells). After 10 min standing to ensure cells sediment to the polycarbonate membrane, medium was added tenderly to just submerge the cells and placed the covers, and then the whole devices were moved to the incubator (5% CO2 at 37 °C) carefully for completed adherence of cells to the membrane. 12 h later, the inserted wells were turned back and placed in a 12-well plate for further growth. The MDCK inverted monolayer could be used when TEER reached 200 Ω /cm2 and Caco-2 cells exceeded 500 Ω /cm2. Cellular uptake investigations of Tf-NG and BSA-NG in two cell models. Before cellular uptake study, the cell viability of nanogranules was firstly detected. CCK-8 assay was used to assess the effect of gold nanogranules on cell viability. Both cell lines were seeded in 96-well-plate and when cells had a 90% convergence rate, nanogranules with the different concentration (125, 250, 500 and 1000 μg/mL) were added to cells and incubated for 24 h. Gold-nanogranules were removed and cells were treated with 100 μL CCK-8 working solution for 2 h at 37 °C and the absorption 35


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was measured at the wavelength of 450 nm via Multiskan FC (Thermo Scientific, USA). The cell uptakes of Tf-NG and BSA-NG in MDCK and Caco-2 cells were investigated by CLSM methods. After cell culture with the confluency up to 90%, 500 μg/mL of nanogranules were added and incubated for 12 h. The cells were washed with cold PBS to terminate cell uptake. The samples were stained with Rhodamine-phalloidine (diluted with the blocking solution as receptor expression study, 30 min at 37 °C) and Hoechst 33258 (20 min at room temperature), respectively. Then the labeled cells were washed, sealed and finally detected by CLSM (LEICA TCS SP8, Germany). To quantify the endocytosis amount of nanogranules, cells (Caco-2, MDCK, HT-29 and HUVECs cells) were first seeded in a 12-well plate. After being incubated with 500 ug/ mL nanogranules for 12 h, cells were washed with cold PBS and digested for obtaining the cell suspension. The cell suspension was centrifuged at 1000 rpm and the substratum cells were collected after washed with PBS three times to remove the nanogranules attached to cell surface and cell junction. In a test of evaluating surface adhesion of nanogranules, the collected cells were washed 1, 3 or 5 times with PBS or a pH3 buffer for 10s followed 3 times with PBS. Then 100 μL RIPA lysis buffer and chloroazotic acid (HCl: HNO3=3:1) were added separately to break the cells and dissolve gold for ICP-MS detection (Elan DRC, Perkin-Elmer 36


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Sciex, USA). For TEM analysis, cells were seeded in 6-well plate as described above. Cells were collected after incubated with nanogranules via digestion and centrifugation at the speed of 1000 rpm. Then cells were fixed by glutaraldehyde for 12 h at 4 °C and then osmification, dehydration, embedding and sectioning in turn. Finally, the ready samples were observed by TEM. Trans-cellular investigations of Tf-NG and BSA-NG across two cell monolayer models. The integrities of tight junction of cell monolayers treated with nanogranules were verified by FITC-Dextran (FD4) permeation method. In brief, 500 μL FD4 solution (50 μg/mL) and 1.5 mL fresh medium were added to the upper and basolateral side, respectively. At determined time points, 100 μL medium was collected from the basolateral chamber and an equal volume of fresh medium without FD4 was added simultaneously. Then the samples as well as a serious of standard solution were measured (EM/EX = 490/515 nm) via FlexStation 3 multifunctional microplate system (Molecular Devices, USA). The apparent permeability coefficient (Papp) was calculated as the following equation: ∆𝑄 1 P𝑎𝑝𝑝 = ∆𝑡 × 𝐴𝐶 0 Where Q is the amount of FD4 collected from basolateral side, t is the time in the experiment, A is the diffusion area (1.12 cm2 for 12-well transwell in the study) and C0 37


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is the initial concentration of FD4 added to the upper side. As for the observation of tight junction in cell monolayer, the polycarbonate membrane covered with cells were cut along the edge carefully and fixed by glutaraldehyde for 12 h at 4 °C after incubated with nanogranules. Then the samples were treated according to the protocol as the cells detected by TEM method as described above. The A-B and B-A cell monolayer models were firstly cultured in transwell membrane. When the TEER value met the demand, 500 μg/mL nanogranules and 1.5 mL fresh medium were added to the apical side and the basolateral chambers sequentially. After 12 h or 24 h incubation, all the basolateral media were corrected and centrifuged at the speed of 13000 rpm for 30 min to collect the nanogranules. Cells of monolayers were treated as the samples of cell uptake. Then the gold nanogranules collected from basolateral chambers and cells were destroyed by chloroazotic acid and detected by ICP-MS. The endocytosis mechanism investigations of Tf-NG and BSA-NG in two cell models. To evaluate the energy dependency of nanogranules transport, the 80% confluent cells were pre-incubated at 37 °C or 4 °C for 30 min, respectively. After nanogranules were added into corresponding wells, the plates continued to incubate at 37 °C or 4 °C for another 12 h. And then the cells were further treated for ICP-MS detection. 38


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The endocytosis mechanism was investigated via pharmacological inhibition strategy. After cells were incubated with various inhibitors (listed in Table 1) for 30 min, the medium was removed and 500 μg/mL nanogranules solution (containing the same concentration of inhibitors) was added for another 12 h incubation. Then the endocytosis amount of Au was determined by ICP-MS. Table 1.The types and concentrations of inhibitors used in the research Inhibitors

