WO2023133529A2

WO2023133529A2 - Glycocalyx vesicles having surface modification of lectins for delivery of cargo to gastrointestinal tract

Info

Publication number: WO2023133529A2
Application number: PCT/US2023/060263
Authority: WO
Inventors: Michael Paul THOMAS; Joseph BOLEN; Agatha Zawadzka; Jungyeon Hwang; Jacob Thomas MARTIN
Original assignee: Puretech LYT Inc
Current assignee: Puretech LYT Inc
Priority date: 2022-01-07
Filing date: 2023-01-06
Publication date: 2023-07-13
Anticipated expiration: 2024-07-07
Also published as: WO2023133529A3

Abstract

Lectin-modified glycocalyx vesicles (GVs) for use in delivering cargos to specific sites or cells in the gastrointestinal tract. The modified GVs may comprise a lipid membrane, to which one or more lectins are attached. The one or more lectins may bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract and may have low binding affinity to the glycocalyx vesicles (GVs).

Description

GLYCOCALYX VESICLES HAVING SURFACE MODIFICATION OF LECTINS FOR DELIVERY OF CARGO TO GASTROINTESTINAL TRACT

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/334,436, filed on April 25, 2022, and U.S. Provisional Application No. 63/297,509, filed on January 7, 2022, the entire contents of each of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION Recent years have seen tremendous development of biologies and related therapeutic agents to treat, diagnose, and monitor disease. However, the challenge of generating suitable vehicles to package, stabilize and deliver payloads to sites of interest remains unaddressed.

Many therapeutics suffer from degradation due to their inherent instability and active clearance mechanisms in vivo. Poor in vivo stability is particularly problematic when delivering these pay loads orally. The harsh conditions of the digestive tract, including acidic conditions in the stomach, peristaltic motions coupled with the action of proteases, lipases, amylases, and nucleases that break down ingested components in the gastrointestinal tract, make it particularly challenging to deliver many biologies orally. The scale of this challenge is evidenced by the number of biologies limited to delivery via non-oral means, including IV, transdermal, and sub-cutaneous administration. A general oral delivery vehicle for biologies and related therapeutic agents would profoundly impact healthcare.

Recent efforts have focused on the packaging of biologies into polymer-based, liposomal, or biodegradable and erodible matrices that result in biologic-encapsulated nanoparticles. Despite their advantageous encapsulation properties, such nanoparticles have not achieved widespread use due to toxicity or poor release properties. Additionally, current nanoparticle synthesis techniques are limited in their ability to scale for manufacturing purposes and are not capable of oral delivery. The development of an effective, non-toxic, and scalable delivery platform thus remains an unmet need.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the development of glycocalyx vesicles (GVs), such as extracellular vesicles (EVs) having surface modification of specific lectins capable of binding to specific sites in the gastrointestinal tract. Such modified GVs allow for delivery of the cargo carried thereby to specific sites in the gastrointestinal tract.

Accordingly, the present disclosure provides, in some aspect, modified glycocalyx vesicle, comprising a lipid membrane, to which one or more lectins are attached. The one or more lectins bind enterocy tes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract. In some embodiments, the modified GVs may further comprise proteins associated with the lipid membrane, wherein optionally the proteins are transmembrane proteins or glycoproteins. In some instances, the lectin may be embedded in the lipids of the lipid membrane. In other instances, the lectin can be attached to one or more of the proteins associated with the lipid membrane. In some embodiments, the one or more lectins bind a site or cells in the GI tract, which is a human GI tract. In some examples, the one or more lectins bind cells in duodenum, upper jejunum, lower jejunum, ileum, cecum, colon, or rectum of the GI tract.

In some embodiments, the one or more lectins have substantially low binding activity to the modified glycocalyx vesicle. Exemplary lectins include ECL, SBA, GSL2, UEA, PNA, GSL1, WGA, PHAL, or DBA. In specific examples, the lectin is ECL, UEA1 , or a combination thereof.

In some embodiments, the one or more lectins are attached to the modified GVs via a receptor-ligand pair. In some examples, the receptor-ligand pair is biotin-streptavidin. For example, the biotin can be conjugated to the GVs (e.g., via a PEG linker) and the streptavidin, which may be monovalent, may form a fusion polypeptide with the one or more lectins. In other examples, the receptor-ligand pair is nitrilotriacetic acid-His tag. In some embodiments, the lipid membrane of the modified GVs comprises phospholipids, cholesterol, and/or tocopherol, which is conjugated to polyethylene glycol (PEG) chains. The PEG chains may have a molecular weight ranging from about 1-10 kDa. In some examples, the PEG chains may have a molecular weight ranging from about 2-5 kDa.

The one or more lectins may form a covalent bond to a functional moiety linked to the PEG chain. Example functional moieties include, but are not limited to, a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANG). In some embodiments, the modified GVs may have modified surface glycocalyx as compared with the wild-type counterpart to prevent interaction of the modified glycocalyx vesicle to the one or more lectins, to mucus, other GVs, or any combination thereof. In some examples, the modified glycocalyx comprises removal of surface sialic acid residues, change of sugar content of glycocalyx, or a combination thereof.

In some embodiments, the modified GVs may have a size of about 20-1,000 nm. For example, the size of the modified GVs is about 80-200 nm. In other examples, the size of the modified GVs is about 100-160 nm.

In some embodiments, the modified GVs comprises one or more of the following features: (1) stability under freeze-thaw cycles and/or temperature treatment; (ii) colloidal stability when the GVs are associated with the biological molecule; (iii) stability under acidic pH; (iv) stability upon sonication; and (v) resistance to enzyme digestion.

Any of the modified GVs disclosed herein may be loaded with a cargo, for example, a therapeutic agent or a diagnostic agent. In some embodiments, the cargo is a peptide, a protein, a nucleic acid, a polysaccharide, a small molecule, or a particle comprising a nucleic acid. In some examples, the particle may be a viral particle. In specific examples, the particle can be an AAV particle.

In another aspect, provided herein is a pharmaceutical composition, comprising any of the modified GVs disclosed herein and a pharmaceutically acceptable carrier. In some instances, the pharmaceutical composition may further comprise an inhibitory sugar. Examples include chitotriose, galactose, N-acetylgalactosamine, lactose, or a combination thereof. In some examples, the lectin attached to the modified glycocalyx vesicle is WGA and the pharmaceutical composition comprises chitotriose. In other examples, the lectin attached to the modified glycocalyx vesicle is ECL and the pharmaceutical composition comprises galactose, N-acetylgalactosamine, and/or lactose. Any of the pharmaceutical compositions disclosed herein may be formulated for oral administration.

Further, the present disclosure features a method for making lectin-displaying GVs, the method comprising: (i) contacting GVs (e.g., extracellular vesicles) with lipid nanoparticles carrying one or more lectins as disclosed herein to allow for fusion of the GVs and the lipid nanoparticle, thereby forming hybrid GVs displaying the one or more lectins, and (ii) collecting the fused GVs. The one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract. In some embodiments, the lipid nanoparticles carrying the one or more lectins can be prepared by a process comprising: (a) providing lipid nanoparticles comprising a lipid conjugated to a first PEG chain, which is further conjugated to a member of a receptor-ligand pair; (b) providing one or more lectins, which are conjugated to the other member of the receptor-ligand pair; and (c) contacting the lipid nanoparticles in (a) with the one or more lectins in (b) under conditions allowing for interaction between the members of the receptor- ligand pair, thereby producing the lipid nanoparticles carrying the one or more lectins. In some examples, the other member of the receptor-ligand pair is conjugated to the one or more lectins via a second PEG chain. In some examples, the receptor-ligand pair is biotin-streptavidin. In other examples, the receptor- ligand pair is nitrilotriacetic acid-His tag.

In some embodiments, the lipid nanoparticles carrying the one or more lectins may be prepared by a process comprising: (a) providing lipid nanoparticles comprising a lipid conjugated to a first PEG moiety, which is further conjugated to a first functional moiety; (b) providing one or more lectins, which is conjugated to a functional agent reactive to the functional moiety; and (c) contacting the lipid nanoparticles in (a) with the one or more lectins in (b) under conditions allowing for reaction between the first functional moiety on the lipid nanoparticles and the functional agent conjugated to the one or more lectins, thereby producing the lipid nanoparticle carrying the one or more lectins. In some examples, the first functional moiety is a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(l)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain- promoted alkyne-nitrone cycloaddition (SPANG). In some examples, the functional agent conjugated to the one or more lectins is a PEG chain, which comprises a second functional moiety that is reactive to the first functional moiety. In other embodiments, the lipid nanoparticles carrying the one or more lectins may be prepared by a process comprising: (a) contacting lipid nanoparticles with a lipid-lectin conjugate under conditions allowing for incorporation of the lipid-lectin conjugate into the lipid nanoparticles, thereby producing the lipid nanoparticles carrying the one or more lectins. In some instances, the lipid-lectin conjugate comprises a PEG chain, which connects the lipid and the lectin.

In yet another aspect, provided herein is a method for making lectin-displaying GVs, the method comprising: (i) incubating hybrid GVs with one or more lectins as disclosed herein to allow for attachment of the one or more lectins onto the hybrid GVs, thereby producing lectin-displaying GVs; and (ii) collecting the lectin-displaying GVs. The one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract. In some embodiments, the method may further comprise, prior to step (1), fusing lipid nanoparticles with GVs to form the hybrid GVs. The lipid nanoparticles comprise a lipid conjugated to a first PEG chain, which is further conjugated to a member of a receptor-ligand pair; wherein the one or more lectins are conjugated to the other member of the receptor-ligand pair. The one or more lectins are displayed on the surface of the hybrid GVs via the interaction between the members of the receptor-ligand pair.

In some examples, the one or more lectins are conjugated to the other member of the receptor-ligand pair via a second PEG chain. Examples include the biotin-streptavidin pair or the nitrilotriacetic acid-His tag pair.

In some embodiments, the method may further comprise, prior to step (i), fusing lipid nanoparticles with G Vs to form the hybrid GVs. The lipid nanoparticles may comprise a lipid conjugated to a first PEG chain, which is further conjugated to a first functional moiety. The one or more lectins are conjugated to a functional agent, which is reactive to the first functional moiety, and the one or more lectins are displayed on the surface of the GVs via the reaction between the functional moiety on the hybrid GVs and the functional agent linked to the one or more lectins.

In some examples, the first functional moiety is a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide -alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANG). In some examples, the functional agent conjugated to the one or more lectins is a PEG chain, which comprises a second functional moiety that is reactive to the first functional moiety.

In some examples, the one or more lectins are conjugated to a lipid; and wherein the lipid-conjugated one or more lectins are incorporated into the hybrid glycocalyx vesicle in step (i). In some instances, the one or more lectins are conjugated to a lipid via a PEG chain. In yet other embodiments, the present disclosure features a method for making lectin- displaying GVs, the method comprising (i) incubating GVs with one or more lectins to allow for attachment of the one or more lectins onto the GVs, thereby producing lectin-displaying GVs; and (ii) collecting the lectin-displaying GVs produced in step (i). The one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract.

In some examples, the GVs comprise a lipid conjugated to a first PEG chain, which is further conjugated to a member of a receptor-ligand pair; wherein the one or more lectins are conjugated to the other member of the receptor-ligand pair. The one or more lectins are displayed on the surface of the GVs via the interaction between the members of the receptor- ligand pair. In some instances, the one or more lectins are conjugated to the other member of the receptor-ligand pair via a PEG chain. Alternatively or in addition, the receptor-ligand pair is biotin-streptavidin or nitrilotriacetic acid-His tag. In other instances, the one or more lectins form a fusion polypeptide(s) with streptavidin, which may be monovalent.

In some examples, the GVs comprise a lipid conjugated to a first PEG chain, which is further conjugated to a first functional moiety; wherein the one or more lectins are conjugated to a functional agent, which is reactive to the first functional moiety. The one or more lectins are displayed on the surface of the GVs via the reaction between the functional moiety on the GVs and the functional agent linked to the one or more lectins. In some instances, the first functional moiety is a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I)-catalyzed azide- alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANG). In some instances, the functional agent conjugated to the one or more lectins is a PEG chain, which comprises a second functional moiety that is reactive to the first functional moiety.

In some examples, the one or more lectins are conjugated to a lipid. In some instances, the lipid-conjugated one or more lectins are incorporated into the GVs in step (i). Alternatively or in addition, the one or more lectins are conjugated to a lipid via a PEG chain.

Any of the methods disclosed herein may further comprise treating the GVs or the hybrid GVs with sialidase, a glycosylation enzyme, a glycosyltransferase enzyme, or a combination thereof to modify surface glycocalyx of the glycocalyx vesicle. For example, the method may further comprise treating the lectin-displaying GVs with sialidase, a glycosylation enzyme, a glycosyltransferase enzyme, or a combination thereof to modify surface glycocalyx of the glycocalyx vesicle. In some embodiments, any of the PEG chains used in any of the methods disclosed herein may have a molecular weight of about 1 kDa to 10 kDa. In some examples, the PEG chains has a molecular weight of about 2 kDa to 5 kDa. In any of the methods disclosed herein, the one or more lectins are selec ted from the group consisting of ECL, SB A, GSI..2, UEA, PNA, GSL1, WGA, PHAL, or DBA. In specific examples, the lectin is ECL and/or UEA1.

In any of the methods disclosed herein, the GVs may be GVs.

Also within the scope of the present disclosure are methods for delivering a cargo to the Gl tract comprising oral administration of any of the modified GVs carrying the cargo or a pharmaceutical compositions comprising such. Further, provided herein are pharmaceutical composition comprising the lectin-modified, cargo-loaded GVs for use in delivering the cargo to a GI tract site via oral administration and uses of such modified GVs for manufacturing a medicament for such proposes.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a chart showing detection of WGA binding to mouse duodenal by an in vivo imaging system (IV IS).

Figure 2 is a schematic illustration of surface functionalization strategies to conjugate lectin molecules to panicle surfaces.

Figures 3A-3D include schematic illustrations of exemplary approaches for incorporation of lectin molecules into GVs. Figure 3 A: a schematic illustration showing attachment of biotinylated lectins to the surface of streptavidin functionalized GVs. STV: lipid PEG-streptavidin. Figures 3B; a schematic illustration showing incorporation of lipid PEG- lectin into liposomes. Figure 3C: a schematic illustration showing incorporation of lipid PEG- lectin into hybrid GV/liposomes. Figure 3D: a schematic diagram depicting a process for surface loading of lectins onto GVs using azide functionalized liposomes or liposome-GV hybrid particles .

Figures 4A and 4B are charts showing fluorescent labeling of lectins. Figure 4A: 10eqVivo Tag645 NHS. Less than 1 dye per biotin-GSL2; Figure 4B: sulfoCy5.5-NHS. ~ 4dyes per biotin-GSL2 and -- 2 dyes per biotin-ECL.

Figures 5A-5C include charts showing streptavidin/biotin-GSL2 loading onto hybrid GV/DOTAP liposomes. Figure 5At absorbance. Figure 5B: fluorescence intensity obtained at the same amount of liposomes in both GV/DOTAP 2k and 2k5kSTV samples. Figure 5C: fluorescence intensity obtained at the condition of more lectin in sample with streptavidin.

Figures 6A-6C include charts showing streptavidin/biotin-ECL loading onto hybrid GV/DOTAP liposomes. Figure 6A: absorbance. Figure 6B: fluorescence intensity obtained at the same amount of liposomes in both samples. Figure 6C: fluorescence intensity obtained at the condition of more lectins in samples with streptavidin.

Figures 7A and 7B include charts showing size distribution of liposomes upon addition of DMPE-PEG5k-STV ( 7A) and upon GV/liposome fusion (7B).

Figures 8A and 8B include charts showing size distribution of particles. Figure 8A: DOTAP Liposomes: Figure 8B : DOTAP Liposome- EV fusion.

Figure 9 is a diagram showing functionalizing lectin with DSPE-PEG5k.

Figures 10A-18C include diagrams showing direct loading of lectins onto liposomes followed by fusion with GVs. Figure 10A: loading of fluorescent dye conjugated ECL onto liposomes. Figures 10B and 70C: loading of fluorescent dye conjugated lectins onto GVs via liposome-GV fusion.

