M cell GP2 mediated lymph targeting drug carrier

Abstract

The present invention provides a delivery agent comprising an Astragalus polysaccharide (RAP) that rapidly modulates the immune system and induces an anti-cancer immune response following oral or aerosol administration. Fluorescently labeled RAP is shown to be efficiently delivered to the small intestine or pulmonary mucosa by endocytosis of microfold (M) cells and to directly contact follicular dendritic cells (FDC) or pulmonary dendritic cells. In addition, glycoprotein 2 (GP2) deficient M cells have been demonstrated not to transport RAP and induce an immune response, which indicates that GP2 of M cells is a specific endocytosis receptor of RAP. The invention also provides that the immunomodulatory polysaccharide can be directly transported into a mucosal immune system through M cells in a GP2-dependent manner. These lymphatic targeting macromolecules are potential delivery agents for poorly bioavailability small molecules, drugs, vaccines, and the like administered via oral or aerosol drugs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 346,903, filed on May 29, 2022, the disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a traditional Chinese medicine-derived macromolecule that triggers anti-tumor immunity via a receptor-mediated lymphatic uptake pathway. In particular, the present invention provides a lymphatic targeting agent to deliver drugs and vaccines with poor bioavailability via oral administration or aerosol drug administration. Background Art

[0004] The intestinal barrier for macromolecules remains an unresolved challenge for the development of orally delivered macromolecular therapeutics. Recent proposed solutions focus on highly engineered systems, such as self-directing microinjectors, but this complexity poses challenges for manufacturing and regulation. The inventors found inspiration in polysaccharides derived from traditional herbs that have a positive effect on the immune system after oral administration. These molecules present a pharmacological paradox in that despite very low oral bioavailability, these molecules can achieve systemic effects in the human body. Understanding how medicinal polysaccharides enter the immune system can open new opportunities for the oral delivery of macromolecules and the modernization of TCM decoctions.

[0005] One potential route by which macromolecules may enter the mucosal immune system is via endocytosis by microfold (M) cells. These specialized epithelial cells, located at the immune sensors of the small intestine, the Peyer's patches (PP), use endocytosis to deliver intact pathogens and food molecules from the intestinal lumen to underlying dendritic cells to control immunity and tolerance. M cells express receptors on their luminal surface that may be targets for drug delivery. Targeted endocytosis by M cells is a promising route for oral vaccines, especially bioactive macromolecules. Given that the small intestine is a major site of polysaccharide accumulation after oral administration, it is of interest to clarify whether and how polysaccharides can be transported across the epithelial cell layer by M cells.

[0006] The development of delivery agents for poorly bioavailable drugs based on delivery routes targeting this M cell receptor-mediated pathway is an unmet need in the field of pharmaceuticals and medicinal chemistry. Summary of the invention

[0007] Therefore, the purpose of the present invention is to provide a macromolecular triggering anti-tumor immunity derived from traditional Chinese medicine. In particular, the present invention provides a targeting agent for delivering macromolecular drugs and vaccines via oral administration or aerosol administration.

[0008] In a first aspect, the present invention provides a use of astragalus polysaccharide for increasing the bioavailability of a drug in a subject.

[0009] In certain embodiments, the astragalus polysaccharide is a branched polysaccharide with an average molecular weight of 100 kDa to 1500 kDa, and the branched polysaccharide comprises Rha, Ara, Glc, Gal and GalA in a molar ratio of 0.01-0.03:1.00:0.20-10.00:0.01-0.50:0.01-0.30.

[0010] In certain embodiments, the branched polysaccharide has an average molecular weight of 1334 kDa and is composed of Rha, Ara, Glc, Gal, and GalA in a molar ratio of 0.03:1.00:0.27:0.36:0.30.

[0011] In certain embodiments, the branched polysaccharide comprises a main chain and one or more side chains, wherein the main chain comprises 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me or β-1,3,6-linked Galp; and the one or more side chains comprise α-T-Araf, α-1,5-linked Araf with O-3, T-linked Araf, T-linked Glcp and T-linked Galp.

[0012] In certain embodiments, the main chain of the branched polysaccharide is composed of 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me, and β-1,3,6-linked Galp, with branches at O-4 of 1,2,4-linked Rhap and O-3 or O-4 of β-1,3,6-linked Galp, and the side chains are mainly α-T-Araf and α-1,5-linked Araf, with O-3 as the branch point, and trace amounts of Glc and Gal; its terminal residues are T-linked Araf, T-linked Glcp, and T-linked.

[0013] In certain embodiments, the astragalus polysaccharide is covalently bonded to the drug via an optional linker to form a covalent conjugate of the astragalus polysaccharide and the drug.

[0014] In certain embodiments, the drug comprises a small molecule, a peptide, an antibody, an antibody fragment, or a vaccine.

[0015] In certain embodiments, the subject is a human or a non-human mammal.

[0016] In certain embodiments, the Astragalus polysaccharide is formulated into an orally administrable composition or an aerosolized composition.

[0017] In certain embodiments, the conjugate of the Astragalus polysaccharide and the drug is formulated into an orally administrable composition or an aerosolized composition.

[0018] In certain embodiments, the admixture is formulated as an orally administrable composition or an aerosolized composition.

[0019] In certain embodiments, the orally administrable composition is formulated as a solid, powder, liquid, gel, capsule, tablet, pill, pellet, or granules.

[0020] In certain embodiments, the aerosolized composition is formulated as particles or an aerosol.

[0021] In certain embodiments, the drug is transported via the lymphatic system at the intestinal mucosa or lung mucosa of the subject by M cell GP2-mediated endocytosis.

[0022] The second aspect of the present invention provides a delivery agent, which includes a polysaccharide that specifically targets microfold (M) cell receptors. When the polysaccharide is covalently bonded to a drug via an optional linker to form a polysaccharide drug conjugate, the resulting polysaccharide drug conjugate can be transported through the subject's lymphatic system via receptor-mediated endocytosis.

[0023] In certain embodiments, the polysaccharide comprises one or more astragalus polysaccharides, wherein the one or more astragalus polysaccharides are branched polysaccharides with an average molecular weight of 100 kDa to 1500 kDa, and the branched polysaccharides include Rha, Ara, Glc, Gal and GalA in a molar ratio of 0.01-0.03:1.00:0.20-10.00:0.01-0.50:0.01-0.30, wherein each of the branched polysaccharides includes a main chain and one or more side chains, wherein the main chain includes 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me or β-1,3,6-linked Galp; and the one or more side chains include α-T-Araf, α-1,5-linked Araf with O-3, T-linked Araf, T-linked Glcp and T-linked Galp.

[0024] Optionally, the polysaccharide may be admixed with a drug to form an admixture, and the resulting admixture may also be capable of being transported through the lymphatic system of a subject via receptor-mediated endocytosis.

[0025] In certain embodiments, the branched polysaccharide has an average molecular weight of 1334 kDa and is composed of Rha, Ara, Glc, Gal, and GalA in a molar ratio of 0.03:1.00:0.27:0.36:0.30.

[0026] In certain embodiments, the main chain of the branched polysaccharide is composed of 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me, and β-1,3,6-linked Galp, with branches at O-4 of 1,2,4-linked Rhap and O-3 or O-4 of β-1,3,6-linked Galp, and the side chains are mainly α-T-Araf and α-1,5-linked Araf, with O-3 as the branch point, and trace amounts of Glc and Gal; its terminal residues are T-linked Araf, T-linked Glcp, and T-linked.

[0027] In certain embodiments, M cells express glycoprotein 2 (GP2).

[0028] In certain embodiments, the drug is transported via the lymphatic system at the small intestinal mucosa or the lung mucosa.

[0029] In certain embodiments, the drug comprises a small molecule, a peptide, an antibody, an antibody fragment, or a vaccine.