Concentration

Inhibitors

Concentration

Holo-transferrin

100 μg/mL

EIPA

40 μM

Chlorpromazine

30 μM

Nocodazole

10 μg/mL

Filipin

0.5 μg/mL

Cytochalasin D

0.5 μM

Bafilomycin A

100 nM

Oligonmycin A

2 μM

Blebbistatin

1 μM

Apical exocytosis detections of Tf-NG and BSA-NG in two cell models. As shown in Figure S2, MDCK and Caco-2 cells were cultured in 12-well plate incubated with 500 μg/mL nanogranules at 37 °C for 12 h. Then the cells of control group were washed three times with PBS and collected cells for intracellular Au detection (as endocytosis control, apical exocytosis for 0 h). The cells of test groups were washed three times with PBS and continued to incubate with fresh media subsequently. After determined time points (apical exocytosis for 2-12 h), the media was removed and intracellular Au was measured by ICP-MS as described before. The retention ratios in 39


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cells were calculated as the following equation:

Retention ratio =

Where m

test

𝑚𝑡𝑒𝑠𝑡 𝑚𝑐𝑜𝑛𝑡𝑟𝑜𝑙

× 100%

is the Au mass of apical exocytosis for 0 h, and m

control

is the Au

mass of apical exocytosis for 2-12 h. As for the inhibitor investigation, endocytosis control (apical exocytosis for 0 h) and apical exocytosis (12 h) were determined as above; For inhibitor groups, brefeldin A (25 μg/mL) and monensin (2.5 μM) were added into the medium contain nanogranules, respectively, to incubate with cells for 12 h，and the cells of control group were collected for intracellular Au detection (as apical inhibitor exocytosis for 0 h). After removing the nanogranules, the cells of test groups were continued to incubate with fresh media containing the same concentration of inhibitors for another 12 h. Then the cells were collected and the intracellular Au was detected (as apical inhibitor exocytosis for 12 h). Then the retention ratios of control and inhibitor groups were calculated. Intracellular colocalization and transportation analyses of Tf-NG and BSA-NG in two cell models. After incubated with 500 μg/mL nanogranules for 12 h, both cells were treated with PBS three times and then stained with cell trackers (ER-tracker, Golgi-tracker) as the protocol from the manufacturer, respectively. Then the cells were observed by CLSM immediately and the data were analyzed by Image-pro Plus. 40


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For the measurement of nanogranules location in endosomes, two cell models were firstly incubated with 500 μg/mL nanogranules for 12 h and then fixed with 4% paraformaldehyde and blocked with BSA solution. After co-incubated with a determined

concentration

antibody

(anti-Rab5

(1:200),

anti-Rab7

(1:200),

anti-Rab11 (1:100) and anti-LAMP1 (1:300)) as the protocol from the manufacturer for 16 h at 4 °C, a Donkey anti-Rabbit IgG H&L was used to line out the first-antibody. With further visualizing nuclei by Hoechst 33258, the stained cells were then investigated by CLSM and the data based on fluorescence and laser scattering intensities were analyzed by Image-pro Plus. Pharmacokinetic study and intestinal sections analysis. Male Wistar rats (250g) were divided into two groups (Tf-NG and BSA-NG, n=5) and fasted overnight but allowed access to water ad libitum before the experiments. Nanogranules (10 mg/kg) were intragastrically administered and blood samples (0.4 mL) were collected from the fossa orbitalis at 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 h. The blood Au level was detected by ICP-MS. Three male Wistar rats were anesthetized and cut along the midline. Duodenum, jejunum and ileum were ligated with cotton thread and injected into Tf-NG and BSA-NG (2 mg), control group was treated with water. After 6 h incubation, almost 1 cm segments of duodenum, jejunum and ileum from three rats were excised from intestine, respectively. Frozen sections of each segment were prepared and detected by CLSM after staining the nucleus. Besides, the treated intestine samples were also prepared as sections for TEM observation after 41


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osmification, dehydration, embedding and sectioning in turn. Proteomics analysis. Cells or cell monolayers were cultured as described above. After incubated with 500 μg/mL nanogranules for 24 h at 37 °C (the control group was treated with blank culture medium), cells or cell transwell monolayers were washed with PBS and then lysed, the cell lysis were collected and centrifuged (13000 rpm, 30 min), the supernatant protein solution were quantified. For the surface absorbed protein analysis, nanogranules (PEG-NG, BSA-NG and Tf-NG, 1 mg/mL) were incubated with blank cell lysate for 6 h. After incubation, nanogranules were centrifuged at 13,000 rpm for 30 min (three times) to remove the excess protein. Then the protein adsorbed onto the surface were eluted ad collected. The same amount of protein (10 μg) for each sample were loaded in the tube gels as protocols (Filter Assisted Sample Preparation, Mann). Then, proteins were incubated with enzyme (protein: enzyme= 50:1) overnight at 37 °C and the obtained peptides were collected and lyophilized for detection based on the nano-LC-MS/MS system (Thermo Scientific, MA, USA) and recorded data at last. During the LC-MS/MS investigation based on label-free strategy, the obtained peptides were loaded on a C18 pre-column (Thermo Scientific, MA, USA) and separated by nano-LC-MS/MS using an Easy-LC nano-HPLC (Thermo Scientific, MA, USA). In the LC separation, H2O/TFA was mobile phase A and ACN/TFA was phase B. The flow rate was adjusted to 300 nL/min, and the gradient 42