Figures 11A-11C include diagrams showing direct loading of lectins onto liposomes/GVs hybrid particles. Figure 1IA: absorbance. Figures 11B and 11C: fluorescent intensities.

Figures 12A-I2C include diagrams showing azide- alkyne cycloaddition reaction of GSL2-DBCO with hybrid GVs. Figure 12 At absorbance. Figures 12B and 12C: fluorescence intensities.

Figures 13A-13C include diagrams showing azide-alkyne cycloaddition reaction of ECL-DBCO with hybrid GVs. Figure I3At absorbance. Figures 13B and 13C: fluorescence intensities.

Figures 14A and 14B include diagrams showing evaluation of the extend of surface functionalization using sulfoCy5.5-DBCO. Figure 14A: a schematic diagram depicting the evaluation process. Figure 74B: a diagram showing retention of the dyes.

Figures 15 is a schematic showing exemplary approaches for surface engineering of lectins, including hydrophobic insertion and covalent modifications. Figures 16A-16C include diagrams showing delivery of reporter proteins via GV- AAV- lectin particles using the Plasma NanoGio® assay. Figure 16A: total flux levels in plasma samples of treated mice on Day 2 post administration of the GV-AAV-lectin particles. Figure 16B: total flux levels in plasma samples of treated mice on Day 4 post administration of the GV-AAV-lectin particles. Figure 16C: total flux levels in plasma samples of treated mice on Day 10 post administration of the GV-AAV-lectin particles.

Figures 17A and 17B include diagrams showing delivery levels of reporter proteins in mice treated by the GV-AAV-lectin particles as indicated. Figure 17A: total flux levels in animals treated by the particles at different time points as indicated. Figure I7B: weight of spleens from the various treatment groups at Day 10 post treatment.

Figures 18A-18D include photos showing staining of mouse duodenal tissues with various lectins as indicated. Figure 18A: biotinylated ECL. Figure 18B: biotinylated Jacalin. Figure 18(7. biotinylated ConA. Figure 18D: biotinylated LEL.

DETAILED DESCRIPTION OF THE INVENTION

Glycocalyx vesicles (GVs) (a.k.a., glycocalyx stabilized vesicles) are vesicles carrying a glycocalyx. The glycocalyx, known as the precellular matrix, is a glycoprotein and glycolipid covering that surrounding the cell membranes of bacteria, epithelial cells, or other cells.

In some embodiments the GVs can be extracellular vesicles (EVs), which are lipid membrane-containing vesicles naturally released by many types of cells. In nature, EVs can carry various types of cargos such as protein, nucleic acids, lipids, metabolites, etc. EVs include various subtypes based mostly on biogenesis, for example, cell pathway, cell or tissue identity, condition of origin, etc.. Examples include ectosomes, microvesicles, microparticles, exosomes, oncosomes, apoptotic bodies, exomeres, etc. “Ectosome,” “microvesicle” (MV), and “microparticle” (MP) are particles released from the surface of cells. Differently, exosome biogenesis begins with pinching off of endosomal invaginations into the multivesicular body (MVB), forming intraluminal vesicles (ILVs). More details regarding differences between exosome and ectosome can be found, e.g., in Mathieu et al., Nature Communications, 12:4389 (2021), the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. EVs are an optimal vehicle for oral delivery of therapeutic agents because of their stability profile at acidic pH and other high-stress or degradative conditions. Int J Biol Sci. 2012 ;8( 1 ): 118 -23. Epub 2011 Nov 29). The present disclosure is based, at least in part, on the identification of specific lectin molecules that have high binding affinity to gastrointestinal (GI) tract cells such as intestinal cells (e.g., to human GI tract cells), for example, the lower part of the GI tract such as duodenum, upper jejunum, lower jejunmn, ileum, cecum, colon, or rectum. The lectins disclosed herein may have low or no binding affinity to GVs as disclosed herein. Such lectin molecules can be used to modify surfaces of GVs for on-site delivery of cargos carried by the GVs to GI cells, which may subsequently be delivered to the whole body. With a low binding affinity to G Vs, use of the specific lectin molecules disclosed herein to modify GVs can minimize the risk of causing GV aggregation. Accordingly, provided herein are modified GVs displaying one or more of the lectins disclosed herein, compositions comprising such, and methods for producing the modified GVs. Also provided herein are methods for using the modified GVs to deliver cargos carried thereby to specific GI sites or tissue/cells mediated by the surface-displaying lectins. I. Glycocalyx Vesicles Having Surface Modification of Lectin

In some aspect, the present disclosure provides modified glycocalyx vesicles (GVs) comprising a lipid membrane, to which one or more lectins are attached. The one or more lectins bind specific sites or cells in the GI tract.

A. Lectins Targeting Gastrointestinal Sites Lectins are a family of proteins capable of binding to carbohydrate molecules or carbohydrate moieties that is a part of other molecules, for example glycoproteins or glycolipids. The sugar binding specificities of exemplary lectins are provided in Tables 1-4 in Example 1 below. Lectins typically do not have enzymatic activity.

The lectins for use in the modified GVs disclosed herein have binding specificity to specific sites or cells in the GI tract, for example, small intestine and large intestine in the GI tract. In some instances, the lectins disclosed herein binds specific sites or cells in the human GI tract, for example, the small intestine or the large intestine in the human tract. In some embodiments, the lectins disclosed herein may bind to enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract, e.g., human GI tract. Exemplary GI tract compartment to which the lectins bind may include duodenum, upper jejunum, lower jejunum, ileum, cecum, colon, or rectum. In some examples, the lectin disclosed herein is erythrina cristagalli lectin (ECL), also known as EGA. ECL consists of two different subunits of approximately 28 kDa and 26 kDa. It binds the carbohydrate structure of Galβ4GlcNAc, which is frequently found in membrane and serum glycoproteins of mammalian origin. In some examples, the lectin disclosed herein is a soybean agglutinin (SBA), also known as SBL, which is a family of lectins found in soybean. SBAs have a molecular weight of 120 kDa and an isoelectric point near pH 6.0. SBAs preferentially bind to oligosaccharide structures with terminal a- or β-linked N -acetylgalactosamine, and to a lesser extent, galactose residues. In some examples, the lectin disclosed herein is Griffonia (Bandeiraea) Simplicifolia

Lectin, including GSL-I (GSL-1), GSL-II (GSL-2), or a combination thereof. GSL 1 is a family of glycoproteins with molecular weights of approximately 114 kDa. There are two types of subunits, termed A and B, with slightly different molecular weights. These subunits combine to form tetrameric structures, resulting In five isolectins. The A-rich lectin preferentially agglutinates blood group A erythrocytes and thus appears to be specific for a-N- acetylgalactosamine residues, while the B-rich lectin preferentially agglutinates blood group B cells and is specific for a-galactose residues. GSL-2 is a dimeric glycoprotein composed of two subunits of nearly identical size with each subunit having di sulfide- linked chains and a binding site for a- or β-linked N-acetylglucosamine residues. In some examples, the lectin disclosed herein is Ulex Europaeus Agglutinin (UEA), for example, UEA-I, UEA II, or a combination thereof. UEA-I consists of two subunits and reacts strongly with a(l,2) linked fucose residues but poorly or not at all with a(l,3) or a(l,6)-linked fucose. UEA-II is a glycoprotein that consists of four 24,000 Da monomer subunits, which require Ca2+ for binding to its ligands through carbohydrate recognition domain. It is specific for di-N-acetylchitobiose, an oligomer of GlcNAc.

In some examples, the lectin disclosed herein is peanut agglutinin (PNA), which binds preferentially to the T- antigen, a galactosyl (β-1,3) N-acetylgalactosamine structure present in many glycoconjugates such as M and N blood groups, gangliosides, and many other soluble and mem brane- associated glycoproteins and glycolipids. The protein is 273 amino acids in length with the first 23 residues acting and a signal peptide, which is subsequently cleaved.

In some examples, the lectin disclosed herein may be wheat germ agglutinin (WGA), which binds N-acetyl-D-glucosamme and Sialic acid. In solution, WGA exists mostly as a heterodimer of 38,000 daltons. It is cationic at. physiological pH. It contains a Carbohydrate- binding module called CBM18.

In some examples, the lectin disclosed herein may be Phaseolus Vulgaris

Leucoagglulinin (PHA), which is a family of lectin each consisting of four subunits. There are two different types of subunits. One appears to be involved primarily in red cell agglutination and has been designated the “E” subunit (PHA-E for erythroagglutinin). The other type is involved in lymphocyte agglutination and mitogenic activity and has been termed the “L” subunit (PHA-L for leucoagglulinin). These subunits combine to produce five isolectins. PHA has carbohydrate-binding specificity for a complex oligosaccharide containing galactose, N- acetylglucosamine, and mannose.

In some examples, the lectin disclosed herein may be Dolichos Biflorus (Horse Gram) agglutinin (DBA), which is a glycoprotein having a molecular weight of about 111 kDa and consists of 4 subunits of approximately the same size. DBA has a carbohydrate, specificity toward a-linked N-acetylgalactosamine. It is commonly used to examine secretor status in blood group A individuals by hemagglutination inhibition assays and in blood typing.

Any of the lectins for use in the modified GVs disclosed herein may be prepared by a conventional method. In specific examples, the lectin may be ECI... In other specific examples, the lectin may be UFA, such as UEA1. In some instances, the lectin may be isolated from a suitable natural source. In other instances, the lectin may be produced by the conventional recombinant technology .

Any of the lectins disclosed herein can be attached to the surface of the modified GVs in any suitable means. The lectin can be displayed directly on the surface of the GVs allowing for its binding to the corresponding sugar moiety. In some instances, at least a portion of the lectin can be embedded in the bilayer of the lipid membrane of the GVs. In some instances, the lectin may be associated with lipids in the lipid membrane of the GVs via, e.g., covalent linkage or non-covalent interaction. In some instances, the lectin can be attached to one or more proteins in the lipid membrane of the GVs. For example, the lectin may be part of a fusion protein with a protein of the GVs. In other examples, the lectin may be associated to a protein of the glycocalyx vesicle via covalent linkage or non-covalent interaction. The lectin may be linked to the protein of the glycocalyx vesicle via a linker, e.g., a peptide linker or a chemical linker. B. Glycocalyx Vesicles Modified with Surface Lectins

The lectin modified GVs disclosed herein comprise GVs, which refer to any lipid bound vesicles secreted by cells into extracellular space. GVs, including microvesicles, typically are in the form of small assemblies of lipids about 20 to 1000 nm in size. The lipids in glycocaly x vehicles often form membrane structures, to which one or proteins are associated (e.g., attached to the surface of the lipid membrane and/or embedded inside the lipid membrane).

The GVs for use in the present disclosure may be ectosomes, microvesicles (MVs), exosomes, and/or apoptotic bodies, which are subtypes of GVs differentiated based on their biogenesis, release pathways, size, content, and function. In some examples, the GVs for use in the present disclosures are ectosomes.

GVs, for example, extracellular vesicles or EVs, can encapsulate or otherwise carry therapeutic cargos such as miRNA species, and can enable oral delivery of a variety of therapeutic agents. The present disclosure harnesses lectin-modified GVs such as lectin- modified GVs to meet the urgent need for suitable delivery vehicles for therapeutics that were previously not orally administrable or suffered from other delivery challenges such as poor bioavailability, storage instability, metabolism, off-target toxicity, or decomposition in vivo.

In some embodiments, the glycocalyx vesicle is approximately round or spherical in shape. In some embodiments, the glycocalyx vesicle is approximately ovoid, cylindrical, tubular, cube, cuboid, ellipsoid, or polyhedron in shape. In some embodiments, the extracellular vesicle may be part of a cluster, collection, or formation of GVs.

In some embodiments, the compositions comprising GVs for use in delivering cargos such as those disclosed herein may have a relative abundance of proteins with a molecular weight of about 25-30 kDa (e.g., casein) no greater than about 40% and/or a relative abundance of proteins with a molecular weight of about 10-20 kDa (e.g., lactoglobulin) no greater than 25%. a. Size of Glycocalyx vesicles

In some descriptions, e.g., where diameter is a relevant measurement, such as in spherical and other shaped vesicles having a measurable diameter, the terms “size” and “diameter” are used interchangeably. The GVs, such as EVs can be about 20 nm - 1000 nm in diameter or size. In some embodiments, the glycocalyx vesicle is about 20 nm to about 200 nm in size. In some embodiments, the glycocalyx vesicle is about 20 nm to about 190 nm or about 25 nm to about 190 nm in size. In some embodiments, the glycocalyx vesicle is about 30 nm to about 180 ran in size. In some embodiments, the glycocalyx vesicle is about 35 nm to about 170 nm in size. In some embodiments, the glycocalyx vesicle is about 40 nm to about 160 nm in size. In some embodiments, the glycocalyx vesicle is about 50 nm to about 150 nm, about 60 nm to about 140 nm, about 70 nm to about 130 nm, about 80 nm to about 120 nm, or about 90 nm to about 110 nm in size. In some embodiments, the glycocalyx vesicle is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, or about 200 nm in size or diameter. In some embodiments, an average vesicle size in a vesicle composition or plurality of vesicles isolated or derived from a suitable source is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, or about 200 nm in average size. In some embodiments, an average vesicle size in a vesicle composition or plurality of vesicles isolated or derived from a suitable source is about 20 nm to about 200 nm, about 20 nm to about 190 nm, about 25 nm to about 190 nm, about 30 nm to about 180 nm, about 35 nm to about 170 nm, about 40 nm to about 160 nm, about 50 nm to about 150, about 60 to about 140 nm, about 70 to about 130, about 80 to about 120, or about 90 to about 110 nm in average size. The size of the GVs disclosed herein is determined by Dynamic Light Scattering (DLS) or nanoparticle tracking analysis (NTA). b. Source of Glycocalyx vesicles

The glycocalyx vehicles described herein can be derived from any suitable source, for example, cultured cells capable of producing GVs, biological samples such as tissue samples or body fluid samples. GVs

In some embodiments, a material for use in purifying the GVs can be lyophilized. Lyophilized materials can be reconstituted using standard procedures as recommended by manufacturer's instruction and/or as known in the art, for example, by mixing distilled water with lyophilized starting material or the GVs derived therefrom at room temperature such that the starting material or the GVs derived therefrom is present at a suitable final concentration by weight relative to water. The GVs described herein can be any types of particles found in a suitable source as disclosed herein. c. Biological Components of Glycocalyx vesicles

In some embodiments, the GVs used in the methods describes herein (e.g., EVs) may comprise one or more of the following molecules: lipid, protein, glycoprotein, glycolipid, lipoprotein, phospholipid, phosphoprotein, peptide, glycan, fatty acid, sterol, steroid, and combinations thereof. Typically, the GVs described herein comprise a lipid-based membrane to which one or more proteins are associated. The proteins may be attached to the surface of the lipid membrane or embedded in the lipid membrane. Alternatively or in addition, the proteins may be encapsulated by the lipid membrane. In some instances, the GVs may contain endogenous RNA, such as miRNA.

Lipid Membrane of GVs

The GVs may comprise one or more lipids selected from fatty acid, sterol, steroid, cholesterol, and phospholipid. In some embodiments, the lipid membrane of the GVs described herein may comprise ceramides or derivatives thereof, gangliosides, phosphatidylinositols (PI) such as alpha- lysophosphatidylinositol (LPI), phosphatidylserine (PS), cholesterol (CHOL), phosphatidic acids (PA), glycerol or derivatives thereof, such as diacylglycerol (DAG) or phosphatidylglycerol (PG), sphingolipids, or combinations thereof. Ceramides are a family of lipid molecules composed of sphingosine and a fatty acid. Examples include, but are not limited to, ceramide (Cer), lactosylceramide (LacCer), hexosylceramide (HexCer), and globotriaosylceramide (Gb3). Gangliosides are a family of molecules composed of a glycosphigolipid with one or more sialic acids, for example, n-acetylneuraminic acid (NANA). Examples include, but are not limited to, GM1, GM2, GM3, GDla, GDlb, GD2, GTlb, GT3, and GQ I. Sphingolipids are a class of lipids containing a backbone of sphingoid bases and a set of aliphatic amino alcohols that includes sphingosine. Examples include sphingomyelin (SM).

Alternatively or in addition, the GVs may contain lipids such as phosphatidylcholines (PC), cholesteryl ester (CE), phosphatidylethanolamine (PE), anchor lysophospha tidylethanol amine (LPE) .