[0030] In certain embodiments, the subject is a human or a non-human mammal.

[0031] In certain embodiments, the delivery agent is formulated as a solid, powder, liquid, gel, capsule, tablet, pill, pellet, aerosol, or granules.

[0032] A third aspect of the present invention provides a pharmaceutical composition comprising a delivery agent as described herein and at least one pharmaceutically acceptable excipient or pharmaceutically acceptable carrier.

[0033] In certain embodiments, the pharmaceutical composition is an orally administrable composition.

[0034] In certain embodiments, the pharmaceutical composition is an aerosolized composition.

[0035] A fourth aspect of the present invention provides a use of a delivery agent described herein in the preparation of a pharmaceutical composition for improving the bioavailability of a drug by inducing M cell GP2-mediated endocytosis in a subject in need thereof.

[0036] In certain embodiments, the delivery agent serves as a pharmaceutically acceptable carrier, vehicle, excipient, adjuvant or additive.

[0037] In certain embodiments, the delivery agent is used as an immunomodulator or active ingredient for preventing, pre-treating, and treating a disease or cancer in the subject.

[0038] In certain embodiments, the pharmaceutical composition further comprises a drug in addition to the delivery agent.

[0039] In certain embodiments, the pharmaceutical composition is formulated for oral administration.

[0040] In certain embodiments, the pharmaceutical composition is formulated as a solid, powder, liquid, gel, capsule, tablet, pill, pellet, or granules.

[0041] In certain embodiments, the pharmaceutical composition is formulated in aerosolized form.

[0042] In certain embodiments, the pharmaceutical composition is formulated as particles or aerosols.

[0043] In certain embodiments, the drug comprises a small molecule, a peptide, an antibody, an antibody fragment, or a vaccine.

[0044] In certain embodiments, the M cell GP2-mediated endocytosis is induced at the small intestinal mucosa or lung mucosa of the subject, and the subject includes humans and mammals other than humans.

[0045] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations thereof (such as "comprises" or "comprising") should be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. It should also be noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising", etc. may have the meanings ascribed to them in the corresponding patent laws; for example, they may mean "includes", "included", "including", etc.; and the infusion of terms such as "consisting essentially of" and "consists essentially of" have the meanings ascribed to them in U.S. patent laws, for example, which allow for elements not expressly cited, but exclude elements that exist in the prior art or affect the basic or novel characteristics of the invention.

[0046] In addition, throughout the specification and claims, unless the context requires otherwise, the word "include" or variations such as "includes" or "including", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0047] Other definitions of selected terms used herein can be found in the detailed description of the entire invention and application. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs.

[0048] Other aspects and advantages of the present invention will become apparent to those skilled in the art from a review of the ensuing description. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The above and other objects and features of the present invention will become apparent from the following description of the present invention when taken in conjunction with the accompanying drawings, in which:

[0050] Figure 1 Antitumor activity and immune response induced by orally administered RAP are shown: (A) Timeline of the 4T1 tumor-bearing mouse model. RAP was pretreated for 7 days, and then 4T1 cells were implanted into the mammary fat pad of BALB / c mice or nude mice (n=10 per group). After 21 days of RAP treatment, mice were sacrificed to collect samples; (B and C) RAP-treated BALB / c mice showed smaller tumor size and weight; (D and E) RAP-treated nude mice showed similar tumor size and weight with no difference; (F) CD8+T cells (CTLs) were efficiently increased in tumors isolated from RAP-treated BALB / c mice; (G) CD4+CD25+T cells (Tregs) were significantly reduced in tumors isolated from RAP-treated BALB / c mice; (H) Total F4 / 80+ macrophages were increased in tumors of the RAP-treated group. M1 macrophages (MTM, F4 / 80+CD11b 高 Macrophages) are induced by RAP, and tumor-associated macrophages (TAM, F4 / 80+CD11 低 (I) IL-10, (J) IFN-γ, (K) MCP-1, and (L) TGF-β in tumors isolated from BALB / c mice. Data are shown as mean ± SD. Significant differences *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.

[0051] Figure 2Different macrophages in tumor tissues induced by RAP are shown: (A) F4 / 80+CD11b+ macrophages in BALB / C mice bearing 4T1 breast tumors by flow cytometry analysis; (B) F4 / 80+CD11b+ macrophages and (C) the percentage of macrophages in nude mice bearing 4T1 breast tumors by flow cytometry analysis. M1 macrophages (MTM), F4 / 80+CD11b 高 Macrophages. Tumor-associated macrophages (TAM), F4 / 80+CD11b 低 Macrophages.

[0052] Figure 3 The distribution of polysaccharides in the bone marrow is shown. Confocal images of bone marrow cells collected from mice treated with orally administered FITC-RAP. PE-F4 / 80 was used to label macrophages, and FITC-RAP was captured by F4 / 80+ macrophages.

[0053] Figure 4 High performance gel permeation chromatography of polysaccharides and polysaccharides labeled with fluorescein isothiocyanate isomer I (FITC) are shown: (A) high performance gel permeation chromatography coupled with fluorescence detector (HPGPC-FLD) chromatography of astragalus polysaccharide (RAP) and FITC-RAP; (B) HPGPC coupled with charged aerosol detector (CAD) chromatography of FITC-RAP and RAP; (C) HPGPC-FLD chromatography of Dendrobium officinale polysaccharide DOP and FITC-DOP; (D) HPGPC-CAD chromatography of FITC-DOP and DOP.

[0054] Figure 5 The dynamic distribution of labeled RAP in the digestive tract and major organs after gavage is shown: (A) Fluorescence images of major organs, including stomach, small intestine, cecum, colon, liver, kidney, spleen and mesenteric lymph nodes (MLN) collected from normal mice (n=6 per group) from 0 to 5 hours after gavage with FITC-RAP (100 mg / kg); (B) Fluorescence intensity of stomach, cecum and colon; (C) HPGPC-FLD chromatogram of FITC-RAP in cecum; (D) HPGPC-FLD chromatogram of FITC-RAP in colon; (E) HPGPC-FLD chromatogram of FITC-RAP in serum; (F) Fluorescence intensity of liver, MLN, kidney and spleen. Data are shown as mean ± SD.

[0055] Figure 6Figure 2 shows the transport of intact polysaccharides into Peyer's patches (PP) to initiate immune responses: (A) Fluorescence intensity and (B) HPGPC-FLD chromatograms of small intestinal contents collected from normal mice at 0 to 5 hours after gavage with FITC-RAP (100 mg / kg); (C) Dynamic carbohydrate content as detected by the phenol-sulfuric acid method in small intestine collected from normal mice at 0 to 5 hours after gavage with unlabeled RAP (10 mg / kg); (D) Fluorescence intensity and (E) HPGPC-FLD chromatograms of small intestine collected from normal mice at 0 to 5 hours after gavage with FITC-RAP and FITC-RAP; (E) Fluorescence intensity and (F) HPGPC-FLD chromatograms of small intestine collected from normal mice at 0 to 5 hours after gavage with unlabeled RAP (10 mg / kg); (F) Dynamic carbohydrate content as detected by the phenol-sulfuric acid method in small intestine collected from normal mice at 0 to 5 hours after gavage with FITC-RAP and FITC-RAP; (D) Fluorescence images and (E) fluorescence intensity of PP isolated from the small intestine collected from normal mice 0 to 5 hours after TC-DOP (100 mg / kg) gavage; (F) HPGPC-FLD chromatogram of PP 2 hours after gavage with FITC-RAP (100 mg / kg); (G) Signaling pathways of NF-κB and MAPK (p38 and ERK) of PP 1 to 4 hours after oral administration with RAP (5 mg / mouse) as determined by Western blot assay. GAPDH as a control; (H) IL-6, (I) TGF-β, (J) IFN-γ and (K) M-CSF production in small intestinal PP collected from mice 24 hours after RAP treatment. PP homogenates were collected and detected with ELISA kits. Data are shown as mean ± SD. Significant differences *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. Astragalus polysaccharide RAP; Dendrobium officinale polysaccharide DOP; high performance gel permeation chromatography coupled with fluorescence detector, HPGPC-FLD; fluorescein isothiocyanate isomer I, FITC.