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was set as from 5% to 30% for 90 min. Mass spectrometric (MS) analysis was performed by using an LTQ Orbitrap Velos pro (Thermo Scientific, MA, USA). MS/MS spectra were obtained in a data-dependent collision induced dissociation (CID) mode, and the full MS was acquired from m/z 350 to 2000 with resolution 60, 000. The top 15 most intense ions were selected to for MS/MS. Raw data were analyzed using MaxQuant. MS/MS spectra were searched against UniProt database. 10 ppm error tolerance in MS and 0.6 Da error tolerance were set in MS/MS. False discovery rates were obtained using percolator selecting identification with a q-value equal or less than 0.01. The data retrieval were carried out via Homo sapiens for Caco-2 cells and Canis lupus families for MDCK cells. The correlation analyses between two independent investigations

were

performed

by

Perseus

software

(http://www.perseusframework.org). By means of the LFQ proteomics quantification, the functional classification and statistical overrepresentation test of abundant proteins

based

on

gene

ontology

(GO)

were

analyzed

via

PANTHER

(http://www.pantherdb.org/). The information of proteins, including molecular function, intracellular location, biological process, signal pathway etc. were carried out by using UniProt database (https://www.uniprot.org/). Feedback pathway validation. Coumarin 6 micelles (C6-M) were selected as a nanoparticle model in the feedback experiment and prepared according to the 43


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following procedures: 10 mg DSPE-PEG (NOF Corporation, Tokyo, Japan) and 10 μg coumarin 6 (Eugene, Oregon, USA) were dissolved in methanol in a pear-shaped flask and rotated evaporation into a film at 60 °C for 40 min. Then 2 mL PBS (60 °C) were added to film and hydrated via vortex. After sonicated for 10 min, coumarin 6 loaded micelles (C6-M) were obtained. After PEG-PCL micelles (carboxy-) were prepared as C6-M, micelles were further activated with 4 mg NHS and 2 mg EDC for 20 min, then pH was adjusted to 8 and 2 mg Tf/BSA were added to react for 4 h. excess protein were removed via ultrafiltration. Then the Tf/BSA modified PEG-PCL micelles (Tf-M and BSA-M) were obtained. Caco-2 and MDCK cell transwell monolayers were cultured as described above. After removing the culture medium, cells were wash by PBS, 0.5 mL five different solutions (PBS, 20 μg/mL free BSA, 20 μg/mL free Tf, 500 μg/mL BSA-NG, 500 μg/mL Tf-NG) and 1.5 mL fresh medium (serum-free) were added into upper or base chambers, respectively. All groups were incubated for 12 h at 37 °C and then terminated by cold PBS. Cells were washed three times and the corresponding amount of full medium was added for recovery for 1 h at 37 °C. Then, 0.5 mL PBS (for control group) or 300 μg/mL C6-M (for four test groups) were added and incubated for 4 h at 37 °C. Finally, the 1.5 mL base medium were collected to detected the coumarin content by HPLC (Detection wavelength, EX/EM: 467/502 nm; Column, ZORBAX Eclipse XDB-C18 (4.6*250 mm, 5 μm); Mobile phase, methanol/water: 95:5; Flow rate, 1 mL/min). 44


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Statistical analysis. All results shown as mean ± standard error (SE) were obtained at least three times independent data. A t-test or one-way analysis of variance (ANOVA) was applied and a p-value less than 0.05 were defined to be statistically significant.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supplementary Information is available: The process of construction of inverted-transwell monolayer based on MDCK and Caco-2 cells; The protocol of apical exocytosis; The preparations and characterizations of nanogranules; Cytotoxicity of nanogranules; Characterizations of nanogranules incubated with cells; The cell uptake and transcytosis results of PEG-NG; The cell uptake of nanogranules in HT-29 cells; The endocytosis studies based on some inhibitors; The evaluation of surface adhesion of nanogranules; The integrality verification of cell monolayers after incubating with nanogranules; The pharmacokinetic data; The relativity between two batches proteomics results; The detailed analysis of function classification based on GO and ORT of up-regulation proteins of cells incubated with nanogranules in Caco-2 and MDCK cells. Supplementary tables: all original proteomics data of the cells or cell

45


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monolayers treated with nanogranules in Caco-2 and MDCK cells.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] (Qiang Zhang) [email protected] (Xing Tang) [email protected] (Bing He) CONFLICTS OF INTEREST 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. H.L.D assisted with the WB experiment in the revision stage. J.Z. assisted with the proteomics analysis. M.M.Q assisted with the preparation of gold nanogranules. X.J.Z. assisted with proteomics detection. L.Y. assisted with the CLSM study. B.S., H.P.L, W.B.D., H.Z. and X.Q.W. assisted with data analysis. All authors discussed the results and commented on the manuscript. The authors disclose no conflicts. ACKNOWLEDGMENTS Supported by the National Basic Research Program of China (2015CB932100), National Key R&D Program of China (2017YFA0205603), the National Natural 46


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Science Foundation of China (81690264, 81703441, 81573359 and 81872809) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81821004).

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Fenton-Reaction-Acceleratable

Magnetic

Nanoparticles for Ferroptosis Therapy of Orthotopic Brain Tumors. ACS Nano 2018, 12, 11355–11365. (44) Gaudin, A.; Tagit, O.; Sobot, D.; Lepetre-mouelhi, S.; Mougin, J.; Martens, T. F.; Braeckmans, K.; Desmae, D.; Smedt, S. C. De; Hildebrandt, N.; Couvreur, P.; Andrieux, K. Transport Mechanisms of Squalenoyl-Adenosine Nanoparticles Across the Blood−Brain Barrier. Chem. Mater. 2015. 27, 3636-3647. (45) Pang, X.; Wang, L.; Kang, D.; Zhao, Y.; Wu, S.; Liu, A. L.; Du, G. H. Effects of P-Glycoprotein on the Transport of DL0410, a Potential Multifunctional Anti-Alzheimer Agent. Molecules 2017, 22, 1–12. (46) Beloqui, A.; Brayden, D. J.; Artursson, P.; Préat, V.; Des Rieux, A. A Human 54