Proteins, polypeptides, and peptides of GVs

The GVs described herein may comprise one or more proteins, which may be associated with the lipid membranes also described herein. A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function.

Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. In some instances, a peptide may contain ten or more amino acids but less than 50. In some instances, a polypeptide or a protein may contain 50 or more amino acids. In other instances, a peptide, polypeptide, or protein may have a mass from about 10 kDa to about 30 kDa, or about 30 kDa to about 150 or to about 300 kDa.

Exemplary proteins may contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these.

In some embodiments, the GVs disclosed herein are EVs purified from a suitable source. Such GVs may comprise ectosomes. In some instances, the GVs disclosed herein comprise one or more proteins of CD9, CD81, BSG, and SLC3A2. Alternatively or in addition, the GVs disclosed herein are free of CD63 and/or LAMP1 , e.g., detection of the involved proteins (e.g., CD63 and LAMP1) by a conventional method or only marginal signal is detected such that presence or absence of the involved proteins cannot be determined.

Any of the protein moieties in the glycocalyx vesicle may be glycosylated, i.e., linked to one or more glycans at one or more glycosylation sites. A glycan is a compound consisting of one or more monosaccharides linked glycosidically, including for example, the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. Glycans can be homo- or heteropolymers of monosaccharide residues and can be linear or branched. Glycans can have O-glycosidic linkages (linked to oxygen in a serine or threonine residue of a peptide chain) or N-Linked linkages (linked to nitrogen in the side chain of asparagine in the sequence Asn-X-Ser or Asn-X-Thr, where X is any amino acid except proline). Glycans bind lectins and have many specific biological roles in cell-cell recognition and cell-matrix interactions.

The glycosylated proteins that can be present in the biological membrane of a glycocalyx vesicle as described herein can include any appropriate glycan. Examples of glycans include, without limitation, N-glycans (e.g., N-acetyl-glucosamines and N-glycan chains), O-glycans, C- glycans, sialic acid, galactose or mannose residues, and combinations thereof. In some embodiments, the glycan is selected from an alpha-linked mannose, Gal β 1-3 GalNAc 1 Ser/Thr, GalNAc, or sialic acid. In some embodiments, the glycocalyx vesicle comprises one or more glycoproteins or glycopolypeptides having a glycan selected from: galactose, mannose, O-glycans, N-acetyl- glucosamines, and/or N-glycan chains or any combination thereof. In some embodiments, the glycocalyx vesicle comprises one or more glycoproteins or glycopolypeptides having a glycan selected from: D- or L- glucose, erythrose, fucose, galactose, mannose, lyxose, gulose, xylose, arabinose, ribose, 2'-deoxyribose, glucosamine, lactosamine, polylactosamine, glucuronic acid, sialic acid, sialyl -Lewis X (SLex), N-acetyl-glucosamine, N- acetyl-galactosamine, neuraminic acid, N-glycolylneuraminic acid (Neu5Gc), N- acetylneuraminic acid (Neu5Ac), an N-glycan chain, an O-glycan chain, a Core 1 , Core 2, Core 3, or Core 4 structure, or a phosphate- or acetate-modified analog thereof or a combination thereof. In some embodiments, the glycocalyx vesicle comprises a glycoprotein having one or more of the following glycans: terminal b-galactose, terminal a-galactose, N- acetyl-D-galactosamine, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine.

In some instances, any of the glycans described herein may exist in free form in the GVs, which are also within the scope of the present disclosure. In some instances, the GVs may be treated by a suitable approach (e.g., enzyme digestion) to reduce the amount of surface sialic acid residues or remove substantially surface sialic acid residues.

In some embodiments, the GVs or a composition comprising such contain proteins having a molecular weight of about 25-30 kDa at a relative abundance of no greater than 40% (e.g., less than about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about

10%, about 5% or less). As used herein, the term “relative abundance” of protein X in the composition refers to the percentage of protein X in the total protein content in the composition. In some instances, the proteins having a molecular weight of about 25-30 kDa are caseins. In some examples, the GVs or the composition comprising such may be substantially free of casein, e.g., cannot be detected by a conventional method or only a trace amount can be detected by the conventional method. Alternatively or in addition, the GVs or a composition comprising such contain proteins having a molecular weight of about 10-20 kDa at a relative abundance of no greater than 25% (e.g., less than about 25%, about 20%, about 15%, about 10%, about 5% or less). In some instances, the proteins having a molecular weight of about 10- 20 kDa are lactoglobulins. In some examples, the GVs or the composition comprising such may be substantially free of lactoglobulins. As used herein, the term “casein” refers to a family of related phosphoprotein having a molecular weight of about 25-30 kDa. Exemplary species include alpha-S 1 -casein (αS1 ), alpha-S2-casein (aS2), β-casein, K-casein. A casein protein may refer to a specific species as known in the art, for example, those noted above. Alternatively, it may refer to a mixture of at least two different species. In some instances, casein can be the population of all casein proteins prepared from a suitable mammalian source, for example, any of those described herein (e.g., cow, goat, sheep, yak, buffalo, camel, or human).

Lactoglobulin, including a-lactoglobulin and β-lactoglobulin, is a family of proteins ha ving a molecular weight of about 10-20 kDa. β-lactoglobulin typically has a molecular weight of about 18 kDa and a-lactoglobulin typically has a molecular weight of about 15 kDa. The term “lactoglobulin” may refer to one particular species, e.g., a-lactoglobulin or |J- lactoglobulin. Alternatively, it may refer to a mixture of different species, for example, a mixture of a-lactoglobulin and β-lactoglobulin.

Besides the other features disclosed herein (e.g., stability), casein and/or lactoglobulin- depleted GVs or compositions comprising GVs have a higher cargo loading capacity, e.g., oligonucleotide loading capacity, as compared with GVs prepared by the conventional ultracen tri fug a ti on m eth od. d. Stability of GVs

The GVs described herein are stable under, for example, harsh conditions, e.g., low or high pH, sonication, enzyme digestion, freeze-thaw cycles, temperature treatment, etc. Stable or stability means that the GVs maintain substantially the same intact physical structures and substantially the same functionality as relative to the GVs under normal conditions. For example, a substantial portion of the GVs (e.g., at least 60%, at, least 70%, at least 80%, at least 90%, or above) would have no substantial structural changes when they are placed under an acidic condition (e.g., pH < 6.5) for a period of time. Alternatively or in addition, the GVs may be resistant to enzymatic digestion such that a substantial portion of the GVs (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above) would have no substantial structural changes in the presence of enzymes such as digestive enzymes. Further, the GVs that are stable after multiple rounds of freeze- thaw cycles (e.g., up to 6 cycles) would have a substantial portion (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above) that has no substantial structural changes and/or functionality changes after the multiple freeze-thaw cycles. Because of, at least in part, the stability of the GVs described herein, such GVs are able to deliver their cargo while withstanding stressed conditions or conditions under which the therapeutic agent would become deactivated, metabolized, or decomposed, e.g., saliva, digestive enzymes, acidic conditions in the stomach, peristaltic motions, and/or exposure to the various digestive enzymes, for example, proteases, peptidases, lipases, amylases, and nucleases that break down ingested components in the gastrointestinal tract.

In some embodiments, the glycocalyx vesicle is stable in the gut or gastrointestinal tract of a mammalian species. In some embodiments, the glycocalyx vesicle is stable in the esophagus of a mammalian species. In some embodiments, the glycocalyx vesicle is stable in the stomach of a mammalian species. In some embodiments, the glycocalyx vesicle is stable in the small intestine of a mammalian species. In some embodiments, the glycocalyx vesicle is stable in the large intestine of a mammalian species. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 1.5 to about pH 7.5. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 2.5 to about pH 7.5. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 4.0 to about pH 7.5. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 1.5 to about pH

3.5. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 2.5 to about pH 3.5. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 2.5 to about pH 6.0. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 4.5 to about pH 6.0. In some embodiments, the glycocalyx vesicle is stable at a pH range of about pH 6.0 to about pH 7.5. In some embodiments, the glycocalyx vesicle is stable at about pH 1.5, pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH

6.5, pH 7.0, or pH 7.5, and increments between about pH of 1.5 and about pH 7.5. In some embodiments, the glycocalyx vesicle is stable in the presence of digestive enzymes, such as, for example, proteases, peptidases, nucleases, pepsin, pepsinogen, lipase, trypsin, chymotrypsin, amylase, bile and pancreatin (digestive enzymes in pancreas). In some embodiments, the glycocalyx vesicle is stable in the presence of pepsin or pancreatin. In particular embodiments, the GVs disclosed herein can protect cargo loaded therein (e.g., oligonucleotides) from enzyme digestion (e.g. , nuclease digestion).

In some embodiments, the GVs disclosed herein are stable after multiple rounds of freeze-thaw cycles. For example, the GVs are stable after at least two freeze-thaw cycles, e.g., at least 3 cycles, at least 4 cycles, at least 5 cycles, or at least 6 cycles. In some instances, the GVs are stable up to 10 freeze-thaw cycles, e.g., up to 9 cycles, up to 8 cycles, up to 7 cycles, or up to 6 cycles.

In some embodiments, the GVs disclosed herein are stable after temperature treatment, e.g., incubated at a low temperature (e.g., at 4 °C) for a period (e.g., 1-3 days) or at a high temperature for period, e.g., at 60-80 °C for 30 minutes to 2 hours or at 100-120 °C for 5-20 minutes.

Further, the GVs disclosed herein have colloidal stability. Colloidal stability refers to the long-term integrity of dispersion and its ability to resist phenomena such as sedimentation or particle aggregation. This is typically defined by the time that dispersed phase particles can remain suspended without producing precipitates. Alternatively or in addition, the GVs may be stable under physical processes, for example, sonication, centrifugation, and filtration. e • GV Modification

In addition to the surface lectin modification, any of the GVs disclosed herein may be further modified to alter one or more lipids, proteins, glycoproteins, glycolipids, lipoproteins, phospholipids, phosphoproteins, peptides, glycans, fatty acids, and/or sterols present in the natural glycocalyx vesicle. In some embodiments, the glycocalyx vesicle is modified by altering the quantity, concentration, or amount of a biomolecule naturally present, e.g., the addition or complete or partial removal of a biomolecule naturally present (e.g., carbohydrate, such as a glycan; fatty acid, lipid). In some embodiments, the glycocalyx vesicle is modified by the addition of a biomolecule not naturally present (e.g., carbohydrate, such as a glycan; fatty acid; lipid, or protein, e.g., a glycoprotein). In some embodiments, the glycocalyx vesicle is modified to alter one or more lipids in the glycocalyx vesicle. In some embodiments, the lipid component of the glycocalyx vesicle is modified or altered, e.g., via the addition of one or more lipids not naturally present in the glycocalyx vesicle or by altering the amount (increasing or decreasing) of one or more lipids naturally present in the glycocalyx vesicle. In some embodiments, the glycocalyx vesicle is modified to increase one or more lipids selected from one or more of the following lipids: LPE, PEO/PEP, Cer, DAG, GM2, PA, Gb3, LacCer, GM1, GM3, HexCer, GDI, PS, Choi, LPI, and SM, In other examples, the glycocalyx vesicle is modified to reduce or remove one or more lipids. For example, methyl -beta-cyclodextrin can be used to extract cholesterol from GVs. The lipid component of the glycocalyx vesicle can be altered or modified by known methods, including, for example, fusion with another vesicle having a lipid bilayer, e.g., liposome and/or lipid nanoparticle.

In some embodiments, the altering the amount or content of the lipids on the glycocalyx vesicle affects the ability of the GV to interact, bind and/or fuse with another vesicle, e.g., a lipid particle encapsulating a particle to which a nucleic acid is attached as those disclosed herein. In some embodiments, altering the amount or content of lipids in the GV alters the overall charge of the GV. For example, the lipid contents of the GV may be altered such that it is negatively charged, which would facilitate its fusion with positively charged lipid particles comprising viral particles. In some embodiments, altering the charge of the vesicle makes the vesicle more attractive for interactions, binding and/or fusion with another vesicle, e.g., a nanoparticle, e.g., a lipid nanoparticle. For example, in some embodiments, lipid nanoparticles and GVs having lipid contents with opposite electrostatic charges are used to promote or improve interactions, binding and/or fusion between the two types of particles. In some embodiments, interactions, binding and/or fusion is achieved between cargo-carrying lipid nanoparticles comprising negatively charged lipids and GVs comprising positively charged lipids. In other embodiments, fusion is carried out between cargo-carrying lipid nanoparticles comprising positively charged lipids and GVs comprising negatively charged lipids.

In some embodiments, the GV comprises one or more glycoproteins. In some embodiments, the GV comprises a biological membrane, wherein the biological membrane comprises one or more glycoprotein(s). In some embodiments, the biological membrane is modified as compared with the natural biological membrane of the GV. In some embodiments, the biological membrane is modified such that it has an increased number of one or more of its native glycoprotein(s). In some embodiments, the biological membrane is modified such that it, has a decreased number of one or more of its native glycoprotein(s). In some embodiments, the GV is modified such that it includes one or more glycoprotein(s) that is not naturally present in the natural biological membrane. In some embodiments, a GV having a decreased number of one or more of its native glycoprotein(s) is produced using an enzyme selected from a serine protease, cysteine protease or metalloprotease. In some embodiments, the enzyme is selected from trypsin, AspN, GluC, ArgC, chymotrypsin, proteinase K, and Lys-C. In some embodiments, the biological membrane is modified such that one or more of its native glycoprotein(s) is eliminated or not present. In some embodiments, the biological membrane is modified such that one or more of its native glycoprotein(s) is reduced.

In some embodiments, the glycocalyx vesicle is modified to alter the amount or content of carbohydrate moieties present on a glycopolypeptide present in or associated with the glycocalyx vesicle. In some embodiments, the glycocalyx vesicle is modified to increase, decrease, or otherwise alter the glycan content of the glycocalyx vesicle, e.g., via the addition of one or more glycans not naturally present in the glycocalyx vesicle or by altering the amount (increasing or decreasing) of one or more glycans naturally present in the glycocalyx vesicle.

In some embodiments, the biological membrane of the glycocalyx vesicle is modified such that one or more of its native glycoprotein(s) is altered. In some examples, the glycocalyx vesicle is modified to decrease or remove one or more glycoprotein(s) having one or more of the sugar moieties, to which the surface displayed lectin binds. For example, the glycocalyx vesicle can be treated by an enzyme capable of removing glycans or sugar residues, e.g., glycosidase, exoglycosidase, endoglycosidase, glycoamidase, neuraminidase, galactosidase, pept,ide:N- glycosidase (PNGase), glycohydrolase, and any combination thereof. In some embodiments, the enzyme is selected from a β-N-acetylglucosaminidase, PNGase F, p (1-4) Galactosidase, O-Glycosidase, N-Glycosidase, N -glycohydrolase, Endo H, Endo D, Endo F2, EndoF3, and any combination thereof.

In some embodiments, the GVs disclosed herein has a modified glycocalyx. Glycocalyx refers to the precellular matrix composed of glycoproteins and/or glycolipids that surrounds naturally-occurring GVs. In some instances, the glycocalyx of the GVs can be modified by removing surface sialic acid residues, e.g., by sialidase treatment. In some instances, the sugar content of glycocalyx of the GVs may be altered via, e.g. , treatment of a glycosylation enzyme, a glycosyltransferase enzyme, or a combination.