[0056] Figure 7 The distribution of Dendrobium officinale polysaccharide DOP in Peyer's patches (PP) and mesenteric lymph nodes (MLN) is shown: (A) HPGPC-FLD chromatogram of FITC-DOP in PP; (B) Fluorescence images of MLN collected from normal mice (n=6 per group) from 0 to 6 hours after gavage with FITC-DOP (100 mg / kg); (C) Fluorescence intensity of MLN; (D) HPGPC-FLD chromatogram of FITC-DOP of MLN. Data are shown as mean ± SD. ns = not significant.

[0057] Figure 8The uptake and endocytosis of intact RAP by M cells into PP are shown: (A) Images of whole-piece PP collected from mice in the ligated intestinal loop mouse model after FITC-RAP treatment using the IVIS Lumina XR small animal imaging system; (B) Fluorescence intensity of the region of interest (ROI) of the ex vivo image taken from above; (C) HPGPC-FLD chromatograms of PP with and without FITC-RAP treatment for 1 hour; (D) Ratio of FITC-RAP uptake between intestinal epithelial cells (IEC) and GP2+ M cells of PP. M cells and IEC were isolated from PP and PP-free small intestine segments of mice treated with FITC-RAP (100 mg / kg), respectively; (E) PP was stained with anti-GP2 mAb and DAPI after ligated intestinal loop assay with FITC-RAP. Uptake of FITC-RAP by GP2+M cells (layers 1 to 9); (F) Serial XY sections of several GP2+M cells are shown from the apical (image 1) to the basal (image 9) domain; (G) Three-dimensional image of (F); (H) XZ images of several M cells indicate that FITC-RAP is transported to the basal layer of GP2 receptors.

[0058] Fig. 9 Design and validation of an in vitro human M cell model are shown: (A) Scheme for establishing an in vitro M cell model and its application; (B) Confocal microscopy images of co-culture transwell inserts were stained with GP2 mAb followed by staining with Alexa 647-conjugated secondary antibodies. DAPI was used as a DNA-specific stain; (C) Flow cytometric analysis of nanoparticles in the supernatant collected from the lower layer of the mono- and co-culture groups (n=3); (D) Percentage of nanoparticles transported through the M cell model. Data are shown as mean ± SD. Significant difference ****p<0.0001.

[0059] Fig.10Transport of intact RAP in an in vitro model of human intestinal follicle-associated epithelium (FAE) is shown: (A) Confocal microscopy images of co-culture transwell inserts were treated with FITC-RAP and then stained with GP2 mAb followed by secondary antibody conjugated to Alexa 647. DAPI was used as a DNA-specific stain. These images show that FITC-RAP is internalized by GP2+M cells; (B) Flow cytometric analysis of FITC-RAP in supernatants collected from the lower layer of monoculture and coculture groups (n=9 per group); (C) HPGPC-FLD chromatograms of FITC-RAP in supernatants collected from the lower layer of monoculture and coculture (n=9 per group); (D) IL-6 and (E) IL-12 secretion of RAW264.7 cells treated with supernatants collected from the lower layer of monoculture and coculture (n=9 per group) 2 hours after loading 200 μl of RAP (2 mg / ml) were compared with cytokines produced by RAW 264.7 cells treated with RAP at different concentrations (0.1 mg / ml, 1 mg / ml, 10 mg / ml and 100 mg / ml). Data are shown as mean ± SD. Significant difference *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0060] Fig.11 Direct contact of M cell-transported RAP with follicular dendritic cells (FDCs) in Peyer's patches is shown: (A) Confocal image of a cryosection (dome area) of PP collected from a ligated intestinal loop assay, treated with FITC-RAP and stained with GP2, CD11c and DAPI. A 3D image of PP indicates that FITC-RAP is transported into the subepithelial dome (SED) and contacts CD11c+ dendritic cells: (B) Confocal image of a cryosection (subepithelial area) in PP.

[0061] Fig.12 GP2 is shown to bind to RAP to promote its endocytosis: (A) Cryosection of PP collected from ligated intestinal loop assay, stained with GP2 and DAPI. It was demonstrated that FITC-RAP was transported into PP by GP2+M cells; (B) Serial XY sections of PP FAE with GP2+M cells from the upper (image 1) to lower (image 10) domains of frozen PP sections. PP were collected from ligated intestinal loop assay by FITC-RAP treatment and stained with GP2 and DAPI. The three-dimensional image of PP shows that FITC-RAP binds to GP2 and is internalized from apical cells to basal cells.

[0062] Fig.13Targeted disruption of the mouse GP2 gene is shown: (A) Genotyping strategy for targeted genes. Wild-type band is 574 bp; mutant band is 557 bp. + / +, wild-type; + / -, heterozygous mice; - / -, homozygous mice; (B) Southern blot analysis of heterozygous cross progeny.

[0063] Fig.14 M cells in the small intestine by identifying specific markers GP2 and NKM 16-2-4: (A) Confocal microscopy of whole-mount PP stained with DAPI. The dome region (i.e., the location of FAE in PP) is marked with a dotted white circle; (BD) Confocal microscopy of whole-mount PP domes stained with anti-NKM 16-2-4 mAb and anti-GP2 mAb, followed by secondary antibodies labeled with Alexa 488 and Alexa 647. DAPI was used as a DNA-specific stain. Three-dimensional image of whole-mount staining of PP; (E) Confocal microscopy of frozen PP tissue isolated from wild-type BALB / c mice stained with anti-NKM 16-2-4 mAb and anti-GP2 mAb, and subsequently stained with secondary antibodies labeled with Alexa 488 and Alexa 647. DAPI was used as a DNA-specific stain; (F) Cryosection of ileal PP stained with H&E and visualized by light microscopy. Abbreviations: FAE, follicle-associated epithelium; SED, subepithelial dome; LF, lymphoid follicle.

[0064] Fig.15 Figure 2 shows that GP2 mediates endocytosis of RAP by M cells: (A) ex vivo images of PP collected from ligated intestinal loop assays and stained with anti-NKM 16-2-4 mAb followed by Alexa 647-labeled secondary antibody and DAPI; (B) images of PP collected from wild-type (WT) C57 mice, WT model mice, GP2 - / - Mice and GP2 - / - PP collected from model mice (n=20 per group) were imaged using the IVIS Lumina XR small animal imaging system in the ligated intestinal loop assay; (C) Fluorescence intensity values ​​of ROIs of the above ex vivo images; (D) HPGPC-FLD chromatograms of PP (n=20 per group) with or without FITC-RAP treatment for 1 hour; (E, F, G, H, I and J) in WT (GP2 + / + ) mice and GP2 - / - After oral administration of RAP (100 mg / kg) to mice for 24 hours (n=10 per group), GP2 - / -Mice failed to induce (E) differentiation of CD11c+DCs, (F) maturation of CD11c+CD80+DCs, (G) activation of CD11c+TLR4+DCs, and upregulated production of (H) IL-6 and (I) TNF-α, and inhibition of (J) IL-10. Data are shown as mean ± SD. Significant differences *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0065] Fig.16 Shown are the 3D images from mice without Peyer's patches, with WT Peyer's patches, and with GP2 - / - GP2+M cells in the epithelial cells of the small intestine segment of Peyer's patch in mice. The cells were stained with PE-GP2 antibody. The data are shown as mean ± SD.

