1,3-and 1,4-linked polysaccharides uptake in intestinal cells relies on clathrin/dynamin 1/Rab5-dependent endocytosis

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: minor_revision

Abstract
It is thought that polysaccharides cannot penetrate the intestinal mucosa into the circulatory system due to their high hydrophilicity and large molecular size. However, we show that different linked and charged polysaccharides can penetrate Caco-2 cell monolayers, and find β−1,3-linked glucan and α−1,4-linked glucan is detectable in rats (male) and mice plasma and liver after oral administration the isotope (99mTc, 3H) and fluorescein labeled polysaccharides. Using gene-knockdown strategies and inhibitors, we further show polysaccharide uptake requires clathrin heavy chain (CLTC) and its associated factors Rab5 and dynamin 1 in intestinal cells. Strikingly, polysaccharide absorption is attenuated in both CLTC intestine deficient mice and RAB5A, DNM1 conventional-knockout mice. Importantly, membrane receptors bone morphogenetic protein receptor type IA (BMPRIA), Dectin-1 and epidermal growth factor receptor (EGFR) are also critical for specific structural polysaccharide internalization. These findings provide novel insight to understand polysaccharide absorption mechanism and lay foundation for oral polysaccharide-based new drugs development.

Introduction
As one of the four fundamental biomolecules in nature, carbohydrates mediate various biological processes such as signal recognition, pathogenesis prevention, immune modulation, and development1. Carbohydrate can be found as simple monosaccharide, oligosaccharide, and polysaccharide or forming more complex glycoconjugates, such structural variability makes carbohydrate hotspots for biomedical intervention2,3. Early in 1940, carbohydrate-based antibiotics, including streptomycin and gentamicin (both are aminoglycosides), were discovered as anti-infection drugs. Then, the ganglioside GM1 was developed for acute stroke treatment, hyaluronic acid and chondroitin sulfate (both are polysaccharides) were investigated as anti-arthritis drug4. Since 2000, tremendous progresses in glycoscience fields bring vast opportunities for novel carbohydrate-based drug discovery5. In 2019, the conditional launch of sodium oligomannate (GV-971) in China further dramatically boosts the innovative carbohydrate drug development to a new climax6. Surprisingly, polysaccharides represent the largest percentage of the recorded carbohydrate drugs7. To date, more than 40 commercial polysaccharides-based drugs have been launched including heparin, which is a high sulfated heterogenous glycosaminoglycan, was approved as an anticoagulant drug for more than 80 years8. Nevertheless, the biological roles of exogenous polysaccharides go further beyond due to their extensive therapeutic properties in antibiotic, antioxidant, antitumor, anticoagulant, antiaging, and immunostimulation fields, etc9. Notably, about 17 approved or clinically developed polysaccharides-based drugs are administrated orally according to the records in CortellisTM (Drug Discovery Intelligence) database8. However, it’s largely unknown whether these bioactive polysaccharides can reach the lesion site after oral administration. Thus, firstly, we are not sure whether compound degradation occurs after intestinal uptake; Secondly, may those polysaccharides enter blood circulation system? More critically, studies on the molecular mechanism of polysaccharide absorption have been limited to date. The main reason is that most researchers believe polysaccharides cannot be absorbed by the gastrointestinal tract due to their large molecular size and high hydrophilicity. However, clinical studies demonstrated the absorption of exogenous high molecular mass chondroitin sulfate in the intestinal lining10. Nevertheless, it is a challenge for clinically used heparin to pass through the gastrointestinal epithelium11. Hence, polysaccharides absorption by the intestinal lining is still a controversial topic.

Clearly, there is a critical need to understand polysaccharide pharmacokinetics to further fully utilize bioactive polysaccharides and develop novel polysaccharide-based drugs. However, owing to their large molecular weight and deficiency of UV-absorption groups, it is challenging to detect plasma polysaccharides, which may be not distinguishable when the exogenous and endogenous polysaccharide consisting of same or similar monosaccharides. During the few last decades, many attempts have been made to address such challenge. The prior method is radioactive isotope labeling (such as 14C, 125I, 35S, 3H and 99mTc), which were widely used in the pharmacokinetics investigations of polysaccharide12–17. Another common method used in pharmacokinetic studies is liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). However, the current chromatographic and mass spectrometric techniques have limited application due to large molecular size and lacking of chromophoric groups of polysaccharides. Luckily, there has been some progress with the development of spectrometric techniques, such as high-performance liquid chromatography (HPLC) followed by post-column fluorescence derivatization or pre-column fluorescein labeled18,19.

Except studying the ability of polysaccharides to permeate the intestinal mucosa, it is critical to study the absorption mechanism. Several pathways were reported involving in extracellular macromolecules uptake, such as macropinocytosis20,21, phagocytosis22 and endocytosis23. Endocytosis, the process by which cells internalize substances via the formation of vesicles from the plasma membrane, is critical for antigen presentation and cell migration, and also regulates many intracellular signaling cascades24. Eukaryotic cells utilize two major endocytic pathways: clathrin-dependent and clathrin-independent. The mechanism of clathrin-mediated endocytosis (CME) has been extensively characterized25,26. In contrast, clathrin-independent pathways comprise a diverse group of processes, including caveolin- and flotillin-dependent endocytosis, as well as phagocytosis27–29. Additionally, CME is required for the uptake of large molecules such as proteins, pathogenic bacteria, and viruses in mammalian cells30,31. Building upon these findings, we sought to determine the endocytic mechanism responsible for intestinal polysaccharide uptake, specifically whether it is primarily mediated by clathrin or occurs via an alternative pathway.

In this study, we explored the ability of various polysaccharides to permeate intestinal cell layers in vitro. Moreover, GFPBW1 (β-1,3-linked glucan from Grifola frodosa32) and WGE (α-1,4-linked glucan from Gastrodia elata Bl33), as two model polysaccharides (two main types of polysaccharides in plant, fungus, and herbal medicine), were employed for the in vivo and mechanism study.

Results
Polysaccharides with different linkage types and charge could be absorbed and internalized into intestinal cells in vitro
Caco-2 cell monolayer is widely used as an in vitro model that mimics human small intestinal mucosa to predict orally administered drugs absorption34. To test the potential whether polysaccharide can be absorbed by intestine epithelial cells, transport studies for GFPBW1 and WGE were firstly performed. The results showed that the retention times of the transported GFPBW1 and WGE were not changed significantly compared with the prototypical polysaccharides in the HPLC chromatograms (Fig. 1a), indicating the molecular sizes of the penetrated polysaccharides did not change much, further suggesting that polysaccharides might be transported by Caco-2 cell monolayer without significant degradation. This result surprised and encouraged us to ask whether this phenomenon occurs widely and to further test the absorption situation of other linkage types and different charged polysaccharides. Hence, the transport experiments of lentinan (clinically used branched 1,3-linked glucan), MDG-1 (1,2-linked fructan), dextran (commercially available 1,6-linked glucan), GFPBW1-NH3 (amino-branched 1,3-linked glucan), WGE-NH3 (amino-branched 1,4-linked glucan), GFPBW1-SO4 (sulfated-branched 1,3-linked glucan), and WGE-SO4 (sulfated-branched 1,4-linked glucan) were then conducted. Surprisingly, the results showed that diversely linked and differently charged polysaccharides could penetrate Caco-2 cell monolayers. In terms of the penetrated amounts, the value for GFPBW1 was close to that for dextran but significantly greater than that for WGE. Moreover, the penetrated amount of MDG-1 (with the smallest molecule size) was the largest, and was similar as that for lentinan (crude polysaccharide). Furthermore, the charged polysaccharides also displayed variation in the penetration values. The amount of GFPBW1-NH3 was dramatically more than that of GFPBW1 and GFPBW1-SO4. Oppositely, the amount of both WGE-NH3 and WGE-SO4 was higher than that of WGE (Fig. 1b). Surprisingly, the apparent permeability coefficient (Papp) values of the tested polysaccharides were more than 1×10−6 cm/s, indicating that they might be absorbed by the cells (Supplementary Table 1).