In some embodiments, two or more native glycoprotein(s) are altered such that at least one glycoprotein has an increased number of glycan residues and at least one other glycoprotein has a decreased number of glycan residues or is missing its glycan residue(s), wherein the glycoprotein(s) having an increased number of glycan residues is different from the glycoprotein(s) having a decreased number of glycan residues or missing glycan residues. In some embodiments, tire one or more native glycoprotein(s) is altered such that it comprises a modified glycan. In some embodiments, the modified glycan comprises at least one carbohydrate moiety that differs from that of the glycan in the native glycoprotein(s). In some embodiments, the modified glycan comprises one or more galactose, mannose, O-glycans, N- acetyl- glucosamines, and/or N-glycan chains or any combination thereof. In some embodiments, the glycan is selected from comprises one or more D- or L- glucose, erythrose, fucose, galactose, mannose, lyxose, gulose, xylose, arabinose, ribose, 2'-deoxyribose, glucosamine, lactosamine, polylactosamine, glucuronic acid, sialic acid, sialyl-Lewis X (SLex), N-acetyl-glucosamine, N- acetyl-galactosamine, neuraminic acid, N-glycolylneuraminic acid (Neu5Gc), N- acetylneuraminic acid (Neu5Ac), an N-glycan chain, an O-glycan chain, a Core 1, Core 2, Core 3, or Core 4 structure, or a phosphate- or acetate-modified analog thereof or a combination thereof. In some embodiments, the modified glycan lacks a portion of one or more of its carbohydrate chain(s). In some embodiments, the modified glycan is missing one or more of its carbohydrate chain(s). In some embodiments, the modified glycan comprises one or more altered carbohydrate chain(s). In some embodiments, the one or more native glycoprotein(s) is altered such that at least one glycan present on the glycoprotein(s) is substituted with a glycan that is not naturally present in the native glycoprotein(s). See also WO2018170332, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

In some examples, the GVs can be treated by neuraminidase and/or sialidase to remove surface sialic acid residues. In specific examples, the GVs used herein may be substantially free of surface sialic acid residues (e.g., not detectable by a conventional method.)

In some embodiments, altering the number or content of the glycan residues on the glycocalyx vesicle affects the colloidal stability of die GV. In some embodiments, altering the number or content of the glycan residues on the glycocalyx vesicle modulates the interaction between GVs and GI cells, e.g., enhances the uptake of GVs in GI cells. In some embodiments, the altering the number or content of the glycan residues on the GV affects the ability of the glycocalyx vesicle to interact, bind and/or fuse with another vesicle, e.g., a lipid particle encapsulating a nucleic acid-attaching particle (e.g., a viral particle) as those disclosed herein. In some embodiments, altering the number or content of the glycan residues alters the overall charge of the GV. In some embodiments, altering the number or content of the glycan residues in the GVs results in a GV with greater positive charge as compared to the unaltered vesicle. In some embodiments, altering the number or content of the glycan residues in the GVs results in a G V with greater negative charge as compared to the unaltered vesicle. In some embodiments, altering the charge of the vesicle makes the vesicle more attractive for interactions, binding and/or fusion with another vesicle, e.g., the lipid particle as disclosed herein. For example, in some embodiments, lipid nanoparticles having lipid contents and GVs having lipid and/or glycan or glycoprotein contents with opposite electrostatic charges are used to promote or improve interactions, binding and/or fusion between the two types of particles. In some embodiments, interactions, binding and/or fusion is achieved between cargo -carrying lipid nanoparticles comprising negatively charged lipids and GVs comprising positively charged lipids and/or glycoprotein or glycan contents. In other embodiments, fusion is carried out between cargo-carrying lipid nanoparticles comprising positively charged lipids and GVs comprising negatively charged lipids and/or glycoprotein or glycan contents. In some embodiments, altering the number or content of the glycan residues on the glycocalyx vesicle improves the ability of the GV and/or the fused vesicle as described herein to be enriched and/or purified. In some embodiments, altering the number or content of the glycan residues on the GV improves the ability of the GV and/or the fused vesicle as described herein to be detected in vitro or in vivo. In some embodiments, anti-glycan antibodies or lectins are used to enrich and/or purify GVs and/or fused vesicles as described herein. In some embodiments, anti-glycan antibodies or lectins are used to detect and/or purify GVs and/or fused vesicles as described herein. Accordingly, methods to enrich and/or purify these GVs or fused vesicles are contemplated which comprise contacting anti-glycan antibodies or lectins with GVs and/or fused vesicles. In some embodiments, methods to detect GVs or fused vesicles using anti-glycan antibodies or lectins are contemplated.

In some embodiments, the GVs are modified to alter one or more proteins in the glycocalyx vesicle. In some embodiments, levels of existing glycocalyx vesicle proteins are reduced. In some embodiments, proteins which do not, naturally occur in the glycocalyx vesicle are added.

In some embodiments, the glycocalyx vesicle can be modified to display a functional agent on the surface. The functional agent may be any molecule having a desired bioactivity, Functional agent: any molecule having a desired bioactivity, for example, targeting a particular tissue, reacting with a cognate ligand for surface modification, or facilitating purification of the GVs disclosed herein. In some examples, a functional agent may facilitate interaction and/or fusion between the glycocalyx vesicle and a lipid particle comprising a particle to which a nucleic acid is attached as those disclosed herein so as to load the nucleic acid-containing particle into the GVs. For example, the functional agent may be a member of a receptor-ligand pair (e.g., biotin-streptavidin pair omitrilotriacetic acid (NTA)-His-tag pair), which can facilitate fusion of the glycocalyx vesicle to a lipid particle that displays the other member of the receptor-ligand pair. In further examples, the functional agent may be a tag commonly used for purification purposes, for example, a protein tag (e.g., His-tag, FLAG, etc.). Modifications to the GVs as described herein can be made via conventional methods.

For example, GVs isolated from a natural source may be subject to extrusion (e.g., once or multiple times) through a filter having a suitable size, e.g., 50 nM, 75 nM, or 100 nM, to change size distribution. In another example, GVs isolated from one or more natural sources may be subject to homogenization (e.g., under high pressure in some instances) to cause fusion of particles. Alternatively, extrusion or homogenization may be performed to GVs isolated from a natural source in the presence of other natural or artificial lipid membrane vesicles or protein micelles or aggregates to produce fused particles. Such fusion may lead to change of protein and/or lipid content of the resultant particles, for example, incorporating non-naturally occurring lipids, which may present in the artificial lipid membrane particles. In another example, additional lipids may be incorporated into GVs isolated from a natural source via saturation of the GVs with specific lipids of interest or incubating the GVs with lipid films, which may contain lipids of interest (e.g., cholesterol, phospholipids, ceramides, and/or sphingomyelins) .

In some instances, any of the functional agents disclosed herein can be conjugated to a glycocalyx vesicle using a conventional method directly. In other instances, the glycocalyx vesicle can be first modified by one or more polyethylene glycol (PEG) chains on the surface. Such PEG chains may have a molecular weight ranging from about 1 kDa to about 10 kDa. In some embodiments, a functional chemical moiety may be added to the PEG chain and one or more of the functional agents may be linked to the PEG chain via the functional moiety, either directly or via a linker. The functional moiety may be a chemical group capable of reacting with another group to form a covalent fond. Examples include, but are not limited to, a thiol group, an amine group, or an azide group.

C. Lectin-Modified. GVs

Also within the present disclosure are GVs, such as EVs having surface modification of one or more lectins and loaded with one or more cargos also disclosed herein. The one or more lectins have binding specificity to specific sites and/or cells in the GI tract, for example, enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract. Exemplary GI compartments include duodenum, upper jejunum, lower jejunum, ileum, cecum, colon, or rectum. Exemplary lectins include ECL, SBA, GSL2, UEA, PNA, GSL1, WGA, PHAL, DBA, or a combination thereof. In some examples, the lectin may be ECL or UEA1. The lectins may be integrated into the lipid bilayer of the lipid membrane in the GVs. Alternatively, the lectins may be attached to one or more proteins in the lipid membrane of the GVs.

In some embodiments, provided herein are fused vesicles or hybrid vesicles derived from fusion of GVs with a lipid particle displaying one or more lectins and optionally carrying a cargo as disclosed herein. See details below. Such fused GVs display the one or more lectins on the surface and carry the cargo in any means as disclosed herein. Comparing to a native counterpart, the fused glycocalyx vesicle may comprise any of the biological components of GVs disclosed above (either naturally existing in GVs or via modification) and additional lipid content, additional protein content, or a combination thereof derived from the lipid particle. In some examples, the fused glycocalyx vesicle is substantially free of surface sialic acid residues.

Such fused GVs would maintain the advantageous features of GVs prior to fusion as disclosed herein, for example, stable under harsh conditions, e.g., low or high pH, sonication, enzyme digestion, freeze-thaw cycles, temperature treatment, etc. For example, a substantial portion of the fused vesicles (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above) have no substantial structural changes when they are placed under an acidic condition (e.g. , pH < 6.5) for a period of time. Alternatively or in addition, the fused vesicles are resistant to enzymatic digestion such that a substantial portion of the fused vesicles (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above) have no substantial structural changes in the presence of enzymes such as digestive enzymes. In addition, the fused vesicles are stable after multiple rounds of freeze-thaw cycles (e.g., up to 6 cycles), for example, having a substantial portion (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above) that has no substantial structural changes and/or functionality changes after the multiple freeze-thaw cycles. The fused vesicles disclosed herein may also be stable after temperature treatment, e.g., incubated at a low temperature (e.g., at 4 °C) for a period (e.g., 1-3 days) or at a high temperature for period, e.g., at 60-80 °C for 30 minutes to 2 hours or at 100-120 °C for 5-20 minutes. Moreover, the fused vesicles disclosed herein may have colloidal stability. In addition, the fused vesicles may be stable under physical processes, for example, sonication, centrifugation, and filtration.

The lectin modified GVs may be loaded with any of the cargos disclosed herein. The cargo can be a therapeutic agent (e.g., peptide, polypeptide, protein, nucleic acid, small molecule, etc.) or can produce a therapeutic agent (e.g., an expression cassette designed for expressing the therapeutic agent or a viral particle carries a nucleic acid (e.g., DNA or RNA, single- strand or double-strand depending upon the type of the virus as disclosed herein) that can produce the therapeutic agent, e.g., a therapeutic nucleic acid or therapeutic protein as also disclosed herein.

The GVs carrying a cargo is also known as “cargo-loaded” GVs, in which the cargo can be any of the therapeutic or diagnostic agents disclosed herein. In some examples, the cargo can be a nucleic acid molecule capable of expressing a therapeutic agent (nucleic acid-based or protein-based) or a viral particle such as an AAV viral particle encapsulating such a nucleic acid. As used herein, the term “cargo-loaded vesicle” is meant to be inclusive of the loading of the cargo disclosed herein. As used herein, the term “loaded” or “loading” as used in reference to a “cargo-loaded vesicle,” refers to a vesicle having cargos that are either (1) encapsulated inside the vesicle; (2) associated with or partially embedded within the lipid membrane of the vesicle (i.e. partly protruding inside the interior of the vesicle); (3) associated with or bound to the outer portion of the lipid membrane and associated components (i.e., partly protruding or fully outside the vesicle); or (4) entirely disposed within the lipid membrane of the vesicle (i.e., entirely contained within the lipid membrane). In some embodiments, the cargo can be present on the interior or internal surface of the lectin-modified glycocalyx vesicle. Alternatively, the cargo can be present on the interior or internal surface of the lectin-modified glycocalyx vesicle are associated with the extracellular vesicle, e.g., via chemical interaction, electromagnetic interaction, hydrophobic interaction, electrostatic interaction, van der Waals interaction, linkage, bond (hydrogen bond, ionic bond, covalent bond, etc.). In other some embodiments, the cargo can be present on the interior or internal surface of the lectin- modi fled glycocalyx vesicle are not associated with the glycocalyx vesicle, e.g., the cargo is unattached to the vesicle. In some embodiments, the lectin-modified glycocalyx vesicle can have a cavity and/or forms a sac, in which the nucleic acid-carrying particle is encapsulated.

II. Methods for Producing GVs Having Surface Modification of Lectins Another aspect of the present disclosure provides methods for preparing GVs having surface modification of one or more lectins, and optionally loaded with a suitable cargo such as those disclosed herein. Various surface functionalization strategies are provided in Figure 2. Such functionalization strategies may be applied individually, or in combination.

For example, surface of lipid particles (e.g., lipid nanoparticles or LNPs) may be functionalized by attaching a functional moiety, for example a member of a receptor/ligand pair, or a PEG chain conjugated to a functional group. In some instances, one or more lectins may be conjugate to the lipid particles via interaction with the functional moiety, either directly or indirectly. In some instances, the one or more of the lectins can be conjugated to the PEG chain via covalent bond with the functional group, directly or via a second functional group attached to the lectins. In other instances, the lectins are conjugated to the other member of the receptor/ligand pair and attach to the lipid particles via receptor-ligand binding. See examples provided in Figure 2. The resultant lectin-carrying lipid particles may then be fused with GVs to form hybrid GVs (fused GVs) having surface modification of lectins.

Alternatively, the surface functionalized lipid particles may be fused with GVs first to form hybrid GVs and the lectins can then be attached to the hybrid vesicles to form lectin- modified GVs, The lectins can be conjugated to the hybrid vesicles via any means disclosed herein. See, e.g., above disclosures.

Accordingly, in some embodiments, provided herein is a method for making lectin- displaying GVs, such as EVs, the method comprising: (i) incubating GVs with one or more lectins to allow for attachment of the one or more lectins onto the GVs, thereby producing lectin-displaying GVs; and(ii) collecting the lectin-displaying GVs produced in step (1). The one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract, e.g., those disclosed herein. In other embodiments, provided herein is a method for making lectin-displaying GVs, the method comprising: (i) contacting GVs (e.g., EVs) with a lipid nanoparticle carrying one or more lectins to allow for fusion of the glycocalyx vesicle and the lipid nanoparticle, thereby forming a hybrid glycocalyx vesicle displaying the one or more lectins, and (ii) collecting the fused GVs. The one or more lectins bind enterocyt.es, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract, e.g., those disclosed herein.

In yet other embodiments, the present disclosure provides a method for making lectin- displaying GVs, the method comprising: (i) incubating a hybrid GVs with one or more lectins to allow for attachment of the one or more lectins onto the hybrid GVs, thereby producing lectin-displaying GVs; and (ii) collecting the lectin-displaying GVs. Prior to step (1), the method may further comprise fusing a lipid nanoparticle with a glycocalyx vesicle to form the hybrid glycocalyx vesicle. The one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract, e.g., those disclosed herein.

In any of the methods disclosed herein, the lipid nanoparticles may carry any of the cargos as disclosed herein to produce cargo-loaded and lectin-modified GVs. In some instances, the GVs, either before cargo-loading and/or lectin-modification or after cargo- loading and/or lectin-modification, may be treated by one or more glycosidases (e.g., sialidase) to reduce surface sugar contents, for example, to remove sialic acid. Doing so could reduce or eliminate binding of the GVs to lectins, e.g., those attached to other GVs.

A. Glycocolyx Vesicle Preparalion

In one aspect, GVs may be harvested from a suitable source as disclosed herein. In some embodiments, the GVs are produced and subsequently isolated from mammary epithelial cells lines adapted to recapitulate the glycocalyx vesicle architecture of that naturally occurring EVs.

In one aspect, the GVs are provided using a cell line one in a batch-like process, wherein the GVs may be harvested periodically from the cell line media. The challenge with cell line-based production methods is the potential for contamination from GVs present in fetal bovine serum (media used to grow cells). In another aspect, this challenge can be overcome with the use of suitable serum free media conditions so that GVs purified from the cell line of interest are harvested from the culture medium. In some embodiments, a filter such as a 0.2 micron filter is used to remove larger debris from a suitable sourse containing GVs. In some embodiments, the method for separation of GVs (for example, in the 80-120 nanometer range) includes separation based on specific glycocalyx vesicle properties such as size, charge, density, morphology, protein content, lipid content, or epitopes recognized by antibodies on an immobilized surface (immuno-isolation). In some embodiments antibodies directed against epitopes located on a polypeptide selected from one or more of CD9, CD81, BSG, and/or SLC3A2 may be used to enrich GV particles.

Alternatively or in addition, antibodies specific to CD63 or LAMP1 may be used for negative selection. In some embodiments, the separation method comprises a centrifugation step. In some embodiments, the separation method comprises PEG based volume excluding polymers.

In some embodiments, the separation method comprises ultra-centrifugation to separate the desired GVs from bulk solution. In some embodiments, sequential steps involving initial spins at 20,000 x g for up to 30 minutes followed by multiple spins at ranges of about 100,000 x g to about 120,000 x g for about 1 to about 2 hours provides a pellet or isolate rich in GVs, such as EVs.

In some embodiments, ultracentrifugation provides GVs, such as EVs that can be re- suspended, for example, in phosphate buffered saline or a solution of choice. In some embodiments, the vesicles are further assessed for desired properties by assessing their attributes when exposed to a sucrose density gradient and picking the fraction in 1.13-1.19 g/mL range.