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Intestinal

M-Cell-like

Model

for

Investigating

Particle,

Antigen

and

Microorganism Translocation. Nat. Protoc. 2017, 12, 1387–1399. (47) Sahay, G.; Querbes, W.; Alabi, C.; Eltoukhy, A.; Sarkar, S.; Zurenko, C.; Karagiannis, E.; Love, K.; Chen, D.; Zoncu, R.; Buganim,Y.; Schroeder, A.; Langer, R.; Anderson, D. Efficiency of siRNA Delivery by Lipid Nanoparticles Is Limited by Endocytic Recycling. Nat. Biotechnol. 2013, 31, 653–658. (48) Ruan, S.; Qin, L.; Xiao, W.; Hu, C.; Zhou, Y.; Wang, R.; Sun, X.; Yu W.; He, Q.; Gao, H. Acid-Responsive Transferrin Dissociation and GLUT Mediated Exocytosis for Increased Blood–Brain Barrier Transcytosis and Programmed Glioma Targeting Delivery. Adv. Funct. Mater. 2018, 28, 1–16. (49) Jin, L.; Huo, Y.; Zheng, Z.; Jiang, X.; Deng, H.; Chen, Y.; Lian, Q.; Ge, R.; Deng, H. Down-Regulation of Ras-Related Protein Rab 5C-Dependent Endocytosis and Glycolysis in Cisplatin-Resistant Ovarian Cancer Cell Lines. Mol. Cell. Proteomics 2014, 13, 3138–3151. (50) Li, Y.; Zhang, X.; Deng, C. Functionalized Magnetic Nanoparticles for Sample Preparation in Proteomics and Peptidomics Analysis. Chem. Soc. Rev. 2013, 42, 8517–8539. (51) Bersuker, K.; Peterson, C. W. H.; To, M.; Sahl, S. J.; Savikhin, V.; Grossman, E. A.; Nomura, D. K.; Olzmann, J. A. A Proximity Labeling Strategy Provides 55


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Insights into the Composition and Dynamics of Lipid Droplet Proteomes. Dev. Cell 2018, 44, 97–112. (52) Schröder, B. A.; Wrocklage, C.; Hasilik, A.; Saftig, P. The Proteome of Lysosomes. Proteomics 2010, 10, 4053–4076. (53) Raesch, S. S.; Tenzer, S.; Storck, W.; Rurainski, A.; Selzer, D.; Ruge, C. A.; Perez-Gil, J.; Schaefer, U. F.; Lehr, C. M. Proteomic and Lipidomic Analysis of Nanoparticle Corona upon Contact with Lung Surfactant Reveals Differences in Protein, but Not Lipid Composition. ACS Nano 2015, 9, 11872–11885. (54) Dang, V. D.; Jella, K. K.; Ragheb, R. R. T.; Denslow, N. D.; Alli, A. A. Lipidomic and Proteomic Analysis of Exosomes from Mouse Cortical Collecting Duct Cells. FASEB J. 2017, 31, 5399–5408. (55) Tharkeshwar, A. K.; Trekker, J.; Vermeire, W.; Pauwels, J.; Sannerud, R.; Priestman, D. A.; Te Vruchte, D.; Vints, K.; Baatsen, P.; Decuypere, J. P.; Lu, H.; Martin, S.; Vangheluwe, P.; Swinnen, J. S.; Lagae, L.; Impens, F.; Platt, F. M.; Gevaert, Kris.; Annaert, W. A Novel Approach to Analyze Lysosomal Dysfunctions through Subcellular Proteomics and Lipidomics: The Case of NPC1 Deficiency. Sci. Rep. 2017, 7, 1–20. (56) Debaisieux, S.; Encheva, V.; Chakravarty, P.; Snijders, A. P.; Schiavo, G. Analysis of Signaling Endosome Composition and Dynamics Using SILAC in 56


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Embryonic Stem Cell-Derived Neurons. Mol. Cell. Proteomics 2016, 15, 542– 557. (57) Lu, K.Y.; Tao, S.C.; Yang, T.C.; Ho, Y.H.; Lee, C.H.; Lin, C.C.; Juan, H.F.; Huang, H.C.; Yang, C.Y.; Chen, M.S.; Lin, Y.Y.; Lu, J. Y.; Zhu, Heng.; Chen, C. S. Profiling Lipid–protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Proteome Microarrays. Mol. Cell. Proteomics 2012, 11, 1177–1190. (58) Han, L.; Kong, D. K.; Zheng, M. Q.; Murikinati, S.; Ma, C.; Yuan, P.; Li, L.; Tian, D.; Cai, Q.; Ye, C.; Holden, D.; Park, J. H.; Gao, X.; Thomas, J. J.; Grutzendler, J.; Carson, R. E.; Huang, Y.; Piepmeier, J. M.; Zhou, J. Increased Nanoparticle Delivery to Brain Tumors by Autocatalytic Priming for Improved Treatment and Imaging. ACS Nano 2016, 10, 4209–4218.