To trace polysaccharide transport, we fluorescein-labeled the polysaccharides35, the labeling positions were verified using raffinose, mannose via NMR (Supplementary Fig. 1a–c), and confirmed that the labeling procedure did not affect the structure (Supplementary Fig. 1d, e) and bioactivity (Supplementary Fig. 1f) of the saccharides referring to our previous report32. Next, the uptake experiment of different linkages and charged polysaccharides was performed. The results clearly showed that all the tested polysaccharides could be internalized into the cells (Fig. 1c), while uptake of the fluorescein alone did not happen in the cells (Supplementary Fig. 1g, h). We then asked whether time and/or concentration affected polysaccharides uptake.

To better understand the details of polysaccharide penetration, GFPBW1 and WGE were employed as the model polysaccharides to do the following exploration. To address the question, human intestinal epithelial cell line (HIEC-6) was treated with GFPBW1 or WGE at different concentrations or for various time points. The results revealed that both GFPBW1 and WGE could be internalized into the cell as early as 5 min, both polysaccharides showed a sharp increase internalization at 10 min, then the internalized polysaccharide decreased at 15 and 30 min. However, the uptake of GFPBW1 had recovered by 1 h (Fig. 1d, e). Moreover, distinct fluorescence could be observed at 1.25 mg/mL for GFPBW1 and 2.5 mg/mL for WGE, respectively (Fig. 1f). The uptake of both polysaccharides exhibited a concentration-dependent manner (Fig. 1g). Subsequently, similar experiment was designed using Caco-2 cells. Similarly, GFPBW1 and WGE showed analogous uptake time dynamics in this cell line (Fig. 1h, i), and both demonstrated a concentration dependent uptake manner (Fig. 1j, k). Furthermore, polysaccharides uptake time dynamics were examined in IEC-6 (rat intestinal cell line), WGE showed a time-dependent pattern. Conversely, GFPBW1 showed a decrease at 15 min and complete recovery at 30 min (Fig. 1l, m). Thus, the uptake dynamics of GPFBW1 and WGE in rat and human cell lines were different. The reason of such difference between the two polysaccharides might be partially due to their structures.

Polysaccharides were absorbed in rat and mice intestinal lining in vivo and could be detected in rodent blood and liver
Next, we sought to ascertain whether the two model polysaccharides could be absorbed in vivo. GFPBW1 and WGE were orally and intravenously administered to rats, while commercially available lentinan was also included as a control. Firstly, the spectrum of blank rat plasma mixed with GFPW (internal standard) or without followed extraction as described in “Methods” were displayed (Supplementary Fig. 2a, b). The HPLC chromatograms showed the retention time and peak pattern of lentinan before and after the pretreatment procedure did not change obviously, indicating good efficiency of the extraction process (Supplementary Fig. 2c). Surprisingly, GFPBW1 was quantifiable in all plasma samples following oral and intravenous administration (Fig. 2a, upper panel). Similar results were obtained for WGE and lentinan, and there were trace changes between the retention time and peak pattern of the plasma polysaccharides and their prototypical ones (Fig. 2a and Supplementary Fig. 2d, e), suggesting that the tested polysaccharides were absorbed at least partially as macromolecular form and underwent minimal degradation in vivo. The pharmacokinetic profiles and parameters of GFPBW1, WGE, and lentinan by oral administration or intravenous injection are shown in Supplementary Fig. 2f–h and Supplementary Tables 2 and 3, respectively. Yet, there were a few differences among them regarding to their pharmacokinetic characteristics. The maximum concentration of GFPBW1 in plasma (Tmax) was at 5 h, while the half time (t1/2) was 1.44 h. However, the clearance rate (CL) was 74.84 mL kg−1 h−1, suggesting that GFPBW1 was absorbed slowly but cleared quickly. The Tmax of WGE occurred at 2 h, the t1/2 was 8.44 h, and the CL was 1.879 mL kg−1 h−1, indicating rapid absorption and slow clearance (Supplementary Table 2).

To further validate these findings, fluorescein-labeled GFPBW1 and WGE were labeled and detected on HPLC with a fluorescence detector. The results showed that both GFPBW1 and WGE demonstrated a typical peak pattern with measurable signals (Supplementary Fig. 2i). Next, C57BL/6J mice were orally administrated by fluorescein labeled WGE, followed by the polysaccharide extraction in mice serum and analysis. HPLC detection of plasma samples collected 1 h after oral administration showed a typical peak for WGE, with a retention time very close to that of the original fluorescein labeled WGE, indicating that WGE was absorbed in its macromolecular form (Fig. 2b).

To more precisely detect the polysaccharide after the oral administration in mice, we next sought to label the polysaccharides by radioactive 3H with the help of QZ Isotech (Shanghai, China) and examined by HPLC connected with β ray detector. Surprisingly, chromatograms showed a typical peak with quantitative signals (Supplementary Fig. 2j). Subsequently, C57BL/6J mice were orally administrated by 3H-GFPBW1 and WGE, and the concentration-time curves of GFPBW1 and WGE in mice serum are displayed in Supplementary Fig. 2k, further confirmed the results we obtained from rats and fluorescein labeled polysaccharides in mice. Further, we were still surprised to observe a peak with quantitative signals in mice plasma after oral administration of both 99mTc labeled GFPBW1 and WGE (Fig. 2c, e, left panels). More importantly, we even obtained canonical peak in mice liver lysate after oral administration of both GFPBW1 and WGE (Fig. 2c, e, right panels), and the retention time of liver lysate was closed to the original labeled ones, further implied that polysaccharides were absorbed partially in the macromolecular form without significantly degradation (similar molecular weights) within the test time and could accumulate in the liver. Moreover, we obtained similar results from the plasma and liver lysate of mice after intravenously injected 99mTc labeled GFPBW1 (Fig. 2d) and WGE (Fig. 2f). The pharmacokinetic parameters of 99mTc-GFPBW1 and WGE in mice were summarized in Supplementary Tables 4 and 5, respectively. Furthermore, fluorescence labeled GFPBW1 (Fig. 2g, h) and WGE (Fig. 2i, j) with Cy5.5 were employed to trace the polysaccharides in vivo using living imaging after orally administration (Fig. 2g, i) or intravenous injection (Fig. 2h, j). Obvious fluorescence was observed in both mice liver and intestine, further indicating the absorption of both polysaccharides.

Next, to investigate when the absorption of the polysaccharides commences in mice intestine, BALB/c mice were orally administered with fluorescein-labeled GFPBW1 and WGE, and we indeed visualized the internalized GFPBW1 and WGE in mice intestine after the oral administration for 20 and 15 min, respectively. Moreover, the maximum internalization of GFPBW1 and WGE occurred at 80 and 30 min after oral dosing, respectively (Fig. 2k). To rule out the possible interference caused by mouse strain, we performed similar experiment in C57BL/6J mice. Microscopic analysis showed that WGE was internalized mostly in the intestinal enterocytes of the duodenum, while less was internalized in the jejunum and only a trace was found in the ileum in WGE-1 h group. Conversely, we observed different absorption pattern in WGE-2 h group. The internalized WGE was increased sequentially in duodenum, jejunum, and ileum segments, and even could be observed in mice ileum crypt (Fig. 2l, m). Collectively, we demonstrated that polysaccharides could be absorbed by intestinal cells in vivo and detectable in rodent blood and liver.

Clathrin mediated endocytosis is critical for polysaccharides uptake in cultured cells
Some macromolecules, such as EGFR and asialoglycoproteins, were reported to be transported into the cells via endocytosis36,37. Inspired by this, we speculated that polysaccharides, which are hydrophilic macromolecules, might be absorbed via vesicular endocytosis. Firstly, we confirmed the localization of internalized polysaccharides using DiI to label cell membrane, the internalized polysaccharides were observed mostly in cytoplasm and partially on cell membrane (Fig. 3a).