[0066] Fig.17 shows that after oral administration of RAP (100 mg / kg) for 1 day (D), the + / + ) mice and GP2 - / - Flow cytometric analysis of immune cells isolated from mouse PP: (A) Percentage of activated CD11c+ dendritic cells; (B) Percentage of CD11c+MHCII+ DCs. Data are shown as mean ± SD.

[0067] Fig.18 shows that after oral administration of RAP (100 mg / kg) for 1 day (D), the + / + ) mice and GP2 - / - Flow cytometry analysis of immune cells isolated from mouse PP: (A) percentage of CD11c+CD80+DC; (B) percentage of CD11c+TLR4+DC. Data are shown as mean ± SD.

[0068] Fig.19 Figure 2 shows the transport of RAP by M cells of the human ileal PP: (A) Section of human distal ileum with the PP indicated by a black arrow; (B) Magnified view of the PP marked by a black arrow (2.5×). Figure 5(A) Tissue section of the distal ileum; (C) Confocal microscopy of a whole-mount PP dome stained with anti-GP2 mAb followed by Alexa 647-labeled secondary antibody. DAPI was used as a DNA-specific stain. The dome domain (i.e., the location of FAE in PP) is marked with a dotted white circle; (D) Confocal microscopy of a whole-mount PP dome stained with anti-human GP2 antibody and DAPI, which shows that FITC-RAP binds to GP2+M cells; (E) Three-dimensional confocal microscopy of FAE. FITC-RAP is internalized by human GP2+M cells; (F) Confocal microscopy of a frozen section of human ileal PP stained with DAPI. FITC-RAP was found in the SEM of PP. Abbreviations: FAE, follicle-associated epithelium; SED, subepithelial dome; LF, lymphoid follicle. DETAILED DESCRIPTION

[0069] The following examples are provided to demonstrate that astragalus polysaccharide (RAP)—a TCM-derived macromolecule with an average molecular weight of 1334 kDa composed of Rha, Ara, Glc, Gal, and GalA in a molar ratio of 0.03:1.00:0.27:0.36:0.30—and 1,2,4-linked Rhap, α-1,4-linked Glc, α-1,4-linked GalAp6Me, β-1,3,6-linked Galp, branched at 1,2,4-linked Rha p and O-3 or O-4 of β-1,3,6-linked Galp; mainly α-T-Araf and α-1,5-linked Araf, with O-3 as the branch point, and trace side chains of Glc and Gal; and the terminal residues of T-linked Araf, T-linked Glcp, and T-linked Galp (Yin et al., Separation, structure characterization, conformation and immunomodulating effect of a hyperbranched heteroglycan from Radix Astragali. Carbohydrate Polymers, 87(1), 667–675) how to trigger antitumor immunity after oral administration. In other embodiments, RAP may have an average molecular weight ranging from 100 kDa to 1500 kDa, including Rha, Ara, Glc, Gal, and GalA in a molar ratio of 0.01-0.03: 1.00: 0.20-10.00: 0.01-0.50: 0.01-0.30, wherein its backbone includes 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me, or β-1,3,6-linked Galp; and one or more side chains include α-T-Araf, α-1,5-linked Araf with O-3, T-linked Araf, T-linked Glcp, and T-linked Galp. RAP may form a chemical conjugate, such as a covalent conjugate with a drug to be delivered or an admixture thereof. RAP may also be formulated as a drug delivery agent or composition to be administered in conjunction with one or more drugs to be delivered or a pharmaceutical composition comprising the same. In addition to oral administration, RAP can also be formulated as an aerosol or an aerosolized composition containing the same for administration via inhalation or respiratory administration routes.The immune-dependent anti-tumor effect can be attributed to the rapid immune response induced by RAP in Peyer's patches (PP).Combined in vivo and in vitro evidence using mouse and human intestinal explants revealed that RAP is transported from the intestinal lumen to follicular dendritic cells (FDC) via endocytosis of microfold (M) cells. In addition to the intestinal mucosa via the intestinal lumen, M cells are also found in the lung mucosa, and therefore RAP can also be considered to be able to trigger the same immune-dependent anti-tumor effects in the respiratory tract as in the intestinal lumen of a subject, which can be a human or non-human mammal. Comparison with a control polysaccharide revealed that this transport pathway is selective, GP2. - / - Loss of transport in mice renders GP2 the transport receptor. M cell-mediated endocytosis of RAP was also validated in human subjects. These results resolve a long-standing paradox in the delivery of pharmaceutical polysaccharides and provide a new targeting strategy for more general oral delivery of macromolecules.

[0070] Experimental Section

[0071] Human subjects, animals, and cells:

[0072] Studies involving human participants were conducted according to established ethical guidelines and approved by the Research Ethics Committee of the School of Medicine, Zhengzhou University, Zhengzhou, China (2019-KY-373).

[0073] BALB / C mice, nude mice, and C57 BL / 6 mice were purchased from the Chinese University of Hong Kong. - / + ) mice (C57 background) were obtained from Cyagen Biosciences (Guangzhou, China). - / + The GP2-deficient mice were crossed and identified by PCR. - / - ) mice. Mice aged 5 to 8 weeks were used in this study. All animal experiments followed the Animals Ordinance guidelines, Department of Health, Hong Kong SAR (DH / HA&P / 8 / 2 / 6 (16-65), DH / HT&A / 8 / 2 / 6 (19-151)). Caco-2 cells, Raji B cells, and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC).

[0074] Antibodies and Reagents:

[0075] PE-conjugated rabbit anti-mouse glycoprotein 2 (GP2) antibody (2F11-C3), rat anti-mouse GP2 antibody (2F11-C3), and mouse anti-human GP2 antibody (3G7-H9) were purchased from MBL International. Rat anti-mouse NKM16-2-4 antibody and APC-conjugated rat anti-mouse NKM 16-2-4 antibody were obtained from Merck. CD11c primary antibody, goat anti-rabbit or rat IgG antibody conjugated with Alexa Fluor 647, and goat anti-rat IgG antibody conjugated with Alexa488 were purchased from Abcam. Flow cytometry antibodies, including CD11c-PE, MHCII-PerCP, CD80-FITC, and TLR4-FITC, were purchased from BioLegend Biotechnology. Phenol-sulfuric acid, 4′,6-diamidino-2-phenylindole DAPI, and fluorescein isothiocyanate isomer I FITC and methyl sulfoxide were obtained from Merck. Multi-fluorescent microspheres 0.20 μm were purchased from Polysciences. All human and mouse ELISA kits were purchased from eBioscience. All primers used for PCR were synthesized by Shenzhen BGI.

[0076] 4T1 Breast Tumor Mouse Model and Treatment:

[0077] Balb / c female mice and nude female mice aged five weeks were used for the tumor model (8 to 10 mice / group). The RAP-treated group or the fecal microbiota transplantation (FMT)-treated group was pretreated for one week before implantation of tumor cells. Feces used in FMT were collected from mice in the control (H2O) and RAP-treated groups and performed according to the established protocol. Implantation of 4T1 cells was performed according to previous studies. Briefly, 4T1 cells (2 × 10 4 / mouse). When the tumor began to develop, the tumor diameter was measured using a vernier caliper and the tumor volume was calculated. After three weeks of tumor development, the animals were sacrificed and samples of interest were harvested for research, including measuring tumor weight and detecting immune cells and cytokines in the tumor and immune system.