FIGURE CAPTIONS Figure 1. TfR exhibited different distribution characteristics, but Tf-modification still promoted the cellular internalizations of nanogranules in both cell models. TfR expressions in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells were analyzed by CLSM. Cell nuclei and tight junctions were labeled with respective markers (Hoechst 33258 and anti-occludin IgG) to define the TfR distributions in two cell models. Red: transferrin receptor. White in the merge images of X-Z were the signals of transwell 57


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membrane (TM). It indicated that Caco-2 cells were bipolar TfR expression and MDCK cells were mainly in basolateral side. c. Western-blot analyses on the TfR expressions of two cell models. Grayscale measurement showed that TfR in Caco-2 cells was expressed almost 1.3 times than that in MDCK cells. Mean ± SE, n=4, *** p < 0.001. d. The schematic diagram showed the construction and analysis strategies for two TfR expression models. In order to eliminate the heterogeneity among cell types, the cellular transportations of nanogranules in two different cells were not directly compared. But the differences between nanogranule groups (Tf-NG/BSA-NG in both bipolar and unipolar cell models) were measured and set as core indicators. This normalization strategy could compare the effects of Tf-modification on the transportation of nanogranules in different cells. Cellular uptakes (500 μg/mL, 37 °C, 12 h) were detected by CLSM in (e) Bipolar (Caco-2) and (f) Unipolar (MDCK) cells. The histograms were the statistical results from corresponding CLSM images (Mean ± SE, n > 5, ** p < 0.01, *** p < 0.001). The white cycles indicated the signals of the intracellular nanogranules by LR technology. Quantitative detections of nanogranule internalization were performed by ICP-MS in (g) Bipolar (Caco-2) and (h) Unipolar (MDCK) cells, Mean ± SE, n=3, * p < 0.05, *** p < 0.001, the statistical differences were computed between Tf-NG and BSA-NG groups.

Figure 2. Tf-conjugation regulated the endocytosis of nanogranules in two models with different mechanisms. Energy dependency mechanisms of nanogranules in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells were evaluated through low 58


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temperature incubation. Different endocytosis inhibitors were coincubated with nanogranules in (c) bipolar (Caco-2) and (d) unipolar (MDCK) models to explore the trafficking pathways. e. TEM detection of Tf-NG in endosomes in two cell models. The vesicular distributions of nanogranules were clearly shown in TEM images. The competitive inhibition analyses by adding free Tf in (f) bipolar (Caco-2) and (g) unipolar (MDCK) models were performed to investigate the effect of Tf-TfR interaction on endocytosis. In (a,b), the intracellular amounts of nanogranules at 37 °C in two groups were normalized as 100% respectively and set as control. In (c,d) and (f,g), the cellular uptakes of nanogranules without inhibitor addition were normalized as 100% and set as control. The statistical differences were all compared between test groups and control sample. # p < 0.05, ## p < 0.005, ### p < 0.001. In (f,g), the statistical differences were further computed between Tf-NG and BSA-NG groups. *** p < 0.001. All data were presented as Mean ± SE, n=3.

Figure 3. Tf-modification altered the intracellular transport pathways (exocytosis and secretion) of nanoparticles in two models. Apical exocytosis analyses in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells were performed at different time points. The retentions of nanogranules in cells during exocytosis were set as indicators. The intracellular amounts of nanogranules at 0 h point in two groups were normalized as 100% respectively and set as control. The relative percentages of test samples were exhibited and the statistical differences were all compared with the control. Data were represented as Mean ± SE, n=3, # p < 0.05, ## p < 0.01. Co-localizations of 59


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nanogranules in bipolar (Caco-2) cells with (c, d) ER and (e, f) Golgi complex were analyzed by CLSM. The confocal images ((c) for ER, (e) for Golgi complex) were shown and the corresponding the co-localization M values ((d) for ER, (f) for Golgi complex) were computed here. Likewise, the locations of nanogranules in unipolar (MDCK) cells with (g, h) ER and (i, j) Golgi complex were detected here. Based on the confocal images ((g) for ER, (i) for Golgi complex), the M values in ER and Golgi complex were shown in (h) and (j) respectively. Scale bar (c, e, g, i): 10 μm. In (d, f, h, j), the statistical differences were compared between Tf-NG and BSA-NG groups. All data were presented as Mean ± SE, n=5. (k) Schematic diagrams showed no difference on the secretion pathway of Tf-NG between bipolar and unipolar cell models, indicating Tf-modification did not exhibit advantage for the ER/Golgi transporation of nanogranules compared to BSA-NG.

Figure 4. Tf-modification altered the intracellular transport pathways (transcytosis and degradation) of nanogranules in two models. Co-localizations of nanogranules in bipolar (Caco-2) cells with (a, b) EE, (e, f) LE, (i, j) RE and (m, n) lysosomes were analyzed by CLSM. The confocal images were shown and the corresponding co-localization M values ((b): EE, (f): LE, (j): RE, (n): lysosomes) were computed here. Similarly, the locations of nanogranules in unipolar (MDCK) cells with (c, d) EE, (g, h) LE, (k, l) RE and (o, p) lysosomes were detected here. Based on the confocal images, the M values in EE, LE, RE and lysosomes were separately shown in (d), (h), (l) and (p). Scale bar (all images): 10 μm. In terms of the M value computations, the 60


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statistical differences were compared between Tf-NG and BSA-NG groups. * p < 0.05, ** p < 0.01 *** p < 0.001. All data were presented as Mean ± SE, n=5. (q) Schematic diagrams showed that Tf-modification caused more nanogranules transport through degradation pathway in unipolar cells, but more through recycling route in bipolar cells, compared to BSA-NG.