Next, we utilized inhibitors to block various endocytosis routes and then measured polysaccharides uptake in intestinal cells. HIEC-6 cells were pretreated with the chloropromazine (CPZ, inhibitor of CME), Nystatin (inhibitor of lipid raft mediated endocytosis), Methyl-β-cyclodexrin (inhibitor of caveolin mediated endocytosis) or cytochalasin D (CytoD, inhibitor of macropinocytosis) for indicated time, then incubated with polysaccharides or endocytosis route markers. Interestingly, the uptake of both GFPBW1 and WGE was dramatically inhibited after CPZ disturbance, while transferrin was used as the marker of CME, suggesting that polysaccharide internalization might be mediated by CME (Fig. 3b, c). Then, similar studies were also performed on IEC-6 cells. Consistently, uptake of GFPBW1 and WGE was reduced in CPZ treated group than that of control cells (Ctrl) in IEC-6 (Supplementary Fig. 3a, b). However, the uptake of both GFPBW1 and WGE in Nystatin treated HIEC-6 cells showed no obvious difference with that of Ctrl and DMSO group, while the uptake of cholera toxin B (marker of lipid raft mediated endocytosis) was nearly abolished (Fig. 3d, e), which indicated the effective inhibition of lipid raft mediated endocytosis. We observed similar phenomenon in IEC-6 cells (Supplementary Fig. 3c, d). Furthermore, the fluorescence images indicated that the internalization of GFPBW1 or WGE was not affected by CytoD (Fig. 3f, g); this observation was confirmed in IEC-6 cells under the same condition while the uptake of FD40 (marker of macropinocytosis or fluid-phase endocytosis) was severely impaired by CytoD (Supplementary Fig. 3e, f). The above data suggested that internalization of GFPBW1 or WGE might be mediated by CME but not through macropinocytosis or lipid raft mediated endocytosis.

It is reported that cell membrane-expressed proteoglycan is internalized via caveolin-containing endosomes38. Thus, we tried to determine whether caveolae-mediated endocytosis was involved in exogenous polysaccharides internalization. A caveolin-free Huh7 cell39 was therefore used to test the hypothesis. However, out of our expectation, the tested polysaccharides could still internalize into Huh7 cells, further implying that polysaccharides uptake did not rely on caveolae mediated endocytosis (Supplementary Fig. 10a). Furthermore, the internalization of GFPBW1 and WGE was unaffected by methyl-β-cyclodextrin, a specific inhibitor that blocks caveolae-mediated endocytosis (Fig. 3h, i). More importantly, we then knockdown CAV1 (caveolin-1, hallmark protein and key regulator of caveolae-mediated endocytosis) in HIEC-6 cells, western blots results confirmed the good efficiency of the siRNAs (Fig. 3l). And the following uptake experiment showed that the internalization of GFPBW1 and WGE in si-CAV1 groups were comparable with that of Sham or Ctrl group, indicating that caveolin-1 did not affect polysaccharide uptake (Fig. 3j, k). Taken together, these data provide strong evidence that caveolae-mediated endocytosis does not contribute to polysaccharide uptake.

As mentioned above, we labeled the polysaccharides using various strategies (radioactive and fluorescent labeling) and detected their presence in rodent plasma following oral administration. This poses the question: how do the polysaccharides enter the systemic circulation? To address this, we employed human microvascular endothelial cells (HMEC-1) as a cell model. We first performed the kinetic uptake assay of GFPBW1 and WGE, fluorescence was detectable as early as 5 min after the treatment and became more pronounced at 30 min and 1 h, revealing that both the two polysaccharides can enter the microvascular endothelial cells (Supplementary Fig. 4). To further investigate the underlying molecular mechanism, we subsequently employed established pharmacological inhibitors (CPZ, inhibitor of CME; dynasore, inhibitor of dynamin; cytochalasin D, inhibitor of actin driven macropinocytosis; amiloride and EIPA, inhibitors for macropinocytosis; Nystatin, inhibitor of lipid/raft mediated endocytosis; methyl-β-cyclodexrin, inhibitor of caveolin mediated endocytosis) as we did in the intestinal cells. The results showed the uptake of GFPBW1 and WGE was markedly decreased by the treatment of both CPZ (Supplementary Fig. 5) and dynasore (Supplementary Fig. 6), however, the internalization of polysaccharides did not affect by cytochalasin D (Supplementary Fig. 7), amiloride (Supplementary Fig. 8a, b), EIPA (Supplementary Fig. 8c, d), Nystatin (Supplementary Fig. 9a, b) or methyl-β-cyclodexrin (Supplementary Fig. 9c, d), suggesting that the uptake of polysaccharides in HMEC-1 cells might rely on clathrin dependent endocytosis. In other words, polysaccharides might enter the bloodstream via CME.

Polysaccharides uptake was impaired in CLTC knockdown intestine cells and CLTC deficient animals
To identify critical proteins during polysaccharide internalization process, HIEC-6 and IEC-6 cells were treated with fluorescein-labeled GFPBW1 and WGE, followed by immunostaining with anti-clathrin heavy chain (CLTC). Intriguingly, we found that the internalized polysaccharides could co-localize with CLTC protein in both HIEC-6 (Fig. 4a, b) and IEC-6 (Fig. 4c, d), indicating that CLTC might play a direct role in polysaccharides uptake. More importantly, we further observed the co-localization of the internalized polysaccharides and CLTC protein in Huh7 cells (Supplementary Fig. 10a). To further ascertain the requirement of CLTC for polysaccharides uptake, we firstly performed CLTC knockdown in HIEC-6 cells and then assessed polysaccharides uptake. The results showed that both the internalization of GFPBW1 and WGE was significantly decreased upon CLTC knockdown. The efficiency of the knockdown was confirmed by immunoblotting (Fig. 4e–g). We next overexpressed CLTC in the cells by transfection with its full-length plasmid and followed by the polysaccharide uptake assay. The results showed that CLTC overexpression significantly enhanced the uptake of both GFPBW1 and WGE (Fig. 4h–j). This result encourages us to ask whether this situation also occurs in animals. Then, we generated intestinal epithelial cell (IEC) -specific CLTC-knockout mice using Cre-loxP conditional gene targeting CLTC (CLTC CKO mice). The fluorescein labeled WGE was orally administrated to both the CLTC flox/flox (Ctrl) and CLTC CKO mice. Surprisingly, we observed trace amount of internalized WGE in the ileum of the CLTC CKO mice, while obvious fluorescence was observed in the ileum of Ctrl mice, suggesting that silencing CLTC sharply blocked the uptake of polysaccharides in vivo (Fig. 4k, l). Hence, our findings revealed a fundamental role of CLTC in polysaccharide uptake with these in vitro and in vivo validations. We then asked whether a direct interaction exists between clathrin and polysaccharides? Surface Plasmon Resonance (SPR) strategy was employed to conduct the exploration. Surprisingly, the SPR results showed that both GFPBW1 and WGE bound directly to clathrin with an estimated equilibrium dissociation constant (KD) of 1.149×10−6 and 3.506×10−6, respectively (Fig. 4m, n).

Dynamin 1 and Rab5 were required for polysaccharides uptake
Although the role of CLTC protein in polysaccharide absorption has been characterized in our above investigation, other proteins involved in polysaccharide uptake are largely unknown. Dynamin, best studied for its role in CME, is a prototypical member of a family of multi-domain GTPases involved in fission and remodeling of multiple organelles40. To examine whether dynamin plays a role in polysaccharide uptake, HIEC-6 cells were treated with dynasore (dynamin inhibitor) in presence of GFPBW1 and WGE. The results showed that the internalized GFPBW1 and WGE were substantially reduced after blockage of dynamin function (Fig. 5a, b), suggesting the extracellular to intracellular transport of the polysaccharide is likely reliant on dynamin. Unexpectedly, uptake of GFPBW1 in IEC-6 cells was not affected by dynamin inhibition, while the internalization of WGE and transferrin were sharply decreased after dynasore treatment (Supplementary Fig. 11), indicating even uptake of the same polysaccharide is cell species-dependent. Since mammalian genomes contain three closely related tissue-specific dynamin genes, siRNAs against DNM1 and DNM2 were employed to determine which isoform was required for the polysaccharide uptake by the cells. Interestingly, uptake of GFPBW1 was impaired by si-DNM1, and the uptake of WGE and transferrin was almost completely abolished by DNM1 depletion, while the knockdown efficiency of DNM1 was confirmed by immunoblot (Fig. 5c–e). Interestingly, although the expression of DNM2 was reduced sharply by si-DNM2, the internalization of GFPBW1 and WGE was not significantly disturbed by DNM2 silencing (Supplementary Fig. 12). Collectively, we speculated that DNM1 rather than DNM2 was responsible for polysaccharides uptake at least in the tested intestine cells. To further confirm this phenomenon in vivo, DNM1 conventional knockout mice were generated firstly, unfortunately, we failed to obtain the homozygous DNM1 KO mice due to that homozygous DNM1 offspring died after birth. Therefore, wild type (Ctrl) and heterozygous mice (DNM1 KO, heterozygote) were employed to do the flowing exploration. Interestingly, both GFPBW1 and WGE were internalized in Ctrl mice intestine after polysaccharides orally administrated, while the intestinal internalized GFPBW1 and WGE were slightly or sharply decreased in DNM1 KO heterozygous mice, respectively (Fig. 5f, g). The above data suggested that dynamin 1 is required for the GFPBW1 and WGE internalized in the intestine cells. More importantly, when we overexpressed dynamin 1, the uptake of GFPBW1 and WGE was dramatically enhanced, further confirming the critical role of dymanin 1 in this process (Fig. 5h-j). Even more interestingly, the SPR revealed that DNM1 could bind to GFPBW1 with the KD of 0.137×10−6 (Fig. 5k), while WGE showed no direct interaction with DNM1 (Supplementary Fig. 13).