[0078] Preparation of polysaccharides and FITC-labeled polysaccharides:

[0079] RAP was prepared from an aqueous extract of the dried roots of Astragalus. It was reportedly labeled with fluorescein isothiocyanate isomer I (FITC). Briefly, RAP (1.0 g) was dissolved in methyl sulfoxide (8 mL) containing a few drops of pyridine. FITC (80 mg) was added to the RAP solution, followed by dibutyltin dilaurate (16 μL). The mixture was heated at 95°C for 2 hours. After repeated precipitation in ethanol (90% v / v) to collect the precipitate and remove the free dye, FITC-RAP was further redissolved in water and further purified by molecular sieves (3 kDa cutoff). The control polysaccharide DOP was prepared from the stems of Dendrobium officinale and labeled with FITC as previously reported.

[0080] Dynamic distribution of FITC-RAP in the mouse gastrointestinal system:

[0081] FTIC-RAP (10 mg in 1 mL of distilled water) was orally administered to each mouse (n=6). Blood and major tissues (including liver, spleen, kidney, stomach, small intestine, mesenteric lymph nodes (MLN), Peyer's patches (PP), cecum and colon) were collected immediately after mice were sacrificed at 0, 1, 2, 3, 4 and 5 hours after oral administration, and imaged using IVIS Lumina XR in vivo imaging system PerkinElmer. High performance gel permeation chromatography was used to monitor the molecular size of FITC-RAP. For Western blotting (WB), PP from different groups were collected at other time points (0, 1, 2, 3 and 4 hours after oral administration) and prepared for WB. For ELISA assay, PP were collected 24 hours after RAP treatment, then homogenized and centrifuged at 15,000 rpm for 10 minutes to obtain supernatant for evaluating cytokine production using ELISA kits. Similarly, the dynamic distribution of FITC-DOP control in MLN and PP was investigated.

[0082] The dynamic distribution of polysaccharides was analyzed by high performance gel permeation chromatography coupled to a fluorescence detector (HPGPC-FLD):

[0083] The tissues collected from the above dynamic distribution analysis were homogenized using 3 volumes of 0.1 mol / L phosphate buffer (pH 7.4) and centrifuged at 15,000 rpm for 10 minutes. In particular, 200 μL PBS was used to homogenize the PP collected from the ligation ring determination model and centrifuged at 15,000 rpm for 10 minutes. The supernatant was collected and stored at -20 ° C for chromatographic analysis. Separation was achieved on a TSK GMPWXL column (164 × 7.8 mm inner diameter, 10 μm) system, which was operated at 40 ° C using an Agilent-1100HPLC system equipped with FLD. An aqueous ammonium acetate solution (20 mM) was used as a mobile phase with a flow rate of 0.6 mL / min. The excitation wavelength and emission wavelength of FLD were 495 nm and 515 nm, respectively.

[0084] Western blotting:

[0085] PP collected at different time points (0 hour, 1 hour, 2 hours, 3 hours and 4 hours) were lysed with RIPA protein extraction reagent containing protease and phosphatase inhibitors for 30 minutes. Protein samples were separated by 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked in 5% blocking agent milk (BioRad) for 1 hour at room temperature (i.e., 20°C-25°C) and then incubated with primary antibodies at 4°C overnight with shaking. According to previous studies, the primary antibodies were GAPDH, P38, p-P38, P65, p-P65, ERK and p-ERK. The membranes were washed three times with PBST (0.1% Tween 20) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour. Protein bands were visualized using enhanced chemiluminescence (ECL) detection reagents and medical X-ray film.

[0086] ELISA assay for cytokines:

[0087] The supernatant collected from the cell culture or PP homogenate was centrifuged at 3000 g for 10 min. The cytokines IL-6, IL-10, IL-12, TNF-α, M-CSF, TGF-β, MCP-1, IFN-γ, VEGF, and MMP-9 were determined using ELISA kits according to the manufacturer's instructions.

[0088] Carbohydrate Detection by Phenol-Sulfuric Acid Method:

[0089] The intestinal contents of BALB / C mice that had received an oral dose of RAP (100 mg / kg / mouse) were collected at 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after oral administration (n=6 per group) for carbohydrate detection by the phenol-sulfuric acid method. Briefly, the intestinal contents collected from the stomach, small intestine, and large intestine of mice treated with and without RAP were homogenized and centrifuged at 15000 rpm for 10 minutes. A maximum of 50 μl of each supernatant in one well of a 96-well plate was quickly mixed with 150 μl of concentrated sulfuric acid. Then 30 μl of a 5% phenol aqueous solution was added to the mixed solution and incubated in a static water bath at 90°C for 5 minutes. The plate was then cooled to room temperature and wiped dry to be analyzed using a microplate reader at A. 490 Glucose was used as the reference standard to establish a standard curve. The experiment was repeated three times.

[0090] Ligated intestinal loop assay:

[0091] C57BL / 6 wild-type (WT) mice and GP2 - / - C57BL / 6 mice. To prepare the ligated intestinal loop, an incision was made near the linea alba of the abdomen to expose the small intestine. FITC-RAP (10 mg / mL) in 100 μl PBS was injected into the ligated intestinal loop. The mice were anesthetized for 1 hour after injection. The PP was excised and rinsed 5 times with PBS. The cleaned PP was photographed using IVIS Lumina XR, followed by HPGPC-FLD analysis, whole-mount staining, sectioning, and further microscopic analysis.

[0092] Immunofluorescence staining and confocal microscopy:

[0093] Cryosections of PP from the ligation ring assay were washed three times with PBS and blocked with 5% normal goat serum in PBS for 1 hour. Sections were incubated with anti-mouse GP2, anti-mouse NKM 16-2-4 antibodies, or anti-mouse CD11c antibodies at 4°C overnight. PP sections were washed three times with PBS and then treated with Alexa Fluor647 secondary goat anti-rabbit, AlexaFluor5 conjugated anti-rat antibodies for 1 hour at room temperature, followed by three consecutive PBS washes. Nuclei were stained with 1ug / mL of 4',6-diamidino-2-phenylindole (DAPI) for 15 minutes. Sections were washed three times with PBS and mounted with anti-fading mounting medium. Images were captured with a Leica TCS SP8 confocal laser scanning microscope.

[0094] Whole mount staining and confocal microscopy:

[0095] For whole-mount staining, mouse PP were isolated from the small intestine and washed 3 times with 1× ice-cold PBS. PP were fixed in 4% paraformaldehyde overnight at 4°C and then permeabilized with 0.1% Triton X-100 for 30 minutes. Blocked with 5% normal goat serum in PBS for 1 hour. PP were incubated with primary rat anti-mouse glycoprotein 2 (GP2) and 1 μg / ml DAPI in PBS for 1 hour at room temperature and then rinsed with PBS for 3×5 minutes. Secondary goat anti-rat IgG antibody conjugated with Alexa647 was further stained for 1 hour at room temperature. PP were washed 3×5 minutes with PBS and fixed with anti-fading reagent. Images were analyzed and captured by confocal laser microscopy (SP8, Leica Microsystems).

[0096] Flow cytometric analysis:

[0097] To detect RAP-induced immune responses, tumor tissues and PP were minced into small pieces and ground with a syringe stopper. To isolate and identify M cells, PP and small intestinal segments collected from FITC-RAP-treated mice were incubated in PBS containing 0.5 mM EDTA and stirred at 37 °C for 20 min. To isolate and detect dendritic cells, PP and small intestinal segments collected from WT mice and GP2 - / - PP collected from mice were ground with a syringe stopper. Tissues and cells were collected and rinsed with ice-cold PBS at 400 g for 5 minutes and then filtered through a 70 μm filter. Single cell suspensions were prepared for antibody staining. Cells were incubated with antibodies or matched isotypes at room temperature for 25 minutes. The stained cells were rinsed twice and resuspended in PBS and analyzed by FACSAria III (BD Biosciences). Data analysis was performed with FlowJo V10 software.