Figure 5. Tf-modification led to a complete transport flow for nanogranules across bipolar monolayer. Three cell monolayer models (a) bipolar (A-B), (b) unipolar (A-B) and (c) unipolar (B-A) models, were constructed for the transcytosis determinations by culturing cells in transwell membranes. (d, e) TEM images of (d) bipolar and (e) unipolar monolayer showed the existences of Tf-NG in apical and basolateral sides in both monolayers, demonstrating the transcytosis mechanism. Scale bar (d, e): 200 nm. Quantitative transcellular detections in (f) bipolar (A-B), (g) unipolar (A-B) and (h) unipolar (B-A) models showed the different transcytosis features between Tf-NG and BSA-NG. The statistical differences were compared between Tf-NG and BSA-NG groups. * p < 0.05, ** p < 0.01, *** p < 0.001. All data were presented as Mean ± SE, n=3. (i, j) The ratios of Tf-NG/BSA-NG based on the quantitative data of uptake, exocytosis (Exo), transcytosis (Trans) and the colocalization (Col) M values with different organelles in (i) bipolar and (j) unipolar cell models. The difference between (i) and (j) indicated the distinct effects of Tf-modification to the cellular transport of nanogranules were related to the cellular polarity. The statistical differences were compared between Tf-NG and BSA-NG groups. * p < 0.05, ** p < 0.01, *** p < 0.001. 61


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All data were presented as Mean ± SE, n=3. (k) Schematic diagrams showed that Tf-modification caused a complete transport flow for nanogranules across bipolar monolayer, but had little effect on the transcytosis through unipolar cells.

Figure 6. Tf-modification induced greater transport-related cell responses during the nanogranule endocytosis in both cell lines. (a, b) The Venn diagrams of up-regulative proteins based on the expression ratios over 1.4 during the endocytosis of nanogranules in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells. (c) The specificity analyses on the proportions of up-regulative proteins after incubations of different nanogranules in bipolar (Caco-2) and unipolar (MDCK) cells. (d) The proportion comparisons of upregulative proteins that classified in ‘receptor activity’ and ‘localization’ between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. (e) The proportion comparisons of ‘catalytic activity’ related proteins between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. The functional subclassification based on the ‘catalytic activity’ class was shown as pie chart after the Tf-NG incubation with unipolar (MDCK) cells. (f, g) The statistical overrepresentation test (ORT) based on the GO classification after nanogranules endocytosis in (f) bipolar and (g) unipolar cells. The p-values of the listed protein classes were all less than 0.05. (h) Heat mapping of the up-regulative proteins that associated with the functional classes including cytoskeleton,

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endosome/exosome, ER-Golgi and protein transport after the endocytosis of Tf-NG and BSA-NG in both cell lines.

Figure 7. Tf-modification triggered positive cellular feedback to nanogranules transcytosis through bipolar monolayer. (a, b) The Venn diagrams of up-regulative proteins based on the expression ratios over 1.4 after the transcytosis of nanogranules across (a) bipolar (Caco-2) and (b) unipolar (MDCK) cell monolayers. (c) The proportion comparisons of up-regulative proteins that classified in ‘receptor activity’ and ‘localization’ between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. (d) The proportion comparisons of ‘catalytic activity’ related proteins between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. The functional sub-classification based on the ‘catalytic activity’ class was shown as pie chart after the Tf-NG transcytosis with unipolar (MDCK) cells. (e) Heat mapping of the up-regulative proteins that associated with the functional classes including cytoskeleton, endosome/exosome, ER-Golgi and protein transport after the transcytosis of Tf-NG and BSA-NG in both cell monolayers. (f) Composite illustration and specificity exhibition of endocytosis and transcytosis enriched proteins after the incubation of Tf-NG with bipolar (Caco-2) cells. (g) Schematic diagram of verifying the cellular response caused by nanogranule incubation and the accelerative effect of Tf-modification to the vesicle mediated transport. The cultured cell monolayer was pre-incubated with different nanogranules 63


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to induce the cellular response. After recovery, the nanogranules were replaced by coumarin-6 (C6) loaded micelles for another 4 h incubation. Finally, the trans-cellular amounts of micelles were determined by measuring the fluorescent intensities of C6. Thus the effect of Tf-modification could be clarified. (h, i) The trans-cellular determination and comparison of C6 loaded micelles through (h) bipolar and (i) unipolar monolayers after the different pre-incubations (Tf-NG, BSA-NG, free Tf, free BSA). The statistical differences were compared between Tf-NG and BSA-NG groups, and between free Tf and free BSA groups. * p < 0.05, ** p < 0.01, *** p < 0.001. All data were presented as Mean ± SE, n=3.

Figure 8. Schematic illustration on the effects of Tf-modification to the cellular transportation of nanogranules. (a) The trafficking comparisons of Tf-NG through Caco-2 (bipolar TfR expression) and MDCK cells (unipolar TfR expression). In bipolar cell model, Tf-modification accelerated nanogranules transported into apical early endosome, recycling endosome, basolateral sorting endosome and then finished the transcytosis based on a whole transport flow. However, due to the absence of a complete transport flow in unipolar cells, Tf-modification could not promote the transcytosis, but triggered nanogranules transfered into apical early endosome, late endosome and finally caused retention in cells. b. The flow diagram for nanogranule trafficking in cells based on the transport flow and feedback. Red and blue stars represented

the

increased

transport

and

protein

expression

caused

by

Tf-modification when nanogranules across bipolar (Caco-2) and unipolar (MDCK) 64


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cells respectively. In bipolar cells, because of the formation of a complete transport flow, the cells themselves also responded to Tf-NG, resulting in up-regulations of intracellular proteins in cytoskeleton, vesicle transport and endosome systems. These cellular responses were further regulated the cells to accelerating the vesicle transportation, finally forming a positive feedback loop.