Rab proteins are a large family of GTPases that regulate vesicle transport in cells. Rab5 is a marker of early endosomes and plays an important role in CME41. To understand the possible role of Rab5 in polysaccharide uptake mediated by clathrin, immunostaining for Rab5 was performed after both HIEC-6 (Fig. 6a, b) and IEC-6 cells (Supplementary Fig. 14) were incubated with GFPBW1 and WGE. Interestingly, Rab5 was partially or completely colocalized with the internalized GFPBW1 and WGE in both cells, suggesting that Rab5 probably involved in polysaccharides uptake. Moreover, fluorescence imaging showed that the uptake of GFPBW1 and WGE was dramatically inhibited by RAB5A silencing, while Western blotting analyses confirmed the knockdown efficiency (Fig. 6c–e), suggesting that Rab5 was also critical for polysaccharides uptake. To further evaluate the role of Rab5 in polysaccharide trafficking in vivo, RAB5A conventional knockout mice were generated. Then the fluorescein-labeled GFPBW1 or WGE were orally administrated to both wild type (Ctrl) and RAB5A−/− (RAB5A KO) mice. Indeed, internalized polysaccharides in both duodenum and jejunum segments of the Ctrl mice were observed obviously, while trace amounts of internalized polysaccharides could be detected in RAB5A KO mice, indicating that littermates of the RAB5A KO mice displayed a marked impairment in their ability to internalize polysaccharides (Fig. 6f–i). Taken together, these data demonstrated that Rab5 was also required for polysaccharides uptake in vitro and in vivo. More importantly, when we overexpressed Rab5, the uptake of GFPBW1 and WGE was dramatically increased, further confirming the important role of Rab5 in this process (Fig. 6j–l). Even more interestingly, the SPR results showed that both GFPBW1 and WGE bound directly to Rab5 with an estimated equilibrium dissociation constant (KD) of 9.193×10−8 and 1.080×10−6, respectively (Fig. 6m, n).

Internalized polysaccharides were detectable in endosome, lysosome, Golgi, and endoplasmic reticulum in intestinal cells
Further, we were wondering how the intracellular fates of the internalized polysaccharides are after the uptake? To address this question, HIEC-6 cells were incubated with fluorescein-labeled GFPBW1 and WGE for the indicated time and then immuno-stained for EEA1 (endosome marker), LAMP1 (lysosome marker), AIF (mitochondria marker), RCAS1 (Golgi marker), or PDI (endoplasmic reticulum marker), respectively. Both GFPBW1 and WGE were mainly colocalized with EEA1 as expected (Fig. 7a, b). Furthermore, significant amount of colocalization between the internalized GFPBW1, WGE with LAMP1 was also observed (Fig. 7c, d). However, it seemed that both polysaccharides were not much in mitochondria, since there was little detectable association between polysaccharides and AIF (Fig. 7e). Moreover, we found that the internalized polysaccharides were significantly colocalized with the marker of Golgi apparatus-RCAS1 (Fig. 7f, g). Finally, the internalized polysaccharides were mainly colocalized with PDI, which is the marker of endoplasmic reticulum (ER) (Fig. 7h, i). Thus, based on the above results, we speculated that polysaccharide might be dynamically transported among organelles. Upon internalization, polysaccharides could move from the early endosomes to lysosomes or/and further moved to other organelles such as Golgi, ER but not mitochondria.

EGFR, dectin-1, and BMPRIA were implicated in polysaccharides uptake
It is reported that transport of some polysaccharides was mediated by specific receptors42. Previously, we found that a sulfation derivative of WGE could bind to BMPRIA, while pectin RN1 (branched l,6-linked galactan with arabinose) could bind to EGFR43, indicating that these receptors might mediate specifical functions of exopolysaccharides. Additionally, dectin-1 was the well-known receptor for β-1,3 glucan, this phenomenon is also demonstrated by this group, evidenced by the binding between GFPBW1 and dectin-1 in our previous work32. To further understand the role of cell membrane receptors in polysaccharides uptake, HIEC-6 cells were treated with fluorescein GFPBW1 and WGE following EGFR knockdown. The results showed that the uptake of both polysaccharides was dramatically diminished, while the internalization of transferrin showed no difference between the control and EGFR knockdown cells, while the efficiency of siRNAs was confirmed by immuno-blotting (Fig. 8a–c). Furthermore, we observed similar phenomenon in T24 cells either by Gefitinib (EGFR inhibitor, Supplementary Fig. 15a, b) or by si-EGFR interference (Supplementary Fig. 15c-e), further validated the important role of EGFR in polysaccharides uptake. Even more critically, overexpression of EGFR led to a marked increase in the uptake of both GFPBW1 and WGE (Fig. 8d–f). Surprisingly, when we assessed the interaction using SPR, no direct binding was detected between the polysaccharides (both GFPBW1 and WGE) and EGFR (Supplementary Fig. 16). Therefore, further investigation is required to elucidate the molecular mechanism by which EGFR mediates polysaccharides uptake. Secondly, siRNAs were utilized to knockdown the expression of CLEC7A in HIEC-6 cells, the fluorescence images revealed that the internalization of GFPBW1 was significantly abolished, while the uptake of transferrin did not affect (Fig. 8g–i). Meanwhile, we validated the importance of dectin-1 in GFPBW1 internalization using anti-dectin-1 treatment in T24 cells (Supplementary Fig. 17). To further confirm the role of dectin-1 in GFPBW1 uptake, we overexpressed this protein in HIEC-6 cells. The subsequent cellular uptake assay demonstrated that overexpression of dectin-1 significantly promoted the uptake of GPFBW1, while the uptake of WGE in overexpressed dectin-1 group was comparable to that of Ctrl and vector groups (Fig. 8j–l). More importantly, the SPR results revealed the direct interaction of GFPBW1 and dectin-1 with the KD value of 1.063×10−8, while WGE could not bind to dectin-1 (Supplementary Fig. 18), further emphasized the importance of dectin-1 in GFPBW1 uptake. Thirdly, the internalization of WGE was indeed dramatically blocked by BMPRIA silence using siRNA in HIEC-6 cells, while transferrin showed no differences (Fig. 8m–o). Moreover, the penetration of WGE was sharply blocked after BMPRIA depletion via anti-BMPRIA antibody treatment or siRNA interference in T24 cells (Supplementary Fig. 19). Next, we overexpressed BMPRIA in HIEC-6 cells, the following uptake experiment showed that the internalization of WGE was significantly augmented by BMPRIA overexpression, while the uptake of GFPBW1 was not affected (Fig. 8p–r). However, the subsequent SPR experiment revealed no direct binding between WGE and BMPRIA. Intriguingly, GFPBW1 showed a strong binding with BMPRIA (Supplementary Fig. 20). Nevertheless, these interesting findings still needed more detailed exploration. Hence, we speculated that the receptors EGFR, BMPRIA, and dectin-1 were involved in the uptake for their corresponding polysaccharides.

Wnt/β-catenin and NF-κB signaling pathways play a role in polysaccharides uptake
To further understand whether some signaling pathways are also implicated in the polysaccharide uptake, HIEC-6 (Supplementary Fig. 21a, b), Caco-2 (Supplementary Fig. 21c, d), or T24 cells (Supplementary Fig. 21e, f) were pretreated with XAV939 (Wnt/β-catenin signaling inhibitor), then incubated with fluorescein labeled GFPBW1 and WGE. The results showed that internalization of the two polysaccharides was significantly attenuated after XAV939 treatment in all the tested cell line (Supplementary Fig. 21a–f), indicating wnt/β-catenin signaling might be implicated in polysaccharides uptake. Interestingly, uptake of GFPBW1 and WGE was also sharply blocked by TPCA-1 (NF-κB inhibitor) (Supplementary Fig. 21g, h). Thus, these findings revealed that wnt/β-catenin and NF-κB signaling pathway might be critical in polysaccharide uptake.