In other embodiments, isolation of vesicles of the present disclosure includes using combinations of filters that exclude different sizes of particles, for example 0.45 μM or 0.22 pM filters can be used to eliminate vesicles or particles bigger than those of interest. GVs may be purified by several means, including antibodies, lectins, or other molecules that specifically bind vesicles of interest, eventually in combination with beads (e.g., agarose/sepharose beads, magnetic beads, or other beads that facilitate purification) to enrich for the desired vesicles. A marker derived from the vesicle type of interest may also be used for purifying vesicles. For example, vesicles expressing a given biomarker such as a surface-bound protein may be purified from cell-free fluids to distinguish the desired vesicle from other types. Other techniques to purify vesicles include density gradient centrifugation (e.g., sucrose or optiprep gradients), and electric charge separation. All these enrichment and purification techniques may be combined with other methods or used by themselves. A further way to purify vesicles is by selective precipitation using commercially available reagents such as ExoQuick™ (System Biosciences, Inc.) or Total Exosome Isolation kit (Invitrogen™ Life Technologies Corporation). Ill some embodiments, isolation of the glycocalyx vesicle is achieved by centrifuging a suitable raw material at high speeds to isolate the vesicle. In some embodiments, GVs, such as EVs can be isolated in a manner that provides amounts greater than about 50 mg (e.g., greater than about 300 mg) of vesicles per 100 mL of the raw material. In some embodiments, the GVs may be prepared by a method comprising the steps of: providing a quantity of the raw material; and performing a centrifugation, e.g., sequential centrifugations, on the raw material to yield greater than about 50 mg of GVs per 100 mL of the row material. In some embodiments, the series of sequential centrifugations comprises a first centrifugation at 20,000 x g at 4 °C for 30 min, a second centrifugation at 100,000 x g at 4 °C for 60 min, and a third centrifugation at 120,000 x g at 4 °C for 90 min. In some embodiments, the isolated vesicles can then be stored at a concentration of about 5 mg/mL to about 10 mg/'mL to minimize coagulation and allow the isolated vesicles to effectively be used for the encapsulation or loading of one or more therapeutic agents. In some embodiments, the isolated vesicles are passed through a 0.22 μm filter to remove any coagulated particles as well as microorganisms, such as bacteria.

In some embodiments, provided here are methods for isolating GVs (e.g., those disclosed herein), wherein the methods involve one or more steps to reduce or eliminate caseins and/or lactoglobulins from the input materials. Briefly, such a method may involve one or more defatting steps to remove abundant such proteins following conventional methods or those disclosed herein. The samples can be subject to one or more steps to disrupt casein micelles, coagulate casein and remove casein from the sample. The casein-depleted sample can thus be subject to steps to enrich GVs, for example, those approached known in the art or disclosed herein, e.g., chromatography-based methods (e.g., for scalable preparation) and ultracentrifugation-ba sed meth ods .

Any approaches known in the art for removing caseins can be used in the methods disclosed herein. In some embodiments, casein removal may be achieved chemically, e.g., by acidification. For example, a suitable acid solution {e.g., acetic acid, hydrochloric acid, citric acid, etc.) or powder of a suitable acid (e.g., citric acid powder) can be added into a suitable sample to cause coagulation of casein or casein micelles, which can be removed by a conventional method, e.g., low-speed centrifugation (e.g., < 20,000 g) or filtration. Alternatively, acidification of the sample may be achieved by saturation of the sample with CO₂ gas.

In other embodiments, casein removal may be achieved using enzymes capable of coagulating or digesting casein, for example, using rennet. As used herein, “rennet” refers to a mixture of enzymes capable of curdling caseins in a casein-containing raw material. In some examples, the rennet used in the methods disclosed herein is derived from an animal, e.g., a complex set of enzymes produced in the stomachs of a ruminant mammal such as calf. Such a rennet may comprise chymosin, which is a protease enzyme that curdles casein in a casein- containing material, and optionally other enzymes such as pepsin and lipase. In other examples, the rennet used in the methods disclosed herein is derived from a plant, e.g., a vegetable rennet.

In some instances, the vegetable rennet used herein can be a commercially available vegetable rennet extracted from a mold such as mucor miehei. Alternatively, one or more recombinant casein coagulation enzymes may be used for casein removal. Such recombinant enzymes may be produced using a suitable host (e.g., bacterium, yeast, insect cell, or mammalian cell) by the conventional recombinant technology.

In yet other embodiments, the method disclosed herein may involve the use of a Ca²⁺ chelating agent such as EDTA or EGTA to disrupt casein micelles, which can be then removed.

After removal of caseins (partially or completely), the resultant sample can be subject to one or more steps to enrich the GVs contained therein, e.g., ultracentrifugation, size exclusion chromatography, affinity purification, tangential flow filtration, or a combination thereof. In some examples, the method disclosed herein may comprise a tangential flow filtration (TFF) step for glycocalyx vesicle enrichment. In some instances, the method may further comprise a size exclusion chromatography following the TFF step. Alternatively, the enrichment may be achieved by a conventional approach such as ultracentrifugation.

Suitable GVs may also be derived by artificial production means, such as from EV- secreting cells and/or engineered as is known in the art.

In some embodiments, GVs can be further characterized by one or more of nanoparticle tracking analysis to assess particle size, transmission electron microscopy to assess size and architecture, immunogold labeling of vesicles or their contents prior to electron microscopy to track species of interest associated with GVs, immunoblotting, or protein content assessment using the Bradford Assay. (B). Preparation of Lipid Particles

As used herein, the term “lipid particle” or “lipid nanoparticle” refers to a particle comprising one or more lipids. In some embodiments, the lipid nanoparticle comprises a monolayer lipid membrane. Examples of such lipid nanoparticles include micelle and reverse micelles. In other embodiments, the lipid nanoparticle comprises one or more bilayer lipid membranes. The lipid nanoparticles may be liposomes. Alternatively, the lipid nanoparticles may be multilamellar vesicles. In other examples, the lipid nanoparticles may be solid lipid nanoparticles. In a solid lipid nanoparticle, the lipid core can be stabilized by surfactants (emulsifiers) and cargos can be distributed into lipid core. The lipid nanoparticles for use herein may have a size of about 40-70 nm, for example, about 40-60 nm, about 40-50 nm, about 40-45 nm, about 50-70 nm, about 50-60 nm, about SO- 55 nm, about 60-70 nm, or about 65-70 nm.

The lipid nanoparticles described herein may be lipidoid-based. The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 2010 21: 1448-1454: Schroeder et al., J Intern Med. 2010267:9-21; Akinc et al., Nat. Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108: 12996-3001)

The lipid nanoparticles may comprise any of the lipid content disclosed herein, e.g., one or more cationic lipids, one or more neutral lipids, cholesterol, phospholipids, PEG-conjugated lipids, or a combination thereof. In some embodiments, the lipid nanoparticle is used for producing lipid particles encapsulating a cargo, for example, any type of cargo disclosed herein (e.g., protein, nucleic acid, particles, etc.). In some instances, the cargo may be a viral particle, for example, an AAV viral particle disclosed herein. Such lipid nanoparticles are preferred to comprise one or more non-ionizable cationic lipids, e.g., those disclosed herein, to facilitate interaction with the viral particle and thus encapsulating the viral particle. In other instances, the cargo may be a nucleic acid comprising an expression cassette for producing a therapeutic agent.

The lipid nanoparticles prepared following any of the methods known in the art or disclosed herein can be analyzed to determine concentration and/or particle size distribution (e.g., by NTA). Alternatively or in addition, the lipid nanoparticles can be fractionated and particles having suitable sizes may be collected for use in the fusion method disclosed herein. a. Lipid Contents

Suitable lipids for use in making the lipid particles include, but are not limited to, cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, conjugated lipids (e.g., PEGylated lipids), and/or structural lipids. In some examples, the lipid nanoparticle may comprise one or more non- ionizable cationic lipids. Since the lipid nanoparticles are used for producing the lipid particles disclosed herein, which carry the nucleic acid for producing the agents of interest, the resultant lipid particles are expected to comprise the same lipid contents as in the lipid nanoparticles.

Some examples of the lipids that present in the lipid nanoparticles and thus lipid particles fire provided below

Cationic Ionizable Lipids and Non-Ionizable Lipids

In some embodiments, the lipid nanoparticle (as well as the lipid particles) comprises a cationic and/or ionizable lipid. As used herein, the term “ionizable lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. An ionizable lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain embodiments, an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipids. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (-t-1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired. It should be understood that the terms “charged” or “charged moiety” does not refer to a

“partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given its ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way. In some embodiments, the ionizable lipid is an ionizable amino lipid, sometimes referred to in the art as an “ionizable cationic lipid''. In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure. In addition to these, an ionizable lipid may also be a lipid including a cyclic amine group. In one embodiment, the ionizable lipid may be selected from, but not limited to, a ionizable lipid described in International Publication Nos. WO2013086354 and WO2013116126. In yet another embodiment, the ionizable lipid may be selected from, but not limited to, formula CLI-CL.XXXXII of US Patent No. 7,404,969. In some embodiments, the lipid nanoparticle may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids. Such cationic and/or ionizable lipids include, but are not limited to, 3- (didodecylamino)-N 1 ,N 1 ,4-tridodecyl- 1 -piperazineethanamine (KL 10) , N 1 -[2- (didodecylamino)etbyl] -N 1 ,N4,N4-tridodecyl- 1 ,4-piperazinediethanamine (KL22) , 14,25- ditridecyl- 15 , 18 ,21 ,24-tetraaza-octatriacontane (KL25), l,2-dilinoleyloxy-N,N- dimethylaniinopropane (DLin-DMA), 2.2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), 2.2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2-( ( 8-1(3 )-cholcst-5-cn-3-yloxy Joctyl } oxy )-N, N-di methyl -3 -I (9Z, 12Z)- octadeca -9, 12- dien- 1 -yloxylprop an- 1 -amine (Octyl-CLinDMA), (2R )-2-( ( 8-1 (3 )-cholcst-5-cn-3-yloxy Joctyl } oxy )-N, N-di methyl -3 -I (9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy ] propan- 1 -amine (Octyl-CLinDMA (2R)), (2S)-2-({ 8-[(3 )-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l -yloxy] propan- 1 -amine (Octyl-CLinDMA (2S)).N,N-dioleyl- N, N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N — N- triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N -dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propy])-N,N,N-trimethylammonium chloride (“DOTAP”); 1,2- Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Q”); 3-b-(N — (N'.N'- dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(l-(2,3-dioleyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoracetate (“DOS PA”), dioctadecylamidoglycyl carboxyspermine ("‘DOGS”), l,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N- dimethyl-2,3-dioleyioxy)propylaniine (“DODMA”), and N-(l,2- dimy ristyloxyprop-3 -yl)-N,N-dimethy 1 -N -hydroxyethyl ammonium bromide (“DMRIE”) .

In some instances, the lipid particles may comprise one or more ionizable cationic lipids. As used herein, an "ionizable cationic lipid" refers to a lipid that carries a net positive charge at a selected pH (e.g. below physiological pH). Such lipids include, but are not limited to, l,2-DiLinoleyloxy-N,N-dimethylaminopro-pane (DLinDMA), 2,2-dilinoleyI-4-(2- dimethylamino-ethyl)-[ 1,3 ]-dioxolane (D Lin-KC2-D MA), heptatriaconta-6, 9 ,28,31-tetraen- 19-y 14-( dimethylamino) butanoate (D Lin-MC3-D MA), dioctadecyl-dimethylainmonium (DODMA), Distearyldimethylammonium (DSDMA), N,N-dioleyl-N,N-dimethylamrnonium chloride (DODAC); N-(2,3-dioley-loxy )propyl )-N,N,N-trimethy I-ammonium chloride (DO TMA); l,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(4-carboxybenzyi)-N,N-di methyl-2,3-bis (oleoyloxy)propan-l-aminium (DOBAQ), YSK05, 4-(((2,3-bis( oleoyloxy )propyl)-(methyl)amino )methyl)benzoic acid (DO BAT), N-( 4-carboxybenzyl)-N,N-dimethyl- 2,3-bis ( oleoyloxy)propan-1-aniinium (DOBAQ), 3-((2,3-bis ( oleoyloxy)propyl)(methyl)amino (propanoic acid (DO PAT), N-(2-carboxypropy 1)-N,N- dimethy 1 -2,3-bis-( oleoy loxy )-propan-l-aminium (DOMPAQ), N-carboxymethyl)-N,N- di-methyl-2,3-bis( oleoyloxy )propan-l-aminium (DOAAQ), Alny-100, 3-( dimethyl amino )- propyl(12Z, 15Z)-3-[ (9Z, 12Z)-octadeca-9, 12-dien-l-yl]-henicosa-l 2, 15-dienoate (DMAP- BLP) and 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol).

In some embodiments the ionizable cationic lipid may be an amino lipid. As used herein, the term "amino lipid" is meant to include those lipids having one or two fatty acid or fatty alkyl chains and an amino head group (includ-ing an alkylamino or dialkylamino group) that may be protonated to form a cationic lipid. In certain embodiments, amino or cationic lipids of the disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that the entire lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwiterionic, are not excluded from use in the disclosure. In some embodiments, the lipids for use in making the lipid particles disclosed herein can be non-ionizable cationic lipids. Such lipids are positively charged at a wide range of pH (e.g., pH of 1-12), as opposed to ionizable cationic lipids, which are positively charged at an acidic and neutral (physiological pH) (e.g., pH of 1 -7.5). Exemplary non-ionizable cationic lipids include, but are not limited to, DOTAP, DOTMA, and DDAB.

Additionally, a number of commercial preparations of cationic and/or ionizable lipids can be used, such as, e.g., LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECT AMINE® (including DOSPA and DOPE, available from GIBCO/BRL). KL10, KL.22. and KL25 are described, for example, in U.S. Patent No. 8,691,750.

Antonie Lipids

In some embodiments, the lipid for use in making the lipid particles can be an anionic lipid. Anionic lipids suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, phosphatidylglycerol, cardiolipin, di acylphosphatidyl serine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.

Neutral Lipids

In some embodiments, the lipid for use in making the lipid particles disclosed herein may be a neutral lipid. Neutral lipids (including both uncharged and zwitterionic lipids) suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, sterols (e.g., cholesterol) and cerebrosides. In some embodiments, the lipid nanoparticle comprises cholesterol. Lapids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains and cyclic regions can be used. In some embodiments, the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine. In some embodiments, the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol. Amphipathic Lipids

In some embodiments, the lipid for use in making the lipid particles disclosed herein can be an amphiphatic lipid. In some embodiments, amphipathic lipids are included in nanoparticles of the disclosure. Exemplary amphipathic lipids suitable for use in nanoparticles of the disclosure include, but are not limited to, sphingolipids, phospholipids, fatty acids, and amino lipids.

The lipid composition in the lipid particles disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

Particular amphipathic lipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-con taining composition (e.g., FNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

Non-natural amphipathic lipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.

For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).

Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidy lethanolaniines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and b-acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.

PEGylated Lipids

In some embodiments, the lipid particles disclosed herein may comprise PEGylated lipid. The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEGylated lipid (also known as a PEG lipid or a PEG-modified lipid) is a lipid modified with polyethylene glycol. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialky Iglycerols. For example, a PEGylated lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:

In one embodiment, PEG lipids useful in the present invention are PEGylated lipids described in International Publication No. WO2012099755. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy- PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention. In some embodiments, the length of the PEG chain comprises about 250, about 500, about 1000, about 2000, about 3000, about 5000, about 10000 ethylene oxide units.

Structural Lipids

The lipid particles disclosed herein can comprise one or more structural lipids. As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol.