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Figure 1. TfR exhibited different distribution characteristics, but Tf-modification still promoted the cellular internalizations of nanogranules in both cell models. TfR expressions in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells were analyzed by CLSM. Cell nuclei and tight junctions were labeled with respective markers (Hoechst 33258 and anti-occludin IgG) to define the TfR distributions in two cell models. Red: transferrin receptor. White in the merge images of X-Z were the signals of transwell membrane (TM). It indicated that Caco-2 cells were bipolar TfR expression and MDCK cells were mainly in basolateral side. c. Western-blot analyses on the TfR expressions of two cell models. Grayscale measurement showed that TfR in Caco-2 cells was expressed almost 1.3 times than that in MDCK cells. Mean ± SE, n=4, *** p < 0.001. d. The schematic diagram showed the construction and analysis strategies for two TfR expression models. In order to eliminate the heterogeneity among cell types, the cellular transportations of nanogranules in two different cells were not directly compared. But the differences between nanogranule groups (Tf-NG/BSA-NG in both bipolar and unipolar cell models) were measured and set as core indicators. This normalization strategy could compare the effects of Tf-modification on the transportation of nanogranules in different cells. Cellular uptakes (500 μg/mL, 37 °C, 12 h) were detected by CLSM in (e) Bipolar (Caco-2) and (f) Unipolar (MDCK) cells. The histograms were the statistical results from corresponding CLSM images (Mean ± SE, n > 5, ** p < 0.01, *** p < 0.001). The white cycles indicated the signals of the intracellular nanogranules by LR technology. Quantitative detections of nanogranule internalization were performed by ICP-MS in (g) Bipolar (Caco-2) and (h) Unipolar (MDCK) cells, Mean ± SE, n=3, * p < 0.05, *** p < 0.001, the statistical differences were computed between Tf-NG and BSA-NG groups.


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Figure 2. Tf-conjugation regulated the endocytosis of nanogranules in two models with different mechanisms. Energy dependency mechanisms of nanogranules in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells were evaluated through low temperature incubation. Different endocytosis inhibitors were coincubated with nanogranules in (c) bipolar (Caco-2) and (d) unipolar (MDCK) models to explore the trafficking pathways. e. TEM detection of Tf-NG in endosomes in two cell models. The vesicular distributions of nanogranules were clearly shown in TEM images. The competitive inhibition analyses by adding free Tf in (f) bipolar (Caco-2) and (g) unipolar (MDCK) models were performed to investigate the effect of Tf-TfR interaction on endocytosis. In (a,b), the intracellular amounts of nanogranules at 37 °C in two groups were normalized as 100% respectively and set as control. In (c,d) and (f,g), the cellular uptakes of nanogranules without inhibitor addition were normalized as 100% and set as control. The statistical differences were all compared between test groups and control sample. # p < 0.05, ## p < 0.005, ### p < 0.001. In (f,g), the statistical differences were further computed between Tf-NG and BSA-NG groups. *** p < 0.001. All data were presented as Mean ± SE, n=3.


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Figure 3. Tf-modification altered the intracellular transport pathways (exocytosis and secretion) of nanoparticles in two models. Apical exocytosis analyses in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells were performed at different time points. The retentions of nanogranules in cells during exocytosis were set as indicators. The intracellular amounts of nanogranules at 0 h point in two groups were normalized as 100% respectively and set as control. The relative percentages of test samples were exhibited and the statistical differences were all compared with the control. Data were represented as Mean ± SE, n=3, # p < 0.05, ## p < 0.01. Co-localizations of nanogranules in bipolar (Caco-2) cells with (c, d) ER and (e, f) Golgi complex were analyzed by CLSM. The confocal images ((c) for ER, (e) for Golgi complex) were shown and the corresponding the co-localization M values ((d) for ER, (f) for Golgi complex) were computed here. Likewise, the locations of nanogranules in unipolar (MDCK) cells with (g, h) ER and (i, j) Golgi complex were detected here. Based on the confocal images ((g) for ER, (i) for Golgi complex), the M values in ER and Golgi complex were shown in (h) and (j) respectively. Scale bar (c, e, g, i): 10 μm. In (d, f, h, j), the statistical differences were compared between Tf-NG and BSA-NG groups. All data were presented as Mean ± SE, n=5. (k) Schematic diagrams showed no difference on the secretion pathway of Tf-NG between bipolar and unipolar cell models, indicating Tf-modification did not exhibit advantage for the ER/Golgi transporation of nanogranules compared to BSA-NG.


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Figure 4. Tf-modification altered the intracellular transport pathways (transcytosis and degradation) of nanogranules in two models. Co-localizations of nanogranules in bipolar (Caco-2) cells with (a, b) EE, (e, f) LE, (i, j) RE and (m, n) lysosomes were analyzed by CLSM. The confocal images were shown and the corresponding co-localization M values ((b): EE, (f): LE, (j): RE, (n): lysosomes) were computed here. Similarly, the locations of nanogranules in unipolar (MDCK) cells with (c, d) EE, (g, h) LE, (k, l) RE and (o, p) lysosomes were detected here. Based on the confocal images, the M values in EE, LE, RE and lysosomes were separately shown in (d), (h), (l) and (p). Scale bar (all images): 10 μm. In terms of the M value computations, the statistical differences were compared between Tf-NG and BSA-NG groups. * p < 0.05, ** p < 0.01 *** p < 0.001. All data were presented as Mean ± SE, n=5. (q) Schematic diagrams showed that Tf-modification caused more nanogranules transport through degradation pathway in unipolar cells, but more through recycling route in bipolar cells, compared to BSA-NG.