Polysaccharide could be internalized in T24 cells via CME, dynamin 1, and Rab5 were require during this process
To determine whether the polysaccharide uptake phenomenon is widespread and to assess its cell-type specificity, we used a cancer cell line of epithelial morphology named T24. This approach also helped to exclude variation between individual cells. Firstly, the uptake dynamics of GFPBW1 and WGE were examined in T24 cells. The results demonstrated that uptake of GFPBW1 showed similar pattern in T24 cells with that in HIEC-6 cells among different time points, while WGE exhibited a different manner with that in intestine cells (Fig. 9a, b). Meanwhile, both GFPBW1 and WGE displayed a concentration-dependent manner as in HIEC-6, IEC-6, and Caco-2 cells (Fig. 9c, d).

To gain insight to the dynamic uptake of polysaccharide, the interaction of GFPBW1 to living and immobilized T24 cells was detected by quartz crystal microbalance (QCM) biosensor, which has proved to be a powerful tool for studying macromolecular interactions44. Interestingly, the binding activity of GFPBW1 to living T24 cells was dramatically stronger than that of the immobilized cells, suggesting that GFPBW1 was dynamically internalized into living T24 cells (Fig. 9e).

Next, similar with what we had done in intestine cells, inhibitors were employed to preliminarily determine the uptake route of polysaccharides in T24 cells. CPZ treatment caused a dramatic decrease in both uptake of GFPBW1 and WGE (Fig. 9f, i), while no obvious changes were observed in neither Nystatin (Fig. 9h, j) nor CytoD (Fig. 9g, k) treated cells compared with Ctrl, revealing that CME might be also critical in polysaccharides uptake in T24 cells.

To further confirm this result, the immunostaining of CLTC was conducted. Most of the internalized polysaccharides were indeed colocalized with CLTC as expected (Fig. 10a, b). Moreover, we found the binding of GFPBW1 to living T24 cells was blocked by anti-CLTC treatment, further confirmed the critical role of clathrin in GFPBW1 uptake (Fig. 10c). Subsequently, we found that knockdown CLTC with siRNA also cause internalization inhibition of both GFPBW1 and WGE (Fig. 10d, f), while the siRNA efficiency was checked by immunoblot in the T24 cells (Fig. 10e). Furthermore, we also utilized QCM to confirm siRNA efficiency (Fig. 10g). Interestingly, the binding curve of the blank group was much higher than that of si-CLTC group after GFPBW1 treatment, indicting the inhibition of GFPBW1 internalization was caused by CLTC knockdown (Fig. 10h). To further confirm, we wondered whether overexpression of clathrin would promote polysaccharide internalization. To test, full-length CLTC cDNA plasmid was transfected in T24 cells, Western blotting analysis confirmed the overexpression of CLTC (Supplementary Fig. 22c). However, the amount of internalized GFPBW1 or WGE was not significantly increased (Supplementary Fig. 22a, b). Interestingly, polysaccharides could not colocalize with caveolin-1 in T24 cells (Supplementary Fig. 10b), further confirmed that polysaccharides uptake did not require caveolin.

Similar as our investigation in intestine cells, the immunostaining for Rab5 was performed and the results showed that Rab5 could colocalize with internalized GFPBW1 and WGE (Fig. 10i, j) in T24 cells. Furthermore, the uptake of GFPBW1 and WGE was blocked after RAB5A depletion, while the knockdown efficiency was examined by immunoblot (Fig. 10k–m). Similarly, T24 cells were cultured with fluorescein labeled polysaccharides, followed by immuno-staining with anti-dynamin antibody. Indeed, the internalized GFPBW1 and WGE showed colocalization with dynamin both on the cell membrane and in the cytoplasm (Fig. 10n, o), indicating that dynamin may also be involved in polysaccharides uptake. Subsequently, the expression of DNM1 was disturbed by siRNA, the microscopy analysis indicated that the uptake of GFPBW1 and WGE was sharply blocked in the DNM1 silence T24 cells (Fig. 10p–r). Meanwhile, the internalization of the polysaccharides was dramatically attenuated by dynamin inhibition caused by dynasore (Fig. 10s, t) in the same cells. Similar as that of intestine cells, we also found that the internalization of polysaccharides did not affect by DNM2 disturbing (Supplementary Fig. 23).

Eps15 (an acronym for EGFR pathway substrate 15) is an important protein involved in CME45,46. To determine the role of Eps15 in polysaccharides uptake, immunostaining assays were performed in IEC-6 and T24 cells after treatment with labeled polysaccharides. The results revealed that GFPBW1 and WGE were co-localized with Eps15 in most areas of the cytoplasm in both cells (Supplementary Fig. 24), suggesting that Eps15 might also be involved in polysaccharide uptake.

The internalized polysaccharides might be examined in endosome, lysosome, and Golgi in T24 cancer cells
The above results outlined extracellular to intracellular transport of polysaccharides in human intestinal epithelial cells, but how is their intracellular feature in cancer cells? Thus, similar strategy was employed as we used in HIEC-6 cells. The immuno-staining experiments revealed that both GFPBW1 and WGE showed strong colocalization with EEA1 (Supplementary Fig. 25a, b) and LAMP1 (Supplementary Fig. 25d, e) as expected. Moreover, we found the internalized polysaccharide were significantly colocalized with RCAS1, consistent with the results in HIEC-6 cells (Supplementary Fig. 25g, i). However, by using AIF and PDI as markers for mitochondria and ER, respectively, we found that there was no obvious association between polysaccharides and AIF or PDI (Supplementary Fig. 25c, f). Thus, we speculated that polysaccharide could transport among organelles in cancer cells, they could move from the early endosomes to lysosomes and might further deliver to Golgi, but not mitochondria or ER in cancer cells.

Methods
Animal treatment
Male Sprague Dawley rats (SD rats, male), BALB/c and C57BL/6J mice (both female and male) were obtained from Shanghai SLAC Laboratory Animal Center and maintained in Animal Center of Shanghai Institute of Materia Medica, Shanghai, China. All procedures involving experimental animals were carried out following protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica (IACUC code: 2020-10-DK-94, 2020-10-DK-95, 2021-10-DK-102, 2021-10-DK-103) and conformed to the Guide for the Care and Use of Laboratory Animals. All animals were maintained under constant humidity and temperature at standard facilities under specific pathogen-free conditions with tap water and standard laboratory rodent chow.

In vivo pharmacokinetics study in rats
Male SD rats (weight, 250–300 g) for orally administered were conducted jugular vein cannulation for blood sample collection; another group of rats for intravenously administered were subjected to intubation in both the jugular (blood sample collection) and femoral vein (for intravenous administration). Following a two-day recovery after the intubation, lentinan, GFPBW1 and WGE were orally administrated (450 mg/kg, 550 mg/kg, and 750 mg/kg, respectively) or intravenously administered (450 mg/kg, 135 mg/kg, and 450 mg/kg, respectively) to rats. Then, blood samples (500 μL) were collected alternately from the jugular vein at the indicated times (n = 5). Plasma was separated from the collected blood immediately by centrifugation (12,000 rpm, 5 min) and stored at −20 °C for analysis.

Polysaccharides were extracted from the plasma before analysis. Shortly, 100 μL GFPW (2 mg/mL) and 200 μL trichloroacetic acid (30%) were added into 100 μL of the plasma for deproteinization (4 °C, 2 h). Then, 2 mL MilliQ-H2O was added, followed by centrifugation (10,000 rpm, 10 min). The supernatant was transferred into the mixture of chloroform and butanol (v/v = 4/1) and vigorously shaken for 7 min. This extract was subjected to two more rounds of extraction followed by centrifugation (12,000 rpm, 10 min). The upper supernatant was collected and desalted twice using the Zeba spin desalt column (Thermo Fisher Scientific, USA) and then lyophilized.