Helper Lipids In some embodiments, the lipid particles may comprise a helper lipid. As used herein,

"helper lipid" refers to stabilizing lipids, including neutral lipids and anionic lipids. Some nanoparticles used in the present disclosure comprise or may be enriched with one or more helper lipids, such as cholesterol and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). A neutral lipid refers several lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative lipids include, but are not limited to, dis-tearoyl- phosphatidylcholine (DSPC), dioleoyl-phosphati-dylcholine (DOPC), dipalmitoyl- phosphatidylcholine (DPPC), dioleoyl-phosphatidylglycerol (DOPGj, dipalmi-toy 1- phosphatidy ’glycerol (D PPG), dioleoy 1-phosphatidy le-thanolamine (DOPE), palmitoyloleoyl-phospha tidylcholine (POPC), palmitoyloieoy 1-phosphatidylethanol amine (POPE) and dioleoyl-phosphatidy-lethanolamine, dipalmi-toy 1-phosphatidy 1 -ethanolamine (D PPE), dimyristoy Iphos-pho-ethanolamine (D MPE), distearoy 1-phosphatidy 1- etha-nolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearioyl-2- oleoyl-phosphatidyethanol amine (SOPE), and 1 ,2-dielaidoyl-sn-glycero-3- phophoe-thanolamine (transDOPE). An anionic lipid is a lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, diacylphosphatidylserine, cardiolipin and neutral lipids modified with anionic modifying groups. Other lipids known in the art for preparing lipid nanoparticles such as liposomes can also be used in the present disclosure. Examples include those disclosed in US20110256175A1, US8642076B2, US20120225434AI , US20150190515A1 , US10195291B2,

US20150165039A1 , US20150306039A1 , US10369226B2, US20130338210A1 , US20190374646A1, US20140308304A1, US9463247B2, US8034376B2, US20130202652A1,

US20180169268A1, US20180170866A1, US20150239926A1 , US9834510B2,

US20180000953A1, US20180085474A1, US20120251618A1 , US20150166462A 1, US20150086613A1, US20160151409A1, US20140288160A1, US9629804B2, US20150366997A1, US20170246319A1, US20170196809A1, US10125092B2, US20180290965A1, US20190358170A1, US10124065B2, US20180296677A1, US20190136231A1, US20170079916A1, US20150140070A1, US20160067346A1, US10086013B2, US20190240349A1, US9840479B2, US9556110B2, US9895443B2, US10086013B2, US9439968B2, US9556110B2, US20170349543A1, US20160220681A1, US20170354672A1, US20120253032A1, US20120149894A1, US20130274523A1, US20130053572A1, US20100048888A1, and US20140162934A1. The relevant disclosures of each of these patents and patent application publications are incorporated by reference for the purpose and subject matter referenced herein.

Any of the lipid disclosed herein may comprise a stabilizing moiety. Examples of stabilizing moieties include but are not limited to compounds comprising polyethylene glycol and other compounds such as, but are not limited to, dendrimers, polyalkylene oxide, polyvinyl alcohol, polycar-boxylate, polysaccharides, and/or hydroxyalkyl starch, which reduce the interaction or binding of the complex to species present in vivo or in vitro, such as serum comple-rnent protein, co-factors, hormones or vitamins. The term "PEG-modified lipid" refers to but is not limited to, a polyethylene glycol chain of up to 20 kDa in length, covalently conjugated to a lipid with alkyl chain(s) of C6-C20 length. In certain embodiments, suitable polyethyl-ene glycol-lipids include PEG-modified phosphatidyletha-nolamine (PEG-PE), PEG- modified ceramides (e.g. PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols and PEG-modified dialkylglycerols. In one embodiment, the polyethylene glycol-lipid is (Methoxy Polyethylene Glycol)2000-dimyristolglycerol (PEG-s-DMG). Further non-limiting examples of PEG-modified lipids include PEG-dialkyloxypropyl (DAA), R-3- [(w-methoxy-poly( ethylene glycol )2000 jcarbamoy 1) ]-l,2-dimyristyloxypropyl-3-amine (PEG-c-DOMG) and N-Acety lga1actosamine-( (R )-2,3-bis( octadecy loxy )propy 1-1- methoxy poly( ethylene glycol)2000)propylcarbamate ))(GalNAc-PEG-DSG).

In addition to lipid contents, the lipid particles disclosed herein may further comprise components such as a pH-responsive polymer, a permeability enhancer molecule, a carbohydrate, polymers, surface altering agents (e.g., surfactants), or other components. The term "pH-responsive polymer" refers to a polymer that at low pH undergoes a change in structure or charge, when compared to their charge or structure at physiological pH (pH of about 7.4), which results in the polymer becoming more fusogenic. In some non- limiting embodiments of the disclosure the polymers can be made of homopolymers of alkyl acrylic acids, such as butyl acrylic acid (BAA) or propyl acrylic acid (PAA), or can be copo-lymers of ethyl acrylic acid (EAA). Polymers of alkyl amine or alkyl alcohol derivatives of maleic- anhydride copolymers with methyl vinyl ether or styrene may also be used. In some embodiments, the polymers can be made as copolymers with other monomers. The addition of other monomers can enhance the potency of the polymers, or add chemical groups with useful functionalities to facilitate association with other molecular entities, including the targeting moiety and/or other adjuvant materials such as poly(ethylene gly-col). These copolymers may include, but are not limited to, copolymers with monomers containing groups that can be cross- linked to a targeting moiety.

In general, the pH-responsive polymer is composed of monomeric residues with particular properties. Anionic monomeric residues comprise a species charged or charge-able to an anion, including a protonatable anionic species. Anionic monomeric residues can be anionic at an approxi-mately neutral pH of 7.2-7.4. Cationic monomeric residues comprise a species charged or chargeable to a cation, includ ing a deprotonatable cationic species. Cationic monomeric residues can be cationic at an approximately neutral pH of 7.2-7.4. Hydrophobic monomeric residues comprise a hydrophobic species. Hydrophilic monomeric residues comprise a hydrophilic species.

A permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No. 2005/0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). Specific combinations of lipids for use in producing AAV-lipid particles may comprise one or more cationic lipids (e.g., DOTAP), one or more helper lipid (e.g., DOPC instead of DSPC), cholesterol, and mPEG-DSPE. In some instances, one or more gangliosides such as GM3 may be used for binding io AAV and imparting hydrophobicity and negative charges. In that case, the amount of cholesterol may be reduced. More details are provided in Examples below. b. Exemplary Processes for Producing Lipid particles A variety of methods are available for preparing lipid nanoparticles such as liposomes.

See, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871 , 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871 , 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO 91/17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976): Fraley, et al., PNAS 76:3348-3352 (1979); Hope et al., Biochim. Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986); Williams et al., PNAS 85:242-246 (1988): Liposomes (Ostro (ed.), 1983, Chapter 1); Hope et al., Chem. Phys. Lip. 40:89 (1986);

Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure, ''homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vehicles and ether fusion methods, all of which are well known in the art. Any of such methods may be performed in the presence of a suitable nucleic acid-carrying particle (e.g., a viral particle such as an AAV viral particle) such that the resultant lipid particle would carry the suitable nucleic acid- carrying particle. In some embodiments, the lipid particle, which may carry a cargo as disclosed herein may be produced by a process comprising incubating lipid nanoparticles with any of the cargo under suitable conditions (e.g., a suitable temperature and/or a suitable pH condition) to produce a lipid particle encapsulating the cargo. In some examples, the suitable temperature ranges from about 25-50 °C, for example 25-45 °C, 25-40 °C, 25-35 °C, 25-530 °C, 30-50 °C, 30-45 °C, 30-40 °C, 30-35 °C, 35-50 °C, 40-50 °C, or 45-50 °C. Alternatively or in addition, the suitable pH condition ranges from pH of about 4.5 to pH of about 8.5 (e.g., 4.5, 5.0., 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5).

In other embodiments, the lipid particle encapsulating the cargo can be produced by a process comprising: rehydrating a lipid film in the presence of the particle to form a mixture, and sonicating and/or extruding the mixture through one or more filters having suitable sizes to produce the lipid particle encapsulating the particle. The lipid film may comprise one or more of the lipids disclosed herein. In some examples, the lipid film comprises one or more non- ionizable cationic lipids, which is particularly suitable for encapsulating viral particles such as AAV viral particles as disclosed herein. In some examples, the extruding step may be performed multiple times through one or more filters having suitable sizes, for example, 200 nm, 100 nm, and/or 50 nm.

In some examples, a lipid particle comprising a cargo such as those disclosed herein may be prepared as follows. One or more suitable lipids as disclosed herein can be dissolved in a suitable solvent (e.g., an organic solvent such as chloroform) to form a solution. The solvent can then be evaporated from the solution using methods known in the art, for example, under a stream of air, and the container containing the solution may be rotated to form a thin lipid film on the wall of the container. If needed, the lipid film may be dried under vacuum for a suitable period for remove any trace amount of the solvent. The lipid film is then rehydrated in a solution containing a suitable cargo. The rehydrated lipid film is then subject to vortexing, sonication, extrusion, or a combination thereof, to allow for formation of lipid particles comprising the cargo.

Extrusion is a technique in which a lipid suspension is forced through a membrane with a defined pore size to yield particles having a diameter near the pore size. The extrusion step may be performed using an extruder having a membrane with a suitable pore size, for example, about 80-200 nm. In some examples, the pore size may be about 80-150 nm or about 80-120 nm. In other examples, the pore size may be about 100-120 nm. In specific examples, the pore size of the extruder may be about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm. In other specific examples, the pore size of the extruder may be about 40 nm, about 45 nm, about 50 nm, or about 60 nm.

Alternatively or in addition, the extrusion step may be performed at a suitable pressure, for example, about 40 Ib/in² to about 300 Ib/in², about 60 lb/in² to about 200 lb/in², or about 80 lb/in² to about 1501b/in'. In some examples, the pressures may be about 40 lb/in², about 60 lb/in², about 80 Ib/in², about 100 lb/in², about 125 lb/in², about 150 lb/in², about 200 lb/in², or about 300 lb/in². In some embodiments, extrusion may be performed for up to about 25 times, for example, for up to about 20 times. In some examples, extrusion can be performed for about 1 time to about 16 times, about 4 times to 14 times, or about 8 times to about 12 times.

In some embodiments, the lipid particle encapsulating the cargo can be produced by a process comprising: mixing a first solution comprising the particle and a second solution comprising lipids dissolved in 10-100% ethanol (e.g., about 10-20% ethanol) in a microfluidic device to produce the lipid particle comprising the cargo. The second solution may comprise any of the lipids as disclosed herein. In some examples, the cargo can be a viral particle (e.g., an AAV viral particle) and the lipids contained in the second solution may comprise one or more non-ionizable cationic lipids to facilitate interaction with the viral particle and thus encapsulating such.

For example, a lipid particle comprising a cargo as disclosed herein is prepared as follows. One or more suitable lipids as disclosed herein are placed in an alcohol solvent (e.g., in ethanol) to form an alcohol solution. A suitable cargo is dissolved in an aqueous solution. The lipld-containing alcohol solution can be mixed with the cargo-containing aqueous solution under suitable conditions, under which lipid particles form with the cargo embedded in the lipid particles. In some embodiments, each of the lipid-containing alcohol solution and the cargo- containing aqueous solution flow through tubes via pumps and the two solutions interact with each other at Y or T junctions of the tubes, wherein cargo-carrying lipid particles form. In some embodiments, the tubes have a diameter of about 0.2-2 mm. In some embodiments, production of cargo-carrying lipid particles are performed using a microfluidic device.

Microfluidics involves manipulating and controlling fluids, usually in the range of microliters (10-6) to picoliters (10-12), in networks of channels with dimensions from tens to hundreds of micrometers. Fluid handling can be manipulated by components such as microfluidic pumps or microfluidic valves. Microfluidic pumps can supply fluids in a continuous way or can be used for dosing. Microfluidic valves can inject precise volumes of sample or buffer. In some instances, the microfluidic device used herein may comprise one or more channels (e.g., of glass and/or polymer materials) having a diameter of about less than 2 mm (e.g., 0.02-2 mm).

In some examples, a high flow rate (e.g., above 100 pl/min, such as above 200 ul/min, above 300 pl/min, or above 500 pl/min) may be used to facilitate better mixing of cargo- carrying particles. In specific examples, the flow rate used herein can be about 500 pl/min to about 1 ,000 pl/min.

Any of the processes for producing lipid particles comprising the cargo-carrying particles such as viral particles (e.g., AAV viral particles) as disclosed herein is also within the scope of the present disclosure, e.g., as part of the methods for producing GVs associated with the lipid particles. c. Surface Functionalization of Lipid Particles

Any of the lipid particles (e.g., loaded with a cargo) may comprise a functional moiety on the surface (surface functionalization) for association with the one or more lectins disclosed herein. In some embodiments, the functional moiety may be linked to a lectin (e.g., those disclosed herein) directly via a covalent bond formed between the functional moiety and a second functional moiety on the lectin. In other embodiments, the functional moiety may be conjugated to a functional agent, which may interact with a lectin, either directly or indirectly (via a second functional agent).

As used herein, a functional moiety refers to any functional group capable of interacting with another moiety by a covalent fond or non-covalent interactions. In some instances, the functional moiety used herein can be a chemically functional group, which refers to an atom or a group of atoms responsible for the characteristic chemical reactions of molecules carrying the functional group. Exemplary functional groups include a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amino group, a sulfhydryl group, and a phosphate group. In some instances, the functional moiety may be a functional group reactive in a click chemistry reaction, for example, an azide group, a Dibenzocyclooctyne (DBCO) group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain- promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANC). In other examples, the functional moiety may be a peptide substrate of a sortase, for example, a peptide comprising a motif of LPETG or a polyG or polyA tail.

Peptide substrates of sortases are known in the art. See, e.g., 20160122707, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

In some instances, the functional moiety is conjugated to a PEG chain connected to one or more lipids in the lipid layer of the lipid particles. The PEG chain may have a molecular weight of about 1 kDa to 10 kDa. In some examples, the PEG chain has a molecular weight of about 2 kDa to about 5 kDa.

In some examples, the functional moiety (e.g., conjugated to the PEG chain) may react with a second functional moiety conjugated to the lectin to form a covalent bond, thereby linking the lectin to the surface of the lipid particles. For example, the reaction may occur between an azide group on the surface of the lipid particle and a DBCO group on the lectin. See, e.g., Figure 2. In some examples, the functional moiety (e.g., conjugated to the PEG chain) may react with a functional agent, e.g., via a covalent bond, to attach the functional agent to the surface of the lipid particle. The functional agent may be a member of a receptor-ligand pair. The other member of the receptor-ligand pair can be conjugated to the lectin. Via interaction between members of the receptor-ligand pair, the lectin can be conjugated on the surface of the lipid particles. Exemplary receptor-ligand pairs include biotin-streptavidin or nitrilotriacetic acid-His tag.

Exemplary approaches for conjugating lectins onto the surface of lipid particles are provided in Figure 2. See also Examples below.

C. Fusion of GVs and Lipid Particles

Any of the lipid particles disclosed herein, which may carry a cargo anchor surface lectin as also disclosed herein, may be brought in contact with any of the GVs also disclosed herein under suitable conditions allowing for association of the lipid particle and the glycocalyx vesicle, e.g., encapsulation, integration partially or completely, or mixture, to form hybrid vesicles. In some embodiments, the lipid particles and GVs can be incubated together under suitable conditions allowing for fusion of the GVs and the lipid particles to produce fused vesicles comprising the particle that carries the nucleic acid for producing agents of interest (e.g., therapeutic nucleic acids or therapeutic proteins) as disclosed herein. In some embodiments, a fusion-based process can be used to produce the GVs associated with the cargo. In some instances, the fusion based process may allow for luminal loading of the cargo into GVs. To perform this method, any of the lipid particles comprising the cargo as disclosed herein may be brought in contact with any of the glycocalyx vesicle as also disclosed herein under conditions allowing for fusion of the two particles to produce a fused vesicle. Optionally, the fused vesicle, in which the cargo is encapsulated, can be collected, for example, by negative selection or by positive selection.

In any of the cargo loading embodiments described herein, the GVs or compositions of GVs used in the loading methods can comprise a relative abundance of casein less than about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or less, e.g., about 4%, about 3%, about 2%, about 1%, or substantially free of any casein. In some embodiments, the GVs or compositions of GVs are substantially free of casein. In some embodiments, the GVs or compositions of GVs comprise lactoglobulin at a relative abundance of no greater than 25% (e.g., less than about 25%, about 20%, about 15%, about 10%, about 5% or less). In some embodiments, the GVs or the composition comprising such may be substantially free of lactoglobulins.