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Figure 5. Tf-modification led to a complete transport flow for nanogranules across bipolar monolayer. Three cell monolayer models (a) bipolar (A-B), (b) unipolar (A-B) and (c) unipolar (B-A) models, were constructed for the transcytosis determinations by culturing cells in transwell membranes. (d, e) TEM images of (d) bipolar and (e) unipolar monolayer showed the existences of Tf-NG in apical and basolateral sides in both monolayers, demonstrating the transcytosis mechanism. Scale bar (d, e): 200 nm. Quantitative transcellular detections in (f) bipolar (A-B), (g) unipolar (A-B) and (h) unipolar (B-A) models showed the different transcytosis features between Tf-NG and BSA-NG. The statistical differences were compared between Tf-NG and BSA-NG groups. * p < 0.05, ** p < 0.01, *** p < 0.001. All data were presented as Mean ± SE, n=3. (i, j) The ratios of Tf-NG/BSA-NG based on the quantitative data of uptake, exocytosis (Exo), transcytosis (Trans) and the colocalization (Col) M values with different organelles in (i) bipolar and (j) unipolar cell models. The difference between (i) and (j) indicated the distinct effects of Tf-modification to the cellular transport of nanogranules were related to the cellular polarity. The statistical differences were compared between Tf-NG and BSA-NG groups. * p < 0.05, ** p < 0.01, *** p < 0.001. All data were presented as Mean ± SE, n=3. (k) Schematic diagrams showed that Tf-modification caused a complete transport flow for


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nanogranules across bipolar monolayer, but had little effect on the transcytosis through unipolar cells.


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Figure 6. Tf-modification induced greater transport-related cell responses during the nanogranule endocytosis in both cell lines. (a, b) The Venn diagrams of up-regulative proteins based on the expression ratios over 1.4 during the endocytosis of nanogranules in (a) bipolar (Caco-2) and (b) unipolar (MDCK) cells. (c) The specificity analyses on the proportions of up-regulative proteins after incubations of different nanogranules in bipolar (Caco-2) and unipolar (MDCK) cells. (d) The proportion comparisons of upregulative proteins that classified in ‘receptor activity’ and ‘localization’ between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. (e) The proportion comparisons of ‘catalytic activity’ related proteins between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. The functional subclassification based on the ‘catalytic activity’ class was shown as pie chart after the Tf-NG incubation with unipolar (MDCK) cells. (f, g) The statistical overrepresentation test (ORT) based on the GO classification after nanogranules endocytosis in (f) bipolar and (g) unipolar cells. The p-values of the listed protein classes were all less than 0.05. (h) Heat mapping of the up-regulative proteins that associated with the functional classes including cytoskeleton, endosome/exosome, ER-Golgi and protein transport after the endocytosis of


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ACS Nano

Tf-NG and BSA-NG in both cell lines.


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Figure 7. Tf-modification triggered positive cellular feedback to nanogranules transcytosis through bipolar monolayer. (a, b) The Venn diagrams of up-regulative proteins based on the expression ratios over 1.4 after the transcytosis of nanogranules across (a) bipolar (Caco-2) and (b) unipolar (MDCK) cell monolayers. (c) The proportion comparisons of up-regulative proteins that classified in ‘receptor activity’ and ‘localization’ between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. (d) The proportion comparisons of ‘catalytic activity’ related proteins between Tf-NG and BSA-NG groups in two cell models. The value on each column presented the percentage and the actual number. The functional sub-classification based on the ‘catalytic activity’ class was shown as pie chart after the Tf-NG transcytosis with unipolar (MDCK) cells. (e) Heat mapping of the up-regulative proteins that associated with the functional classes including cytoskeleton, endosome/exosome, ER-Golgi and protein transport after the transcytosis of Tf-NG and BSA-NG in both cell monolayers. (f) Composite illustration and specificity exhibition of endocytosis and transcytosis enriched proteins after the incubation of Tf-NG with bipolar (Caco-2) cells. (g) Schematic diagram of verifying the cellular response caused by nanogranule incubation and the accelerative effect of Tf-modification to the vesicle mediated transport. The cultured cell


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monolayer was pre-incubated with different nanogranules to induce the cellular response. After recovery, the nanogranules were replaced by coumarin-6 (C6) loaded micelles for another 4 h incubation. Finally, the trans-cellular amounts of micelles were determined by measuring the fluorescent intensities of C6. Thus the effect of Tf-modification could be clarified. (h, i) The trans-cellular determination and comparison of C6 loaded micelles through (h) bipolar and (i) unipolar monolayers after the different pre-incubations (Tf-NG, BSA-NG, free Tf, free BSA). The statistical differences were compared between Tf-NG and BSA-NG groups, and between free Tf and free BSA groups. * p < 0.05, ** p < 0.01, *** p < 0.001. All data were presented as Mean ± SE, n=3.


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Figure 8. Schematic illustration on the effects of Tf-modification to the cellular transportation of nanogranules. (a) The trafficking comparisons of Tf-NG through Caco-2 (bipolar TfR expression) and MDCK cells (unipolar TfR expression). In bipolar cell model, Tf-modification accelerated nanogranules transported into apical early endosome, recycling endosome, basolateral sorting endosome and then finished the transcytosis based on a whole transport flow. However, due to the absence of a complete transport flow in unipolar cells, Tf-modification could not promote the transcytosis, but triggered nanogranules transfered into apical early endosome, late endosome and finally caused retention in cells. b. The flow diagram for nanogranule trafficking in cells based on the transport flow and feedback. Red and blue stars represented the increased transport and protein expression caused by Tf-modification when nanogranules across bipolar (Caco-2) and unipolar (MDCK) cells respectively. In bipolar cells, because of the formation of a complete transport flow, the cells themselves also responded to Tf-NG, resulting in up-regulations of intracellular proteins in cytoskeleton, vesicle transport and endosome systems. These cellular responses were further regulated the cells to accelerating the vesicle transportation, finally forming a positive feedback loop.


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Graphic for TOC only. Transferrin functionization elevates transcytosis of nanogranules across epithelium by triggering polarityassociated transport flow and positive cellular feedback loop 220x137mm (300 x 300 DPI)


Transferrin Functionization Elevates Transcytosis of Nanogranules

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