[0098] In vitro M cell-like model:

[0099] Caco-2 cells were grown in 10% (v / v) fetal bovine serum DMEM at 37°C and 10% CO2 in a water-saturated atmosphere. Caco-2 cells were grown on Transwell polyester inserts (0.3 μm pore size, 12 mm diameter, Corning Costar) coated with 100 μg / mL phenol red-free Matrigel basement membrane matrix (Becton Dickinson) prepared in pure cold DMEM without phenol red. Raji B cells (5×10 5) were resuspended in DMEM and added to the basolateral chamber of a 7-day-old Caco-2 cell monolayer. The co-culture was maintained for 5 days. A monoculture of Caco-2 cells cultured as described above (but without Raji B cells) was used as a control. The inserts were used for all of the following experiments, including the assessment of M cell function by nanoparticle transport measurements using flow cytometry, M cell display in co-cultured monolayers using immunofluorescence staining assays, and the determination of transported RAP using chromatographic analysis and ELISA assays. The transport function of this in vitro M cell model was first verified using nanoparticles. FITC-RAP (2 mg / mL) in 400 μl DMEM was added to the upper layer of the monoculture and co-culture inserts, followed by incubation for 2 hours at 37°C and 5% CO2. All inserts were removed and the lower layer solution was collected for chromatographic analysis. The lower solution collected from the co-culture group treated with unlabeled RAP was further used to treat RAW264.7 cells in 96-well plates for 24 hours, and the supernatant was sampled for IL-6 and IL-12 detection using ELISA kits.

[0100] Identification of M cells in co-culture monolayers:

[0101] Three co-culture and mono-culture inserts were used to detect the presence of M-like cells by immunofluorescence. All inserts were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 15 minutes, then blocked in 5% normal goat serum in PBS for 1 hour. Rat anti-human GP2 antibody was used as the primary antibody and incubated overnight at 4°C. After washing three times with PBS, all inserts were incubated with Alexa Fluor647-conjugated goat anti-rat secondary antibodies in the dark for 1 hour. DAPI was used to stain the nuclei for 15 minutes in the dark. All inserts were washed three times with PBS, and polyester membranes were cut and fixed using anti-fading mounting medium. Sectioning was performed with a Leica TCS SP8 confocal laser scanning microscope.

[0102] Research on Human Subjects:

[0103] Twenty volunteers who required surgery participated in the study. The age of the participants ranged from 20 to 50 years old. The distal ileum of all small intestines was collected after surgery. Twenty small intestine samples with PP were used in this study. Briefly, after surgery, the small intestine with PP was stored in ice-cold PBS with 5% FBS. The small intestine segment was ligated with surgical sutures for ligation loop determination. Then 10 ml of FITC-RAP solution (5 mg / ml) was injected and then incubated for 2 hours at 37°C, 5% CO2. PP was collected for whole-mount staining, preparation of frozen sections, and immunofluorescence staining.

[0104] Statistical analysis:

[0105] GraphPad Prism 7.0 software was used to assess statistical significance. Static analysis of the data was performed using standard t-test, one-way ANOVA followed by Tukey post hoc analysis, and two-way ANOVA followed by Bonferroni post hoc analysis. Statistical significance was determined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. All data are expressed as mean ± SD. Each experiment was performed three times or as indicated in the legend.

[0106] result

[0107] Anticancer activity of immune-dependent RAP

[0108] It has been widely reported that the various beneficial effects of astragalus polysaccharide (RAP) are related to its immunomodulatory effects. The present inventors evaluated the effects of RAP on tumor growth and related immune responses in tumor tissues in a 4T1 tumor mouse model ( Figure 1 A). The results showed that RAP significantly inhibited tumor growth ( Figure 1 B and Figure 1 C) and induce anti-tumor immune responses in tumor tissues, including an increase in CD3+CD8+ cytotoxic T lymphocytes (CTLs) ( Figure 1 F), CD4+CD25+ regulatory T cells (Treg, Figure 1 G) and F4 / 80+ macrophages ( Figure 1 H) decrease, from F4 / 80+CD11b 低 Tumor-associated macrophages (TAMs) differentiate into F4 / 80+CD11b 高 M1 macrophages (MTM) Figure 1 H and Figure 2 A), as well as changes in anti-tumor related cytokines IL-10 (I), IFN-γ (J), MCP-1 (K), and TGF-β (L). The inventors also tested the anti-tumor effect of RAP on immunodeficient nude mice, and no tumor inhibition ( Figure 1 D and Figure 1 E), and no differentiation of macrophages in tumor tissue was observed ( Figure 2 B and Figure 2 C). All these above results indicate that the anticancer activity of RAP is immune-dependent.

[0109] Intact RAP is delivered specifically to Peyer's patches (PP) to directly induce an immune response:

[0110] The inventors' previous results (Bao et al., "Radix Astragali polysaccharide RAP directlyprotects hematopoietic stem cells from chemotherapy-induced myelosuppression by increasing FOS expression", Int J Biol Macromol. 2021 Jul 31;183:1715-1722. doi:10.1016 / j.ijbiomac.2021.05.120. Epub 2021 May 24. PMID: 34044030) showed that the positive signal of fluorescently labeled RAP (FITC-RAP) bound to CD34+ cells in the bone marrow. The inventors also screened other immune cells in the bone marrow and found that F4 / 80+ macrophages could also bind to FITC-RAP ( Figure 3 ). This suggests that RAP is able to enter the host and directly induce an immune response.

[0111] The intestinal mucosal immune system is the part of the host immune system most likely to be affected by orally administered polysaccharides. Before studying the connection between RAP and immune cells, it is first necessary to figure out the dynamic distribution of oral RAP in the host. The inventors' previous research (Li et al., "Destiny of Dendrobium officinale Polysaccharide after Oral Administration: Indigestible and Nonabsorbing, Ends in Modulating Gut Microbiota", Journal of Agricultural and Food Chemistry (J. Agric. Food Chem.) 2019, 67, 21, 5968–5977, https: / / doi.org / 10.1021 / acs.jafc.9b01489) showed that Dendrobium officinale polysaccharide (DOP) could not be delivered to the host, providing a perfect control polysaccharide here. To track the localization of orally administered polysaccharides in mice, the inventors first labeled RAP and DOP with fluorescein isothiocyanate isomer I (FITC). High performance gel permeation chromatography showed that all fluorescent signals were covalently bound to their corresponding polysaccharides ( Figure 4 Whole-body imaging allows visualization and quantification of the passage of labeled RAP through the intestine ( Figure 5A to Figure 5 B). Gel permeation chromatography showed that labeled RAP remained intact in the small intestine for several hours ( Figure 6 A to Figure 6 C), but is immediately degraded in the cecum and colon ( Figure 5 C to Figure 5 D). Fluorescence measurements in the liver, kidney, spleen, mesenteric lymph nodes (MLN), and circulation showed no detectable increase ( Figure 5 E to Figure 5 F), thus confirming the low oral bioavailability of the macromolecule.

[0112] To measure the delivery of RAP to the mucosal immune system, the inventors dissected and imaged PP at different times after oral administration. RAP, but not DOP, transiently accumulated in these areas where intestinal immune cells were concentrated ( Figure 6 D). Gel permeation chromatography showed that most of the RAP in PP was not degraded ( Figure 6 E), which demonstrates the transport of intact polysaccharides across the intestinal wall. Figure 6 D to Figure 6 E and Figure 7 A) or MLN( Figure 7 B to Figure 7 The lack of DOP accumulation in D) demonstrates that RAP transport is site-specific.