The analysis of the samples was performed on an Agilent 1200 high performance liquid chromatography (HPLC) equipped with an Agilent G1362A refractive index (RI) detector and a serially linked combination of an UltrahydrogelTM 2000 and UltrahydrogelTM 500 column (Waters, USA). The mobile phase was MilliQ-H2O, and column temperature was set at 25 °C. By comparing the peak height of the polysaccharides extracted from the plasma in the chromatograms with that of the original samples in the chromatograms (standard curve), the concentrations of the serum polysaccharides were obtained. Then, the concentration-time curves of the polysaccharides were achieved. The curves were analyzed using the WinNonlin software (Scientific Consulting Inc., USA) to determine the pharmacodynamic of polysaccharides in rats.

Synthesis and characterization of fluorescein-labeled polysaccharides
Saccharides were labeled using a modified version of Glabe’s35. Briefly, 40 mg polysaccharides dissolved in 20 mL MilliQ-H2O were initially activated by saturated CNBr solution (Fluka, USA) for 7 min (the pH of the reaction system throughout the process was monitored and maintained above 10.0 by adding 0.25 M NaOH intermittently). The mixture was then dialyzed against distilled water to remove redundant CNBr. 48 h later, the retentate was concentrated and dissolved in Na2B4O7 (0.2 M, pH 8.0, Sinopharm, China), followed by the addition of fluorescein (dansylethylenediamine (DED, TRC Inc., Canada), 5-FITC cadaverine (Anaspec, USA) or Texas red (Biotium, USA) and vortexed in dark at room temperature for 18 h. Subsequently, the mixture was centrifugated, and the supernatant was then dialyzed against distilled water for 48 h in dark, then the retentate was concentrated and lyophilized, designated as fluorescein labeled polysaccharides. For fluorescein labeling maltose and raffinose, saccharides were subjected to Bio-Gel-P2 column for further purification after activation with CNBr.

Saccharides and their fluorescein-labeled derivatives (30 mg) were deuterium-exchanged and dissolved in 0.5 mL D2O (99.8% D, Sigma-Aldrich, USA). The 1H and 13C NMR spectra were measured at 25 °C with the Bruker AM-400 NMR spectrometer (Karlsruhe, Germany), and acetone was used as the internal standard (δ 31.50 ppm for 13C-NMR and δ 2.29 ppm for 1H-NMR).

Preparation of 99mTc-labeled polysaccharides
The labeling process were performed according to the previous report17. Shortly, about 40 mg polysaccharides were dissolved in DMSO, then 4 mg of DTPAA and 2 mg of DMAP were added and reacted at 40–45 °C. 24 h later, the solution was further precipitated with ethanol and dialyzed with distilled water for 48 h, followed by the purification of Sephadex G50 column and gave the DTPA-polysaccharide intermediate. Subsequently, 10 μL of freshly prepared SnCl2·2H2O was mixed with 1 mL of Na99mTcO4 solution for stirring for 5 min. 1 mL of DTPA-polysaccharide intermediate and 50 μL saturated sodium bicarbonate were then added and incubated for 1 h at 60 °C, purification on a PD-10 column (General Electric, Fairfield CT, USA) was then conducted to obtain 99mTc-labelled polysaccharides.

Generation of RAB5A and DNM1 conventional knockout mice by transcription activator-like effector nucleases (TALENs)
The RAB5A heterozygous KO mice were produced in SiDanSai Biotechnology Co., Ltd (Shanghai, China), then the heterozygous mice were crossed to generate the wild type (Ctrl) and homozygous RAB5A deficient mice (RAB5A KO mice). 8-10 weeks of the male or female mice were used in the following experiments. Similarly, the DNM1 heterozygous KO mice were produced in SiDanSai Biotechnology Co., Ltd (Shanghai, China), then the heterozygous mice were crossed to generate the wild type and homozygous DNM1 deficient mice. Unfortunately, the homozygous DNM1 KO mice died shortly after birth in our study; thus, the heterozygous mice and wild type mice were used to do the polysaccharides absorption experiment.

Generation of intestinal epithelial cell specific CLTC knockout mice
CLTC loxP heterozygous (CLTC flox/wt) mice were produced by SiDanSai Biotechnology Co., Ltd (Shanghai, China). Then the CLTC heterozygous were crossed to obtain a CLTC flox/flox genetic background, subsequently, the CLTC flox/flox mice were crossed with C57BL/6 mice expressing Cre recombinase driven by the villin promoter, which was extensively in intestine. Next, resulting progeny (CLTC flox/wt mice heterozygous for villin-Cre-ERT2) were mated with CLTC flox/flox mice to generate CLTC flox/flox mice (Ctrl mice) and intestine-specific CLTC knockout mice (CLTC CKO mice). Male and female mice aged 8-10 weeks were used for the experiments.

In vivo absorption studies in mice
For the absorption experiments in BALB/c or C57BL/6J mice, mice (weighted 20–25 g) were fasted overnight except for water. Mice were orally administrated with fluorescein-labeled polysaccharides for the indicated time points, the intestine loops were removed and rinsed with saline. After washing, frozen sections of the intestine samples were obtained using a Leica CM1950 cryostat (Leica, Germany): 6-μm thick tissues sections were snap-frozen in Tissue-Tek® O.C.T. compound (Sakura, USA) and then affixed onto gelatin-coated glass slides. Then, the sections were fixed and stained, imaged by confocal microscopy (FV1000, Olympus, Japan).

For the in vivo absorption experiment in KO and CKO mice, 8-10 weeks old male and female mice (Ctrl mice (CLTC flox/flox) and CLTC CKO mice; RAB5A KO mice and wild type mice (Ctrl); DNM1+/− mice and wild type mice (Ctrl)) were orally administrated with fluorescein-labeled polysaccharides for the indicated time intervals, the mice were euthanized, and the small intestine was taken for analyses. Small intestine was embedded in Tissue-Tek OCT cryostat molds (Leica) and frozen at −80 °C, then 6-μm thick sections were generated in a cryostat (Leica CM1950, Germany). Then the tissue sections were stained with DAPI and imaged.

For the absorption experiments in C57BL/6J mice with 3H-labeled polysaccharides, C57BL/6J mice were orally administrated with 3H-labeled GFPBW1 or WGE (1200 μCi/kg), the blood samples were collected after dosing for the indicated time intervals (1 h, 2 h, 4 h, 8 h for GFPBW1, 0.5 h, 1 h, 2 h, 4 h for WGE). The radioactivity of the dosing formulation and plasma samples was determined with a liquid scintillation counter (LSC) 600 SL (Hidex, Lemminkäisenkatu, Turku, Finland).

For the in vivo absorption experiment with analysis with HPLC, C57BL/6J mice for orally administrated with fluorescein-labeled WGE 100 mg/kg, and after dosing, the blood samples were obtained after centrifugation at 3000 rpm for 5 min, plasma samples were subsequently quantified and analyzed by HPLC.

For the absorption experiments in BALB/c mice with 99mTc-labeled polysaccharides: mice were dosing by the polysaccharides at 111 MBq (3 mCi/0.5 mL) via oral administration. The blood samples and the liver were collected at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h, stored properly until analysis.

Analytical conditions and sample pretreatment procedure
Sample pretreatment (for fluorescein-labeled polysaccharides): 400 µL of ethanol was added into a 100 µL portion of plasma sample, then the mixture was vortexed for 2 min and then centrifuged at 12,000 rpm for 20 min to precipitate the polysaccharides. The precipitate was then dissolved with distilled water and vortexed. After the centrifugation for 20 min at 12,000 rpm, the supernatant was collected and lyophilized, then subjected to HPLC analysis.

Sample pretreatment (for 99mTc-labeled polysaccharides): the plasma or the liver homogenate was mixed with the saturated (NH4)2SO4 with the same volume, then stirred for 10 min, followed by centrifugation at 12,000 rpm for 15 min, the supernatant was collected and subjected to Radio-HPGPC analysis.

For fluorescein-labeled polysaccharides analysis: the assay system consisted of an Agilent 1200 series HPLC with a fluorescence detector set at λex 492 nm and λem 516 nm. Samples were separated by HPGPC using a Shodex KS-804HQ column. The eluent was 0.1 M phosphate buffer (pH 7.4), delivered at a flow rate of 0.5 mL/min; the chromatographic procedures were performed at 35 °C.