In some embodiments, the size of the GVs is about 20-1,000 nm. In some embodiments, the GVs are not modified from their naturally occurring state. In some embodiments, the GVs are modified from their natural state. In some embodiments, the GVs are modified by altering the quantity, concentration, or amount of a biomolecule naturally present, e.g., the addition or complete or partial removal of a biomolecule naturally present (e.g., carbohydrate, such as a glycan and/or glycan residue; fatty acid, lipid). In some embodiments, the glycocalyx vesicle is modified by the addition of a biomolecule not naturally present (e.g., carbohydrate, such as a glycan; fatty acid; lipid; or protein, e.g., glycoprotein). In some embodiments, the size of the GVs is about 100-160 nm. In some embodiments, the GVs comprise a lipid membrane to which one or more proteins described herein are associated.

In some embodiments, the GVs comprise one or more proteins selected from BTN1A1, CD81 and XOR. In some embodiments, one or more proteins associated with the lipid membrane of the GVs are glycosylated. In some embodiments, the GVs demonstrate stability under freeze-thaw cycles and/or temperature treatment. In some embodiments, the GVs demonstrate colloidal stability when loaded with the biological molecule. In some embodiments, the GVs demonstrate stability under acidic pH, e.g., pH of < 4.5 or pH of <2.5.

In some embodiments, the GVs demonstrate stability upon sonication. In some embodiments, the GVs demonstrate resistance to enzyme digestion, e.g., resistance to one or more digestive enzymes described herein and/or resistance to nuclease treatment. In any of these embodiments, the beneficial properties of the glycocalyx vesicle can be conferred to the fused vesicle produced by the methods described herein, and accordingly make the fused vesicle suitable to be used for oral delivery of a cargo, e.g., a cargo encapsulated in the fused vesicle. In some embodiments, the fused vesicles are formulated to form a suitable composition for use in oral delivery of the cargo encapsulated therein to a subject, for example, a human patient. In some embodiments, the cargo can be a peptide, a protein, a nucleic acid, a polysaccharide, or a small molecule.

Fusion of the cargo-carrying lipid particles and GVs can be performed following methods known in the art or those disclosed herein, e.g., incubation under suitable conditions for a suitable period, extrusion, sonication, and/or PEG-facilitated fusion. In some embodiments, fusion of the cargo-carrying lipid nanoparticle and GVs can be performed by incubating the two types of particles under a suitable temperature for a suitable period. It is reported herein that heating could facilitate fusion of the particles. In some embodiments, the two types of particles are incubated for at least one hour (e.g., for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours or longer) at a temperature of about 4°C to about 50°C. In some embodiments, the incubation temperature is about 10°C to about 40°C. In some embodiments, the incubation temperature is about 15 °C to about 35°C. In some embodiments, the incubation temperature is about 20°C to about 40°C. In some embodiments, the incubation temperature is about 25°C to about 40°C. In some embodiments, the incubation temperature is about 35°C to about 45°C. In some embodiments, the incubation temperature is about 40°C to about 50°C. In some embodiments, the two types of particles are incubated for at least one hour and the incubation temperature is at least 35°C and no more than 50°C. In one embodiment the two types of particles are incubated for at least one hour and the incubation temperature is at least 35°C and no more than 40°C. In any of the methods disclosed herein, the fusion step may be performed in a solution comprising polyethylene glycol (PEG) having a suitable molecular weight (e.g., about 2 kD to about 50 kD) and a suitable concentration (e.g., about 2% to about 50%) to improve fusion efficiency. In some embodiments, the PEG solution comprises PEG molecules having a molecular weight ranging from about 5% to about 40%, for example, about 10% to about 35%, about 15% to about 35%, about 20% to about 40%, or about 20% to about 35%. In specific embodiments, the PEG concentration is about 25%. In other embodiments, the PEG concentration is about 30%. In yet other embodiments, the PEG concentration is about 35%. Alternatively or in addition, in some embodiments, the suitable molecular weight of the PEG ranges from about 5 kD to about 20kD, e.g., about 5kD to about 18kD, about 5 kD to about 15kD, or about 5kD to about 12kD. In some embodiments, the PEG concentration is about 6 kD, about 8kD, about 10kD, or about 12 kD.

In some embodiments, the fusion reaction is performed in a solution comprising PEG having a molecular weight of about 6 kD to about 12 kD and a PEG concentration for about 10% to about 35%. In some embodiments, the fusion step is performed for at least 1 hour (e.g., 2 hours or 3 hours) at a temperature of about 25 ( to about 50°C (e.g. , about 35°C to about

45°C). In specific embodiments, the fusion reaction is performed in a solution comprising PEG having a molecular weight, of about 8 kD to about 12 kD (e.g., about 8 kD) and a PEG concentration for about 20% to about 30% (e.g., about 30%).

In some embodiments, fusion of the cargo-carrying lipid particles and the glycocalyx vesicle is achieved by extrusion. For example, a suspension comprising the cargo-carrying lipid particle and the glycocalyx vesicle can be prepared via routine methodology and subject to extrusion for one or multiple times through a suitable filter under pressure. The ratio between the cargo-carrying lipid particle and the glycocalyx vesicle in the suspension may range from 5:1 to 1 :5. For example, in some embodiments, the ratio between the cargo-carrying lipid particle and the glycocalyx vesicle in the suspension is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5. In one embodiment, the ratio is 1 :1, In some embodiments, the filler comprises a polycarbonate membrane. Alternatively or in addition, in some embodiments, the membrane of the filter has a pore size of about 50 nm to about 200 nm (e.g., about 50 nM to about 150 nm, about 50 to about 100 nm, about 100 to about 200 nm, or about 150 nm to about 200 nm). In some embodiments, the filler comprises more than one membrane, each having a different pore size. For example, in some embodiments, the filter comprises three membranes having pore sizes of 50 nm, 100 nm, and 200 nm. During extrusion, the suspension goes through the three membranes sequentially to form the fused vesicles. In some embodiments, the extrusion step is repeated, for example, for 2-10 times (e.g., 2-8 times, 2-6 times, or 2-5 times).

In some embodiments, the fusion step disclosed herein is performed using a device containing multiple tubes forming a Y junction or a T junction. In some embodiments, the cargo-carrying lipid particles and the GVs flow through tubes via pumps and the two types of particles interact with each other at Y or T junctions of the tubes, wherein fused vesicles encapsulating the cargo form. In some embodiments, the tubes have a diameter of about 0.2-2 mm. In some embodiments, the fusion step utilizes a microfluidic device as disclosed herein. In some embodiments, the microfluidic device used herein comprises one or more channels (e.g., of glass and/or polymer materials) having a diameter less than 2 mm, for example, about 0.02-2 mm. In some examples, the one or more channels may have a diameter of about 0.05-2 mm. In some examples, the one or more channels may have a diameter of about 0.1-2 mm. In some examples, the one or more channels may have a diameter of about 0.2-2 mm. In some examples, the one or more channels may have a diameter of about 0.5-2 mm. In some examples, the one or more channels may have a diameter of about 0.8-2 mm. A schematic illustration is provided in Figure 1C. In any of the fusion methods disclosed herein (e.g., extrusion -mediated or PEG- niediated fusion), lipid particles and GVs capable of binding to each other may be selected to enhance fusion efficiency. In some examples, the lipid particles may be modified to cany a surface moiety that is capable of binding to the glycocalyx vesicle so as to enhance fusion efficiency. For example, the lipid particles may be modified to display a binding moiety capable of binding to another binding moiety that is conjugated to the surface of the GVs. Such binding moiety pairs may be any receptor-ligand pairs such as biotin-streptavidin. Alternatively, lipid particles and GVs having lipid contents with opposite electrostatic charges may be used. For example, fusion may be carried out between cargo-carrying lipid particles comprising negatively charged lipids and GVs comprising positively charged lipids. In other examples, fusion may be carried out between cargo-carrying lipid particles comprising positively charged lipids and GVs comprising negatively charged lipids. In some embodiments, the glycan residues and/or glycoproteins provide a charge on the glycocalyx vesicle that is opposite to the electric charge of the lipid particles. For example, fusion may be carried out between cargo-carrying lipid particles comprising negatively charged lipids and GVs comprising positively charged lipids and/or glycan residues and/or glycoproteins. In other examples, fusion may be carried out between cargo-carrying lipid particles comprising positively charged lipids and GVs comprising negatively charged lipids and/or glycan residues and/or glycoproteins.

In some embodiments, the fused vesicles encapsulating the cargo have substantially similar physical and/or chemical features as the glycocalyx vesicle used in the fusion such that the resultant fused vesicle would retain the advantageous features as GVs for oral delivery of the cargo to a subject. This goal may be achieved by using lipid particles having similar’ lipid contents and/or protein contents as the GVs for fusion. Accordingly, in some embodiments, lipid particles and GVs employed for fusion have similar lipid contents and/or protein contents. Alternatively, one may use lipid particles that are much smaller than the GVs such that the lipid and/or protein contents of the GVs would not have significant change after being fused with the lipid particles.

D. Surface A ttachment of Lectins

The one or more lectins disclosed herein may be conjugated to the surface of the GVs, such as EVs (e.g., cargo-loaded), to the lipid particles (e.g., cargo-loaded), or to the hybrid GVs after fusion with the lipid particles. In some instances, lectin-carrying lipid particles can then be fused with GVs to form hybrid GVs having surface modification of the one or more lectins. Such hybrid GVs may also be loaded with a suitable cargo such as those disclosed herein.

In some instances, the one or more lectins may be attached to GVs, lipid particles, or hybrid GVs via hydrophobic integration. For example, the one or more lectins can be conjugated to a lipid via a PEG linker. The lipid can be integrated into the lipid membrane of the GVs, such as EVs, the lipid particles or the hybrid GVs. See, e.g., Figure 15, top panel. In some instances, the PEG linker may have a molecular weight of about IkDa to about 10 kDa, for example, about 3kDa to about 8 kDa. In some examples, the PEG linker may have a molecular weight of about 5 kDa. In some instances, the one or more lectins may be attached to GVs, lipid particles, or hybrid GVs via covalent bonding. For example, the one or more lectins can be conjugated to a first functional moiety, which can react with a second functional moiety attached to the surface of the GVs, the lipid particles, or hybrid GVs. Exemplary functional moieties are provided elsewhere in the present disclosure. See, e.g., Figure 2. The one or more lectins can be incubated with the GVs, the lipid particles, or hybrid GVs under suitable conditions allowing for reactions between the two functional moieties to form a covalent bond, thereby attaching the one or more lectins on the surface of the GVs, the lipid particles, or the hybrid GVs.

In yet other instances, the one or more lectins can be conjugated to a member of a receptor-ligand pair. The other member of the receptor -ligand pair can be conjugated to the surface of the GVs, the lipid particles or the hybrid GVs. Upon incubating such lectins and the GVs, the lipid particles, or hybrid GVs, the two members of the receptor-ligand pair bind to each other, thereby producing GVs, lipid particles, or hybrid GVs having surface modification of the one or more lectins. In some examples, the receptor-ligand pair is biotin-streptavidin. In other examples, the receptor-ligand pair is nitrilotriacetic acid-His tag. In other instances, the one or more lectins form a fusion polypeptide(s) with streptavidin, which may be monovalent.

In some embodiments, the one or more lectins may be conjugated to the GVs via an adhesion adapter. In some instances, the adhesion adapter may comprise a polypeptide comprising a member of a ligand-receptor pair (e.g., those disclosed herein). Such a polypeptide may be fused to the lectin, which can then be associated to the GV via binding to the other member of the ligand-receptor pair linked to the GVs. In some instances, the other member of the ligand- receptor pair may be conjugated to a PEG linker, which can be associated to a lipid of the GVs. In some examples, the GVs may be functionalized by a member of a ligand-receptor pair, which refers to a pair of molecules that specifically interact with each other. Exemplary ligand-receptor pairs include biotin/streptavidin, avidin, or NeutrA vidin, or nitrilotriacetic acid-His tag. In one example, the GVs may be functionalized with biotin, which can bind streptavidin. In that case, the one or more lectins can form fusion polypeptides with the streptavidin, which may be a monovalent streptavidin. Via the interaction between biotin and streptavidin, the lectins can be attached to the GVs.

In some embodiments, the GVs disclosed herein may be further modified by one or more lectins such as the lectins capable of binding to GI compartments as disclosed herein. The one or more lectins may be conjugated directly to the GVs, e.g., via a covalent bond. In other embodiments, the GVs may be modified to contain a functional moiety, e.g., those disclosed herein

E. Enrichment of Lectin-Modified GVs

After the incubation step allowing for the one or more lectins to attach to the GVs, or after the fusion step and optionally lectin conjugation, the resultant GVs or fused vesicles having surface modification of lectins may be enriched by conventional methods or approached disclosed herein, e.g., ion-exchange chromatography, affinity chromatography, or a combination thereof. For example, the lectin- modified vesicles may be selectively collected by negative selection (e.g., excluding lipid nanoparticles) or positive selection (e.g., collecting specifically the fused vesicles). In some examples, the lectin-modified vesicles may be enriched by fractionation based on particle size, for example, SEC. In other examples, the fused vesicles may be enriched via an affinity binding approach, using a target molecule that specifically binds lectin-modified, cargo-carrying vesicles. Such target molecule may be a lectin, for example, Con A, RCA, WGA, DSL., Jacalin, and any combination thereof. In yet other examples, the lectin-modified vesicles may be enriched using one or more columns (e.g., an ion-exchange column and/or an affinity column) that selectively bind unfused lipid nanoparticles and/or GVs. Alternatively, the lectin-modified vesicles may be enriched using one or more columns (e.g., an ion-exchange column and/or an affinity column) that selectively bind the lectin-modified, cargo-carrying vesicles. In some embodiments, the lectin-modified, optionally cargo-carrying, GVs, either enriched or not enriched, may be treated by a suitable approach (e.g., enzymatic digestion) to reduce or remove surface sialic acid residues. In some examples, one or more of neuraminidase and/or sialidase may be used for removing surface sialic acid residues. III. Lectin-Medicated Cargo Delivery to Gastrointestinal Sites

Any of the GVs having surface modification of one or more lectins disclosed herein, e.g., lectin-modified GVs, may be used for delivering one or cargos carried by the lectin- modified GVs to a specific GI tract site or cells based on the binding specificity of the one or more lectins, e.g., for treatment of relevant diseases and/or disorders targeted by the cargos.

(A) Pharmaceutical Compositions

Any of the GVs (e.g.,EVs) having surface modification of lectin(s) and carrying any of the cargos disclosed herein may be mixed with one or more pharmaceutically acceptable carrier, adjuvant, or vehicle to form a pharmaceutical composition, which is also within the scope of the present disclosure. In some embodiments, the pharmaceutical composition as disclosed herein is formulated for oral administration to a patient. Tire term “patient,” as used herein, means an animal, for example a mammal, such as a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to a non- toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the cargo-loaded vesicle, with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used In the compositions of this Invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene -polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Compositions of the present disclosure may be administered orally. Pharmaceutically acceptable compositions of this disclosure can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. The pharmaceutical compositions for oral administration as described herein may be administered to a subject with or without food. In some embodiments, pharmaceutically acceptable compositions disclosed herein are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food. To aid in delivery of the lectin modified GVs carrying a cargo as disclosed herein, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.

Other commonly used surfactants, such as polysorbates (Tween® compounds), sorbitan esters (Span® compounds) and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

Alternatively, pharmaceutically acceptable compositions of this disclosure may be administered in the form of suppositories for rectal administration. These can be prepared by- mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The amount of the cargo-loaded GVs having surface modification of lectins as disclosed herein that is to be combined with the carrier materials to produce a composition in a single dosage form may vary depending upon the host treated, the particular mode of administration, and other factors known to one of ordinary skill. Preferably, provided compositions should be formulated so that a dosage of between 0.01 - 100 mg/kg body weight/day of the therapeutic agent can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific therapeutic-loaded glycocalyx vesicle employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a therapeutic- loaded glycocalyx vesicle of the present disclosure in the composition may also be depend upon the particular therapeutic-loaded vesicle in the composition.

In some embodiments, any of the compositions comprising the lectin-modified, cargo- loaded GVs may further comprise one or more inhibitory sugars, i.e., sugars that could block binding of the GVs to each other via the surface lectin and the corresponding sugar moieties on the surface of the GVs. Selection of the inhibitory sugar is based on the type of lectin displayed on the surface of the GVs, which is within the knowledge of those skilled in the art. For example, when an ECL lectin is used, the inhibitory sugar can be galactose, N- acetylgalactosamine, lactose, or a combination thereof. In another example, when the lectin is a WGA, the inhibitory sugar can be chitotriose.