[0113] As expected, intact RAP is directly transported into PP, providing an opportunity for RAP to directly contact immune cells. The inventors next tested the effect of RAP on the immune response in PP by measuring in vitro the key signaling pathways affected by RAP. The nuclear factor kappa-light-chain-enhancer of activated B (NF-κB), p38, and extracellular signal-regulated kinase (ERK) pathways were significantly activated by oral administration of RAP within a few hours ( Figure 6G), which indicates that RAP induces a rapid immune response by entering PP. Consistent with the inventors' previous in vitro report (Wei et al., "TLR-4 may mediate signaling pathways of Astragalus polysaccharide RAP induced cytokine expression of RAW264.7 cells", Journal of Ethnopharmacology, Vol. 179, 2016, pp. 243-252, ISSN 0378-8741, https: / / doi.org / 10.1016 / j.jep.2015.12.060), it was found that cytokines IL-6, TGF-β and IFN-γ were induced. Figure 6 H to Figure 6 J) and inhibited the expression of M-CSF ( Figure 6 K). These results suggest that FITC-RAP rapidly enters PP and triggers immune signaling, which may play a central role in the therapeutic effects of RAP.

[0114] M cells mediate endocytosis of intact RAP into epithelial cells:

[0115] To evaluate how RAP is transported across the intestinal wall, the inventors tracked FITC-RAP in a ligated intestinal loop mouse model in C57BL / 6 wild-type mice. The use of a whole animal imaging system showed that FITC-RAP was concentrated in the area associated with PP ( Figure 8 A and Figure 8 B), where it is present in an undegraded form ( Figure 3 C). Further flow cytometric analysis of enterocytes showed that GP2+M cells, but not other enterocytes, accumulated FITC-RAP in the ligation ring model. Figure 8 D). Specific transport of FITC-RAP by GP2+ M cells. Confocal microscopy images of fixed and stained whole mount preparations of PP isolated from mice 1 hour after FITC-RAP treatment ( Figure 8 E to Figure 8 To verify the M cell-mediated polysaccharide endocytosis, the inventors also established and verified an in vitro M cell-like model by co-culturing human intestinal epithelial cell line Caco-2 and Raji B cells on filter inserts ( Fig. 9 A to Fig. 9D) Only after differentiation into M-like cells can epithelial cells transport RAP into the lower layer of the insert ( Fig.10 A to Fig.10 E).

[0116] M cell-mediated endocytosis of RAP is GP2 receptor-dependent:

[0117] Transport of luminal molecules through M cells is thought to occur through a combination of nonspecific endocytosis, receptor-mediated endocytosis, and extension of DC processes through transcellular pores of M cells. Because the inventors did not observe transport of the control polysaccharide DOP, it is unlikely that nonspecific endocytosis makes a major contribution to the system. Confocal microscopic images of cryosections of PP isolated from mice 1 hour after oral administration of FITC-RAP show that follicular dendritic cells (FDC) receive FITC-RAP after endocytosis by M cells, but not by extending long processes to capture the material ( Fig.11 A to Fig.11 B). These observations suggest receptor-mediated endocytosis. Consistent with receptor-mediated uptake, the inventors observed that FITC-RAP colocalized precisely with GP2 receptors on the apical surface of M cells in cryosections ( Fig.12 A). A series of confocal microscopic images of whole mount staining from the apical to the basal cells on the surface of M cells shows that FITC-RAP accumulates around GP2 and is internalized into M cells ( Fig.12 B) GP2 is a known receptor for targeted endocytosis by M cells, and the present inventors therefore hypothesized that the GP2 receptor of M cells might mediate specific endocytosis of polysaccharides.

[0118] To test this hypothesis, the present inventors developed a GP2-deficient (GP2 - / - ) Mouse Model( Fig.13 A and identified by PCR assay ( Fig.13 B). The present inventors have demonstrated for the first time that both GP2 and NKM16-2-4 can be used to label M cells in wild-type (WT) mice ( Fig.14 A to Fig.14 F). Separation from GP2 - / - Whole-mount staining of mouse PP showed that NKM 16-2-4 positive signals were still detected ( Fig.15 A), but no GP2 signal was detected ( Fig.16 ). This indicates that M cells are still present in GP2 - / - The inventors then used a ligation ring assay to compare the expression of FITC-RAP in PP of WT mice and GP2 - / - Accumulation between mice. IVIS Lumina XR small animal images show that in GP2- / - In mice, the positive signal of FITC-RAP in PP was significantly reduced (compare Fig.15 B and Fig.15 C). This decrease was further confirmed by HPGPC-FLD analysis of PP-related fluorescence ( Fig.15 D).

[0119] To determine whether GP2 deficiency affects the immunomodulatory activity of RAP, the present inventors compared WT and GP2 - / - Signaling responses in immune cells between mice Fig.15 E to Fig.15 J. Fig.17 A to Fig.17 B and Fig.18 A to Fig.18 B) Among the pathways measured, RAP induced strong signaling in WT mice but not in GP2 - / - There were no statistically significant changes in mice. These data strongly suggest that GP2-mediated endocytosis of M cells is required for RAP to exhibit therapeutic activity.

[0120] M cell-mediated endocytosis of RAP in the human intestine:

[0121] To investigate whether this finding could be extended to humans, the present inventors collected live distal ileum explants from surgical patients and subjected them to PP dome screening ( Fig.19 A and Fig.19 B). The inventors incubated the PP dome with FITC-RAP and then stained the whole piece. The dome area of ​​PP is represented by a white circle ( Fig.19 C). GP2 was used as an M cell marker. FITC-RAP clustered puncta, most of which were positive for GP2 ( Fig.19 D). The two signals are not completely consistent, which may be attributed to the dynamic endocytosis process. The 3D images also confirmed and demonstrated the dynamic transport of FITC-RAP from the apical surface of the PP to the subepithelial dome in M ​​cells ( Fig.19 E). Confocal microscopy images of PP cryosections confirm that FITC-RAP can be transported into PP ( Fig.19 F) Taken together, these findings demonstrate that RAP can be transported into PP by human M cells.

[0122] discuss

[0123] The inventors' data provide the first example of orally administered polysaccharide-based pharmaceutical macromolecules being delivered to the mucosal immune system via targeted endocytosis of M cells, which is of great significance to the pharmacology of pharmaceutical polysaccharides and the oral delivery of future macromolecular drugs. Polysaccharides are one of the most important active ingredients of many TCMs, and despite their extremely low oral bioavailability, how they can achieve systemic therapeutic effects has always been a mystery. The present invention shows that the representative pharmaceutically active polysaccharide RAP enters the immune system via receptor-mediated endocytosis of M cells. Other orally administered TCM macromolecules can also use similar pathways.

[0124] This targeted delivery mode successfully bridges the gap between the in vitro and in vivo bioactivities of orally administered polysaccharides. Many in vivo studies have shown various immune-targeted bioactivities of oral polysaccharides, such as anti-tumor and diabetes alleviation, and many in vitro studies have addressed the underlying molecular mechanisms. However, due to the intestinal barrier, few in vivo and in vitro studies can be linked together to form a convincing fact. The present invention shows that intact polysaccharides can directly enter the host in the small intestine through M cells, thus supporting the possibility of direct contact between polysaccharides and cells in vivo. In fact, the inventors have previously observed that orally administered RAP appears in the bone marrow and is associated with CD34+ stem cells, which helps protect them from chemotherapy-induced bone marrow suppression. Therefore, M cell-mediated delivery reveals the targeted channel for polysaccharides to directly encounter immune cells in vivo, which provides scientific evidence for the significance of in vitro studies of polysaccharides.