For 99mTc-labeled polysaccharides analysis: the 99mTc-labeled polysaccharides and the biological samples were analyzed by the Thermo ScientificTM DionexTM UltiMateTM 3000 HPLC (Thermo, USA) system equipped with a Berthold LB514 radiodetector. Samples were separated by HPGPC using a Waters Ultrahydrogel Liner column (300 mm × 10 μm). The eluent was 0.1 M NaNO3, delivered at a flow rate of 1.0 mL/min, the chromatographic procedures were performed at 50 °C. Data was analyzed by the Thermo ScientificTM ChromeleonTM chromatography data analysis system (Thermo, USA).

Plasmids and siRNA
Clathrin heavy chain cDNA clone and the vector control (pcmv-6) plasmids were purchased from OriGene (Rockville, MD, USA). PGMLV-CMV-H_DNM1-3×Flag-PGK-Puro, PGMLV-CMV-H_CLEC7A-3×Flag-PGK-Puro and GMLV-PGK-Puro plasmids were bought from Genomeditech (Shanghai) Co., Ltd. (Shanghai, China). pCMV-BMPR1A (human)-3×FLAG-Neo, pCMV-T7-MCS-3×FLAG-Neo, pLV3-CMV-EGFR (human)-3×FLAG-CopGFP-Puro, pLV3-CMV-RAB5A (human)-3×FLAG-CopGFP-Puro and pLV3-CMV-MCS-3×FLAG-CopGFP-Puro plasmids were purchased from miaoling plasmid (Wuhan, China). siRNA sequences were provided in table S6 in the Supplementary Information.

Cell culture and transfection
Human intestine epithelial cells (HIEC-6 cells, CRL-3266) and rat intestine epithelial cells (IEC-6, CRL-1592) were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Caco-2 (human colorectal cancer cell), HEK-293, Huh7 cells (human hepatoma cell), human microvascular endothelial cells (HMEC-1), and T24 cells (human bladder cancer cell) were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HIEC-6 cells were cultured in Opti-MEM Reduced Serum Medium (31985, Gibco, USA) supplemented with 20 mM HEPES (15630080, Gibco, USA), 10 mM GlutaMAX (35050, Gibco, USA), 10 ng/mL Epidermal Growth Factor (EGF, Sino Biological, Beijing, China), 4% fetal bovine serum (FBS, Gibco, USA), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Gibco, USA). The other cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning, USA) supplemented with 10% FBS (Gibco) and antibiotics. HMEC-1 cells were cultured in MCDB-131 medium (Meilun Bio, Dalian, China) supplemented with 2 mM L-glutamine (Meilun Bio, Dalian, China), 10 ng/mL EGF, 15% FBS, and antibiotics. All cells were cultured at 37 °C in an atmosphere of 5% CO2. Cells were routinely transfected with plasmids and siRNAs using Lipofectamine 2000 (Invitrogen, USA) or Rfect (Baidai biotech, Changzhou, China), respectively, according to the manufacturer’s instructions.

In vitro transepithelial transport studies
The transepithelial transport studies were conducted according to the literature51. All solutions used were pre-warmed (37 °C). The Caco-2 cell monolayers were firstly washed with phosphate-buffered saline (PBS). Then, the test polysaccharide solutions (5 mg/mL, 0.5 mL) were added into the apical chamber, while 1.5 mL of PBS was added into the basolateral side. Then, 0.5 mL solutions were collected from the basolateral side every 2 h up to 8 h and replaced by 0.4 mL of fresh pre-warmed PBS. The collected samples were stored at -20 °C prior to analysis. The amount of polysaccharide that permeated the Caco-2 cell monolayers was calculated using the formula: equation (1) Q n=0:5×C n+Q n−1, where C represents the concentration of the polysaccharides in the permeated samples, Q means the cumulative amounts of permeated polysaccharides in mg, while n represents the time at which the sample was collected (2, 4, 6, and 8 h). Papp (cm/s) was calculated using the equation: equation (2) Papp = (ΔQ/Δt) / (A×C0), where ΔQ/Δt is the linear appearance rate of the samples on the receiver side in mg/s, A is the surface area in cm2, and C0 is the donor concentration in mg/mL.

In vitro cellular uptake studies of polysaccharides
To estimate the uptake of polysaccharides in HIEC-6, IEC-6, Caco-2, Huh7, HMEC-1 or T24 cells, the cells under various conditions were seeded in a 12-well plate for 24 h, then the cells were incubated with fluorescein-labeled polysaccharides for 1 h. Then, the cells were fixed with 4% paraformaldehyde for 15 min followed by staining with DAPI (#4083, Cell Signaling Technology, USA) and scanning using confocal microscopy (FV1000, Olympus, Japan; Lecia TCSSPSCFSMP, Leica, Germany).

For the immunostaining assays, cells were blocked with 5% bovine serum albumin (BSA) (with 0.3% Triton X-100) for 1 h after the fixation with 4% paraformaldehyde (w/v, in PBS). Next, the cells were incubated with primary antibodies at 4 °C overnight. After washing with PBS, the cells were incubated with fluorescein-conjugated secondary antibody. After washing to remove the redundant secondary antibodies, the cells were stained with DAPI and imaged using confocal microscopy (FV1000, Olympus, Japan; Lecia TCSSPSCFSMP, Leica, Germany).

Intracellular transportation studies
To expound the intracellular transportation of polysaccharides, HIEC-6 and T24 cells were cultured with fluorescein labeled GFPBW1 and WGE for 1 h or 1.5 h. Then, the organelle localization IF antibody sample kit (#8653, Cell Signaling Technology, USA) was used to stain the subcellular organelles. Antibodies including AIF rabbit mAb, EEA1 rabbit mAb, LAMP1 rabbit mAb, PDI rabbit mAb and RCAS1 rabbit mAb were employed to stain mitochondria, endosome, lysosome, endoplasmic reticulum, and Golgi, respectively. The colocalization of polysaccharides with the subcellular organelles was imaged by CLSM and analyzed by Fiji ImageJ.

Western blotting analysis
The treated cells or mouse intestine tissues were harvested and lysed with the cell lysis buffer (#9803, Cell Signaling Technology, USA) on ice for 30 min followed by denaturation for 30 min at 95 °C and centrifugation. The supernatants were subjected to SDS-PAGE for separation, and the gels were then transferred to nitrocellulose membranes (Pall, USA), and the membranes were blocked with 5% w/v nonfat milk in TBST buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.1% Tween-20, v/v). This was followed by incubation with the relevant primary antibody at 4 °C overnight, and further incubated with an appropriate horseradish peroxidase-conjugated secondary antibody. Finally, SuperSignal West Dura Substrate (Thermo, USA) and Pierce ECL Western blot substrate (Pierce, USA) were used for signal detection, GAPDH, β-tubulin, and β-actin were employed as the internal control. All antibodies are listed in Table S6 in the Supplementary Materials.

QCM analysis of polysaccharide—cell interaction
The interaction measurements of polysaccharides—cell were performed using an Attana Cell 200 System QCM Biosensor (Attana AB, Stockholm, Sweden) according to the literatures. Shortly, T24 cells were seeded on the COP-1 chips for 24 h and then subjected to the various treatments. The cells were fixed with 4% paraformaldehyde (fixed cells) or were not fixed (living cells). Next, the cell crystals were placed in a chip holder and docked into QCM channels. Before the measurement, the chips needed to be equilibrated under a continuous flow of running buffer (PBS, pH 7.4). The flow rates were 1 and 25 μL/min for the living and fixed cells, respectively. The resonant frequency of the interaction was recorded using the Attester software (Attana AB, Stockholm, Sweden). After the experiment, the chips were stained by DAPI and the fluorescent images were taken under a fluorescent microscope (Olympus BX53, Japan).

Surface plasmon resonance (SPR) analysis
A Biacore T200 instrument was used to measure the binding kinetics of human CLTC 981-1100aa (abmart, Shanghai, China), human DNM1 1-271aa (Bioss, Beijing, China), human DNM1 (TP306284, OriGene, USA), human Rab5a (NBC1-18504, Novusbio, USA), human EGFR 1-645aa, human Dectin-1, human BMPRIA 1-152aa (Sino Biological Inc, Beijing, China) to the polysaccharides. The above proteins were immobilized on the sensor chip (CM5, GE Healthcare) using the amine coupling method according to standard protocols. Measurements were performed at 25 °C, different concentrations of polysaccharides (GFPBW1, WGE) were injected into the chips. All the procedures were performed using the HBS-EP, pH 7.4 (GE Healthcare) running buffer. Data were processed using standard double-referencing and fit to a 1:1 binding model using Biacore T200 Evaluation software.

