Oral SMEDDS promotes lymphatic transport and mesenteric lymph nodes target of chlorogenic acid for effective T-cell antitumor immunity

Background Mesenteric lymph nodes (MLNs) are critical draining lymph nodes of the immune system that accommodate more than half of the body’s lymphocytes, suggesting their potential value as a cancer immunotherapy target. Therefore, efficient delivery of immunomodulators to the MLNs holds great potential for activating immune responses and enhancing the efficacy of antitumor immunotherapy. Self-microemulsifying drug delivery systems (SMEDDS) have attracted increasing attention to improving oral bioavailability by taking advantage of the intestinal lymphatic transport pathway. Relatively little focus has been given to the lymphatic transport advantage of SMEDDS for efficient immunomodulators delivery to the MLNs. In the present study, we aimed to change the intestinal lymphatic transport paradigm from increasing bioavailability to delivering high concentrations of immunomodulators to the MLNs. Methods Chlorogenic acid (CHA)-encapsulated SMEDDS (CHA-SME) were developed for targeted delivery of CHA to the MLNs. The intestinal lymphatic transport, immunoregulatory effects on immune cells, and overall antitumor immune efficacy of CHA-SME were investigated through in vitro and in vivo experiments. Results CHA-SME enhanced drug permeation through intestinal epithelial cells and promoted drug accumulation within the MLNs via the lymphatic transport pathway. Furthermore, CHA-SME inhibited tumor growth in subcutaneous and orthotopic glioma models by promoting dendritic cell maturation, priming the naive T cells into effector T cells, and inhibiting the immunosuppressive component. Notably, CHA-SME induced a long-term immune memory effect for immunotherapy. Conclusions These findings indicate that CHA-SME have great potential to enhance the immunotherapeutic efficacy of CHA by activating antitumor immune responses.


Fig. S1
Endocytosis and exocytosis pathways of CHA-SME in Caco-2 cells. (A) The influence of different endocytosis inhibitors on the endocytosis of CHA-SME monitored by flow cytometry. Each value represents the mean ± SEM (n = 3). ***p < 0.001 compared with the control group. (B) The effect of cholesterol on the endocytosis of CHA-SME monitored by flow cytometry. Each value represents the mean ± SEM (n = 3). **p < 0.01. (C) The influence of various endocellular transport inhibitors on the exocytosis of CHA-SME monitored by flow cytometry. Each value represents the mean ± SEM (n = 3). *p < 0.05 and ***p < 0.001 compared with the control group.
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Fig. S2
The in vitro cellular uptake profile of Cou-6-labeled CHA-SME in lymphocytes derived from MLNs determined by flow cytometry. Each value represents the mean ± SEM (n = 3). ***p < 0.001 compared with the Cou-6. The in vitro cellular uptake profile of Cou-6-labeled CHA-SME in lymphocytes derived from MLNs determined by flow cytometry. The lymphocytes derived from MLNs were stained with CD3 and CD11c, which represented as T cells and DCs, respectively. Each value represents the mean ± SEM (n = 6). ***p < 0.001. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Fig. S4
Transport mechanism of CHA-SME across Caco-2 cell monolayers. (A) The impact of the microtubule inhibitor nocodazole on the CHA-SME transportation across Caco-2 cell monolayers. Each value represents the mean ± SEM (n = 3). **p < 0.01. (B and C) CLSM images of Caco-2 cell monolayer. The Caco-2 cell monolayer was treated with Cou-6-labeled CHA-SME, washed with cold HBSS, and then incubated with the microtubule inhibitor nocodazole. The cell nuclei and cytoskeleton were stained with DAPI (blue) and phalloidin (red), respectively. The cell nuclei and cytoskeleton were stained with DAPI (blue) and phalloidin (red), respectively. "SME" represents the mice that are orally administered Cou-6-labeled CHA-SME. "SME+Cyc" represents the mice that are pretreated with cycloheximide and orally administered Cou-6-labeled CHA-SME.
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Fig. S6
In vivo antitumor efficacy of CHA-SME in a subcutaneous G422 glioma murine model. (A) The schematic of treatment schedule. The images of excised tumors (B) and tumor weight (C) of subcutaneous murine G422 glioma tumor-bearing mice after treatment of blank CHA-SME. Each value represents the mean ± SEM (n = 6).The images of excised tumors (D), tumor weight (E), tumor growth inhibition ratio (TGI%) (F), and body weight changes (G) of subcutaneous murine G422 glioma tumor-bearing mice after treatment of CHA-SME. Each value represents the mean ± SEM (n = 7). n.s., not significant. ***p < 0.001 compared with the control group. i.p., intraperitoneally; p.o., orally.  (K) Flow cytometric dot plots of cytokine production of IFN-γ and TNF-α induced by CHA-SME in tumor. Each value represents the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)