[0125] Although the intestinal microbiota plays an important role in the biological activity of many polysaccharides, RAP here is independent of the intestinal microbiota. Unlike the initiation of immune responses induced by intestinal microbiota located in the large intestine, the data of the inventors show that oral administration of RAP can directly trigger the immune response of PP within a few hours. The rapid immunomodulatory effect supports the research showing that M cell-mediated antigen sampling can be completed in about 15 minutes. Combined with the results that FMT does not affect the growth of the 4T1 tumor model, the inventors speculate that the immune response in PP induced by RAP is independent of the intestinal microbiota, which is consistent with the function of polysaccharides on bile acid signaling and immunomodulation. Although some polysaccharides have also been found to enter PP and directly induce immune responses, their entry is considered to be through nonspecific endocytosis mediated by M cells. The present invention shows that the endocytosis of RAP is selective, which is GP2-dependent. Therefore, the selective M cell-mediated delivery of RAP also suggests a new in vivo polysaccharide immunomodulatory mechanism driven by an intestinal microbiota-independent manner.

[0126] Perhaps an exciting implication of the inventors' disclosure is for the development of future oral drug formulations. Oral drug delivery is the most preferred route for the administration of therapeutic agents. Despite these benefits, oral delivery of macromolecular therapeutics is limited by the physicochemical conditions in the stomach, including acidic pH, gastric enzymes, mucus layers, and epithelial cell layers, which are incompatible with the physical properties of the therapeutic agent molecules (e.g., surface charge and molecular size). Nanomaterials have been used to deliver macromolecules; however, they also have disadvantages, such as the need to use organic solvents during synthesis, which may denature proteins, toxicity at high doses, limited loading capacity, and no specificity. The field of oral vaccination has long recognized the need for M cell targeting functions to guide vaccine molecules through M cells to the mucosal immune system. In this context, several targeting molecules have been explored. So far, most are proteins or peptides, which may undergo degradation during passage through the stomach and intestines. RAP efficiently targets M cells in the present invention and is known to be non-toxic and is actually beneficial to intestinal health. The inventors propose that intact RAP or chemically defined fragments can be used as efficient, safe, and chemically stable targeting agents to deliver macromolecular drugs and vaccines via oral administration.

[0127] in conclusion

[0128] The present invention provides a delivery target site for polysaccharides in the small intestine: polysaccharides are endocytosed into Peyer's patches by M cells in a receptor-dependent manner. These results clarify the long-standing mystery of how polysaccharides are delivered after oral administration and highlight a method that allows non-digestible macromolecules to function in vivo independent of blood and intestinal microbiota. The present invention also provides a receptor-dependent strategy for the delivery of future macromolecular drugs and vaccines targeting the mucosal immune system.

[0129] Industry Applications

[0130] The present invention provides a macromolecule derived from traditional Chinese medicine as a delivery agent for triggering anti-tumor immunity. In particular, the present invention provides a targeting agent astragalus polysaccharide to deliver macromolecular drugs and vaccines via oral administration.

Claims

1. Use of astragalus polysaccharide for increasing the bioavailability of a drug in a subject.

2. The use according to claim 1, wherein the astragalus polysaccharide is a branched polysaccharide with an average molecular weight of 100 kDa to 1500 kDa, and the branched polysaccharide comprises Rha, Ara, Glc, Gal and GalA in a molar ratio of 0.01-0.03:1.00:0.20-10.00:0.01-0.50:0.01-0.

30.

3. The use according to claim 2, wherein the branched polysaccharide comprises a main chain and one or more side chains, wherein the main chain comprises 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me or β-1,3,6-linked Galp; and the one or more side chains comprise α-T-Araf, α-1,5-linked Araf with O-3, T-linked Araf, T-linked Glcp and T-linked Galp. The use according to claim 1 , wherein the astragalus polysaccharide is covalently bonded to the drug via an optional linker to form a covalent conjugate of the astragalus polysaccharide and the drug.

5. The use according to claim 1, wherein the drug comprises a small molecule, a peptide, an antibody, an antibody fragment or a vaccine.

6. The use according to claim 1, wherein the subject is a human or a non-human mammal.

7. The use according to claim 1, wherein the astragalus polysaccharide is formulated into an orally administrable composition or an aerosolized composition.

8. The use according to claim 4, wherein the covalent conjugate of the astragalus polysaccharide and the drug is formulated into an orally administrable composition or an aerosolized composition.

9. The use according to any one of claims 7 or 8, wherein the orally administrable composition is formulated as a solid, powder, liquid, gel, capsule, tablet, pill, pellet or granule.

10. The use according to any one of claims 7 or 8, wherein the aerosolized composition is formulated as particles or aerosol.

11. The use according to claim 1, wherein the drug is transported via the lymphatic system at the small intestinal mucosa or lung mucosa of the subject by M cell GP2-mediated endocytosis.

12. A delivery agent comprising a polysaccharide that specifically targets microfold (M) cell receptors, and when the polysaccharide is covalently bonded to a drug via an optional linker to form a polysaccharide drug conjugate, the resulting polysaccharide drug conjugate is capable of being transported through the subject's lymphatic system via receptor-mediated endocytosis.

13. The delivery agent of claim 12, wherein the polysaccharide comprises one or more astragalus polysaccharides, wherein the one or more astragalus polysaccharides are branched polysaccharides having an average molecular weight of 100 kDa to 1500 kDa, and the branched polysaccharide comprises Rha, Ara, Glc, Gal and GalA in a molar ratio of 0.01-0.03:1.00:0.20-10.00:0.01-0.50:0.01-0.

30.

14. A delivery agent according to claim 13, wherein each of the branched polysaccharides comprises a main chain and one or more side chains, wherein the main chain comprises 1,2,4-linked Rhap, α-1,4-linked Glcp, α-1,4-linked GalAp6Me or β-1,3,6-linked Galp; and the one or more side chains comprise α-T-Araf, α-1,5-linked Araf with O-3, T-linked Araf, T-linked Glcp and T-linked Galp.

15. The delivery agent of claim 12, wherein the M cells express glycoprotein 2 (GP2). The delivery agent according to claim 12 , wherein the drug is transported via the lymphatic system at the small intestinal mucosa or the lung mucosa.

17. The delivery agent of claim 12, wherein the drug comprises a small molecule, a peptide, an antibody, an antibody fragment, or a vaccine.

18. The delivery agent of claim 12, wherein the subject is a human or a non-human mammal.

19. The delivery agent of claim 12, wherein the delivery agent is formulated as a solid, powder, liquid, gel, capsule, tablet, pill, pellet, aerosol, or granules.

20. A pharmaceutical composition comprising the delivery agent according to any one of claims 13 to 19 and at least one pharmaceutically acceptable excipient or pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to claim 20, wherein the pharmaceutical composition is an orally administrable composition.

22. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is an aerosolized composition.

23. Use of a delivery agent according to any one of claims 13 to 19 in the preparation of a pharmaceutical composition for improving the bioavailability of a drug by inducing M cell GP2-mediated endocytosis in a subject in need thereof.

24. The use according to claim 23, wherein the delivery agent is used as a pharmaceutically acceptable carrier, vehicle, excipient, adjuvant or additive.

25. The use according to claim 23, wherein the delivery agent is used as an immunomodulator or active ingredient for preventing, pre-treating and treating a disease or cancer in the subject.

26. The use according to claim 23, wherein the pharmaceutical composition further comprises a drug in addition to the delivery agent.

27. The use according to claim 23, wherein the pharmaceutical composition is formulated for oral administration.

28. The use according to claim 27, wherein the pharmaceutical composition is formulated as a solid, powder, liquid, gel, capsule, tablet, pill, pellet or granule.

29. The use according to claim 23, wherein the pharmaceutical composition is formulated into an aerosolized form.

30. The use according to claim 29, wherein the pharmaceutical composition is formulated as particles or aerosol.

31. The use according to claim 23 or 26, wherein the drug comprises a small molecule, a peptide, an antibody, an antibody fragment or a vaccine.

32. The use according to claim 23, wherein the M cell GP2-mediated endocytosis is induced at the small intestinal mucosa or lung mucosa of the subject, and the subject includes humans and mammals other than humans.

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