Statistical analysis
Statistical analysis was performed using the GraphPad Prism software package. Results are expressed as mean ± standard deviation (SD) or mean ± standard error of mean (SEM). Significance (p value) of the difference between two groups was determined using Unpaired two-tailed t-test, and p < 0.05 was considered to statistically significant (*, p < 0.05; **, p < 0.01, ***, p < 0.001, ****, p < 0.0001). Differences between multiple groups were analyzed using one-way or two-way ANOVA, followed by Tukey’s or Sidak’s multiple-comparison test.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Liao et al. presents a compelling and rigorously conducted study that challenges the long-standing belief that high molecular weight polysaccharides cannot be absorbed through the gastrointestinal tract. The authors provide strong in vitro and in vivo evidence that β-1,3- and α-1,4-linked glucans can cross intestinal epithelial barriers and reach systemic circulation, with uptake mediated by clathrin-dependent endocytosis and specific cell surface receptors.\nThe study employs a broad array of techniques—including fluorescent and radiolabeling, gene silencing, knockout models, and subcellular localization—to provide a comprehensive view of polysaccharide uptake. These findings offer valuable insight for the development of orally administered polysaccharide-based therapeutics and advance our understanding of carbohydrate pharmacokinetics.\nWhile the manuscript is of high quality and scientific merit, several key issues remain that should be addressed to strengthen the conclusions and enhance the manuscript's impact.\nMajor Comments\n1. Mechanism of Entry into the Bloodstream\nWhile the authors convincingly show that polysaccharides can be absorbed by intestinal cells, the mechanism by which these macromolecules subsequently enter the bloodstream remains unclear. This is a critical gap in the current work. Investigating whether polysaccharides exit the basolateral side of intestinal epithelial cells via transcytosis, transporter-mediated efflux, or paracellular leakage would provide essential pharmacokinetic context. Without addressing this, the conclusion regarding systemic availability remains incomplete.\n2. Lack of Evidence for Direct Interaction with Clathrin\nThe study identifies clathrin-mediated endocytosis (CME) as a major route of polysaccharide uptake, yet it does not demonstrate a direct physical interaction between polysaccharides and clathrin. The current reliance on knockdown and inhibition data is supportive but insufficient to confirm a mechanistic link. Incorporating direct binding assays, such as co-immunoprecipitation, surface plasmon resonance (SPR), or pull-down assays, would substantiate the central claim and significantly reinforce the mechanistic conclusions.\n3. Specificity of Polysaccharide–Receptor Interactions\nEGFR, BMPRIA, and Dectin-1 are identified as receptors involved in polysaccharide uptake. However, the specificity and binding affinity of these interactions are not thoroughly characterized. To understand receptor selectivity and the structural basis of recognition, further experiments such as isothermal titration calorimetry (ITC), competition assays, or structural modeling would be beneficial. Additionally, it remains unclear whether other receptors may be involved; a broader screening of receptor–polysaccharide interactions would improve the generalizability of the findings.\nMinor Comments\nReproducibility across cell types: The manuscript describes cell line-dependent differences in uptake dynamics but does not explore the underlying causes (e.g., differential receptor expression, cell polarity). A more in-depth discussion or data on receptor expression profiles could provide useful context.\nExclusion of other endocytic pathways: The conclusion that polysaccharide uptake is independent of lipid raft- or caveolin-mediated endocytosis is primarily based on pharmacological inhibition. Including genetic knockout models or more specific inhibitors would provide stronger evidence.\nStatistical presentation: In several figures, error bars and statistical annotations lack clarity. Please ensure that sample sizes (n), error types (e.g., SEM or SD), and exact p-values are clearly described in both figure legends and the main text.\nLanguage and grammar: Minor grammatical errors and awkward phrasing (e.g., \"relay on\" should be \"rely on\") detract from the manuscript’s clarity. A thorough language edit is recommended to improve overall readability.", "reviewer_2": "In this manuscript, the authors attempted to elucidate polysaccharide absorption mechanisms in intestinal mucosa. The authors showed that some polysaccharide samples can permeate intestinal cell layers in vitro, and found that the isotope- or fluorescein-labeled polysaccharides are detectable in blood and liver after their oral administration to rodents. Using gene-knockdown strategies, pharmacological inhibitors, and specific knockout mice, the authors demonstrated that the polysaccharide absorption requires clathrin-mediated endocytosis (CME) machineries in both intestinal cells in vitro and intestinal mucosa in vivo. Interestingly, the authors also showed the functional involvement of plasma membrane receptors such as BMPRIA, Dectin-1, and EGFR in the cellular uptake of distinct polysaccharides.\nExperiments are carefully performed, and the information presented in this manuscript is invaluable as a foundation for oral polysaccharide-based drag development.\nMajor point #1\nIn this study, in vitro polysaccharide uptake was evaluated by measuring fluorescent intensity of the polysaccharide samples used. Considering the involvement of membrane receptors in the polysaccharide internalization, does the detected fluorescence truly reflect those of the internalized polymers only? Can the influence of those bound to the cell surface be excluded? Similar concerns exist in the results using tissue sections. The authors should clarify these points.\nMajor point #2\nUnlike a beta-1,3-linked glucan GFPBW1 and an alpha-1,4-linked WGE, are there any polysaccharides that are not absorbed in the authors' experimental systems? Such examples may reinforce the functional importance of cell membrane receptors in polysaccharide absorption.\nMajor point #3\nIn most of experiments, the polysaccharide uptake was examined by functional inhibition of CME-related molecules and some membrane receptors. Therefore, some complement results showing the increased polysaccharide uptake by functional augmentation of the target molecules are required to strengthen the author’s conclusions.\nMajor point #4\nThe authors should also show whether the transport of orally administrated polysaccharides (GFPBW1 and WGE) to serum and liver is actually restricted in mice deficient in CME-related molecules.\nMinor point #1\nThis manuscript contains a large amount of data and many repetitive explanatory expressions, making it difficult to understand the important points. It would be better to reconstruct the manuscript during the revision process.\nMinor point #2\nThis manuscript contains many misspellings and citation errors for figures. Check them carefully.", "reviewer_3": "The full article has a clear logic, a clear purpose, a good layout, and sufficient experimental data. It is a very valuable article . Accept as current format"}, "author_response": "Nov. 19th, 2025\nDear Editor and Reviewers,\nThank you very much for taking your time to review our manuscript entitled “1,3-and 1,4-linked polysaccharides uptake in intestinal cells relies on clathrin/dynamin 1/Rab5-dependent endocytosis” (NCOMMS-25-34919). Appended to this letter is our point-by-point response to your comments and questions. Our responses are given directly afterward. Furthermore, we have also uploaded files of the revised manuscript, and the changes in the revised manuscript are marked in red. We sincerely hope that our revisions will meet your expectations.\nWarm regards,\nKan Ding, Ph.D.\n[... Author response text truncated for brevity as per instructions to parse the provided text ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The additional experiments and detailed clarifications regarding polysaccharide uptake, including the validation of fluorescence specificity, exclusion of cell surface-bound signal, the use of multiple polysaccharide types, and functional overexpression of CLTC, substantially strengthen the manuscript. The manuscript is now significantly improved in scientific rigor, clarity, and completeness.\nConsidering the comprehensive revisions and the robust experimental evidence provided, I am satisfied with the responses and recommend the manuscript for acceptance.", "reviewer_2": "The authors have addressed my concerns in this revised manuscript, and I am satisfied with their responses."}, "author_response": "Dec. 29th, 2025\nDear Editor and Reviewers,\nThank you very much for taking your time to review our manuscript entitled “1,3-and 1,4-linked polysaccharides uptake in intestinal cells relies on clathrin/dynamin 1/Rab5-dependent endocytosis” (NCOMMS-25-34919A). We are so happy to receive your email that in principle, our manuscript will be able to publish on Nature Communications under an open access license. Appended to this letter is our response to your comments and questions. Our responses are given directly afterward. Furthermore, we have also uploaded files of the revised manuscript, and the changes in the revised manuscript are marked in red. We sincerely hope that our revisions will meet your expectations.\nWarm regards,\nKan Ding, Ph.D."}]

Decision letter