(B). Therapeutic Applications

Any of the cargo-loaded GVs (e.g.,EVs) having surface modifications of lectins or compositions comprising such may be used to deliver the nucleic acid to a subject to an intestinal site via oral administration based on the binding specificity of the lectins on surface of the GVs. The GVs can then enter into host cells and release therein the cargo (e.g., any of the therapeutic agents disclosed herein) carried by the GVs. In some instances, the host cells (e.g., enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches) can release the cargo into the circulation system of the body, thereby achieving systemic delivery of the cargo. Using a glycocalyx vesicle as a carrier enhances desirable properties of the biological molecule such as improving oral bioavailability, for example by minimizing destruction of the cargo in the gut or minimizing liver first-pass effect; or improving delivery of the cargo to specific sites or cells in the GI tract, based on binding specificity of the lectins displayed on the surface of the GVs; or increasing the solubility and stability of the cargo in vivo. Having passed through the stomach protected, the therapeutic cargos can act either directly in the GI tract, or transit through the mucosa to the underlying lymphatic vascular network. In the case of cargos that yield mRNAs, complex biologies such as antibodies can be produced within mucosal cells and then be secreted into the mucosal lymphatic vascular network for subsequent systemic distribution. Consequently, the lectin-modified EVs, such as lectin modified G Vs as described herein can be harnessed to provide new treatments for diseases, such as rheumatoid arthritis, diabetes and cancer for which the standard of care requires intravenous infusion or subcutaneous injection of monoclonal antibodies (e.g., anti- PD1, anti-TNF) or protein/ peptides (e.g., GLP-1, β-glucocerebrosidase, Factor IX, Erythropoietin). As such, the lectin- modified GVs disclosed herein hold promise for expanding a variety of modalities, such as messenger RNA and antisense, to new disease areas and treatment regimens. Within the context of infectious disease as another example, the lectin-modified GVs as described herein can support oral administration of neutralizing monoclonal antibodies or antibody combinations to supply passive immune therapies for infected individuals and passive immune protection for healthcare and first responder professionals. In some instances, time required to produce sufficient supplies of such monoclonal antibodies by standard manufacturing processes, accompanied by the significant manufacturing cost as well as the need for intravenous monoclonal antibody infusion, render this approach difficult. Moreover, in some instances, more than one anti-virus antibodies may need to be combined in order to achieve virus control. Using the GVs, such as EVs as a delivery strategy may allow for rapid transfer of the DN A sequences or other nucleic acid expression systems coding for the monoclonal antibodies into the GVs, such as EVs, thereby enabling the body to make its own “drug” (e.g., through oral administration of mRNA or other gene delivery system) and permitting oral administration at significantly lower cost than traditional approaches.

Importantly, this approach will permit the generation of multiple antibody combinations where needed for more optimal therapeutic efficacy. Oral administration of modified GVs, such as modified EVs as those made according to the methods described herein, to a subject in need of treatment in certain instances will permit the subject's own G1 tract cells to make therapeutic protein. This approach also has the potential to provide a more convenient and significantly less expensive means to deliver biological medicines. To practice the methods described herein, an effective amount of any of the lectin modified, cargo-loaded GVs can be administered to a subject in need of the treatment via a suitable route, e.g., those described herein. In one example, the cargo-loaded glycocalyx vesicle is administered orally. The cargo-loaded glycocalyx vesicle would be effective in treating or diagnosing target diseases of interest, depending upon the cargo loaded into the glycocalyx vesicle. Any of the various therapeutic agents disclosed herein may be compatible with association and/or encapsulation in a glycocalyx vesicle according to the present disclosure. In some embodiments, the cargo carried by the lectin modified GVs disclosed herein can be or can produce an autoimmue antigen. Such cargo-loaded glycocalyx vesicle can be used to treat, prevent, or ameliorate an autoimmune disease, such as Rheumatoid Arthritis, Diabetes Mellilus, Insulin-Dependent Lupus Erythematosus (Systemic), Multiple Sclerosis, Psoriasis, Pancreatitis, Inflammatory Bowel Diseases, Crohn's disease, ulcerative colitis, Sjogren's Syndrome, autoimmune encephalomyelitis, experimental Graves' Disease, Sarcoidosis, Scleroderma, primary biliary cirrhosis, Chronic lymphocytic thyroiditis, Lymphopenia, Celiac Disease, Myocarditis, Chagas Disease, Myasthenia Gravis, Glomerulonephritis, IGA, Aplastic Anemia, Lupus Nephritis, Hamman-Rich syndrome, Hepatitis, Chronic Active Dermatomyositis, Glomerulonephritis, Membranous Mucocutaneous Lymph Node Syndrome, Pemphigoid, Bullous Behcet Syndrome, Spondylitis, Ankylosing Hepatitis, Autoimmune Cushing Syndrome, Guillain- Barre Syndrome, Cholangitis, Sclerosing Anti phospholipid Syndrome, Vitiligo, Thyrotoxicosis, Wegener's Granulomatosis, idiopathic purpura, Raynaud's Thrombocytopenia, Autoimmune hemolytic anemia, Cryoglobulinemia, Mixed Connective Tissue Disease, Temporal Arteritis, Pemphigus Vulgaris, Addison's Disease, Rheumatic Fever, pernicious anemia, Alopecia Areata, Lupus Erythematosus, Discoid Narcolepsy, Takayasu's Arteritis, autoimmune neuritis, Experimental Polyarteritis Nodosa, Polymyalgia Rheumatica, Dermatitis Herpetiformis, Autoimmune Myocarditis, Meniere's Disease, Chronic Inflammatory Demyelinating Polyneuropathy, Lambert-Eaton Myasthenic Syndrome, Lichen Sclerosus et Atrophicus, Churg-Strauss Syndrome, Erythematosis, Reiter Disease, Anti-Glomerular Basement Membrane Disease, autoimmune polyendocrinopathies, Felty's Syndrome, Goodpasture Syndrome, Achlorhydria, Autoimmune Lymphoproliferative Polyradiculoneuropathy, Uveomeningoencephalitic Syndrome, Polychondritis, Relapsing Atopic Allergy, Idiopathic thrombocytopenia, Stiff-Person Syndrome, AutoimmunePolyendocrinopathy -Candidiasis -Ectodermal -Dystrophy, Epidermolysis, Bullosa

Acquisita, Autoimmune orchitis, Oculovestibuloauditory syndrome, Ophthalmia, Sympathetic Myelitis, Transverse Diffuse Cerebral Sclerosis of Schilder, Neuromyelitis Optica, Still's Disease, Adult Onset Autoimmune oophoritis, Mooren's ulcer, Autoimmune Syndrome Type II, Polyglandular Autoimmune hypophysitis, Lens-induced uveitis, pemphigus foliaceus, Opsoclonus-Myoclonus Syndrome, Type B Insulin Resistance, Autoimmune Atrophic Gastritis, Lupus Hepatitis, Autoimmune Hearing Loss, Acute hemorrhagic leukencephalitis, autoimmune hypoparathyroidism, or Hashimoto's Thyroidosis, Additional examples include Addison's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Axonal & neuronal neuropathy (AMAN), Behcet's disease, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Cicatricial pemphigoid/benign mucosal pemphigoid, Cogan's syndrome. Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressier's syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis. Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis. Glomerulonephritis, Goodpasture's syndrome. Granulomatosis with Polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis. Hemolytic anemia, Henoch- Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG),

Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease. Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis. Juvenile diabetes (Type 1 diabetes). Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, chronic Lyme disease, Meniere's disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha- Habermann disease, Multiple sclerosis (MS), Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid. Optic neuritis, Palindromic rheumatism (PR), PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome. Pars planitis (peripheral uveitis), Parsonnage- Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis. Pernicious anemia (PA), POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes), Polyarteritis nodosa, Polymyalgia rheumatics, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis. Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's syndrome. Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis (RA), Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm and testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, or Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis (GPA). In some embodiments, the cargo loaded in the lectin modified glycocalyx vesicle may- be or may produce a therapeutic agent (nucleic acid or protein) for modulating an immune response, and/or for treating hyperproliferative disease, disorder, or condition such as cancer. In some embodiments, the disease, disorder, or condition is selected from a hyperproliferative disorder, viral or microbial infection, autoimmune disease, allergic condition, inflammatory disease, cardiovascular disease, metabolic disease, or neurodegenerative disease.

Any therapeutic nucleic acids and/or therapeutic proteins known in the art can be delivered to a subject by the approach disclosed herein.

In some examples, lectin modified GVs loaded with any of the cargos disclosed herein are administered to a subject in need of the treatment. The cargo can be a therapeutic agent or can produce a therapeutic agent (e.g., an expression cassette designed for expressing the therapeutic agent or a viral particle carries a nucleic acid (e.g., DNA or RNA, single-strand or double-strand depending upon the type of the virus as disclosed herein) that can produce the therapeutic agent, e.g., a therapeutic nucleic acid or therapeutic protein as also disclosed herein.

In some specific examples, the cargo is a viral particle. In some instances, the viral particle is an AAV viral particle, for example, an AAV viral particle of a particular serotype that can infect enterocytes (e.g., AAV1, AAV2, AAV2.5, AAV2.5T, or AAV8). In some instances, the DNA molecule carried by the AAV viral particle is a standard AAV vector. In other instances, the DNA molecule carried by the AAV viral particle is a self-complementary AAV vector for fast expression of the encoded agents of interest. Besides the nucleotide sequence(s) coding for one or more agents of interest, the DNA molecule in the A AV viral particle may further comprise a 5' inverted terminal repeat (ITR), a 3' ITR, and one or more gene expression regulatory elements, for example, an enhancer, a silencer, 5 '-untranslated region (5'UTR), 3' -untranslated region (3'UTR), a miRNA binding site, or a combination thereof. See also above descriptions. The coding sequences are in operable linkage to a suitable promoter. In some examples, the promoter is tissue specific, for example enterocyte-specific. Examples include, but are not limited to, intestinal alkaline phosphatase promoter, an epithelial-specific ETS-1 promoter, or a Kruppel-like factor 4 (KLF4) promoter.

In some instances, the cargo may be an antibody, a nucleic acid(s) encoding the antibody, or a viral particle carrying such a nucleic acid(s). An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term

“antibody” encompasses not only intact (e.g., full-length) antibodies, but also antigen- binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., nanobody), single domain antibodies (e.g., a VH only antibody), multi- specific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

In some instances, the cargo can be or can produce a multi-chain protein such as a multi-chain antibody. The coding sequences for the multiple chains of the multi-chain protein may be located in one nucleic acid molecule carried by a viral particle (e.g., in a polycistronic setting). Alternatively, each of the coding sequences may be located in one nucleic acid molecule carried by a viral particle (e.g., in a monocistronic settling). Such vector designs would depend on multiple factors, such as packaging capacity of a particular viral particle, which are known to those skilled in the art.

Any of the lectin modified GVs loaded with a suitable cargo (e.g., an antibody, a nucleic acid(s) encoding such, or a viral particle such as an AAV particle encapsulating the nucleic acid) described herein or pharmaceutically acceptable composition thereof, may be administered to a patient in need thereof in combination with one or more additional therapeutic agents and/or therapeutic processes.

A cargo-loaded, lectin-modified glycocalyx vesicle of the present disclosure can be administered alone or in combination with one or more other therapeutic compounds, possible combination therapy taking the form of fixed combinations or the administration of a cargo- loaded glycocalyx vesicle of the disclosure and one or more other therapeutic compounds being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic compounds. A cargo-loaded, lectin-modified glycocalyx vesicle of the present disclosure can besides or in addition be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after tumor regression, or even chemo-preventive therapy, for example in patients at risk.

Such additional agents may be administered separately from a provided therapeutic- loaded glycocalyx vesicle-containing composition, as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together with a therapeutic-loaded glycocalyx vesicle of the present disclosure in a single composition. If administered as part of a multiple dosage regime, the two active agents may be submitted simultaneously, sequentially or within a period of time from one another.

As used herein, the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this disclosure. For example, a cargo-loaded glycocalyx vesicle of the present disclosure may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present, invention provides a single unit dosage form comprising a cargo-loaded glycocalyx vesicle of the present disclosure, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In some embodiments, the additional agent is encapsulated in the same glycocalyx vesicle as the first therapeutic agent. In some embodiments, the additional agent is encapsulated in a different glycocalyx vesicle than the first therapeutic agent. In some embodiments, the additional agent is not encapsulated in a glycocalyx vesicle. In some embodiments, the additional agent is formulated in a separate composition from the therapeutic-loaded glycocalyx vesicle. The amount of both cargo-loaded, lectin-modified glycocalyx vesicle and additional therapeutic agent (in those compositions which comprise an additional therapeutic agent as described above) that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. In certain embodiments, compositions of this disclosure should be formulated so that a dosage of between 0.01-100 mg/kg body weight/day of a disclosed cargo-loaded GVs can be administered. In those compositions which comprise an additional therapeutic agent, that additional therapeutic agent and the cargo-loaded, lectin-modified glycocalyx vesicle of the present disclosure may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent. In such compositions a dosage of between 0.01—1 ,000 pg/kg body weight/day of the additional therapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositions of this disclosure will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

Examples of agents, with which the cargo-loaded, lectin-modified GVs of the present disclosure may be combined include, without limitation: treatments for Alzheimer's Disease such as Aricept® and Excelon®; treatments for HIV such as ritonavir; treatments for Parkinson's Disease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine, pergolide, trihexephendyl, and amantadine; agents for treating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex® and Rebif®), Copaxone®, and mitoxantrone; treatments for asthma such as albuterol and Singulair®; agents for treating schizophrenia such as zyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agents such as corticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory and immunosuppressive agents such as cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids, cyclophophamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anti-convulsants, ion channel blockers, riluzole, and anti- Parkinsonian agents; agents for treating cardiovascular disease such as beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents for treating liver disease such as corticosteroids, cholestyramine, interferons, and anti-viral agents; agents for treating blood disorders such as corticosteroids, anti-leukemic agents, and growth factors; agents that prolong or improve pharmacokinetics such as cytochrome P450 inhibitors (i.e., inhibitors of metabolic breakdown) and CYP3A4 inhibitors (e.g., ketokenozole and ritonavir), and agents for treating immunodeficiency disorders such as gamma globulin. In certain embodiments, combination therapies of the present invention, or a pharmaceutically acceptable composition thereof, include a monoclonal antibody or a siRNA therapeutic, which may or may not be encapsulated in a disclosed glycocalyx vesicle.

In another embodiment, the present disclosure provides a method of treating an inflammatory disease, disorder or condition by administering to a patient in need thereof a cargo-loaded glycocalyx vesicle and one or more additional therapeutic agents. Such additional therapeutic agents may be small molecules or a biologic and include, for example, acetaminophen, non-steroidal anti-inflammatory drugs (NS AIDS) such as aspirin, ibuprofen, naproxen, etodolac, and celecoxib, colchicine, corticosteroids such as prednisone, prednisolone, methylprednisolone, hydrocortisone, and the like, probenecid, allopurinol, febuxostat, and sulfasalazine. Other examples include monoclonal antibodies such as tanezumab, anticoagulants such as heparin and warfarin, antidiarrheals such as diphenoxylate, and loperamide, bile acid binding agents such as cholestyramine, alosetron, and lubiprostone, anticholinergics or antispasmodics such as dicyclomine, beta-2 agonists such as albuterol and levalbuterol, anticholinergic agents such as ipratropium bromide and tiotropiuni, inhaled corticosteroids such as beclomethasone dipropionate and triamcinolone acetonide.

A lectin-modified, cargo-loaded glycocalyx vesicle as disclosed herein may also be used in combination with an antiproliferative compound. Such antiproliferative compounds include, but are not limited to, aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule active compounds; alkylating compounds; histone deacetylase inhibitors; compounds which induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity and further anti- angiogenic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds used in the treatment of hematologic malignancies; compounds which target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors such as 17- AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17- dimethyl aminoethylamino- 17- demethoxy-geldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010 from Coniorma Therapeutics; temozolomide (Temodaf’®); kinesin spindle protein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine/chlorpromazine from CombinatoRx; MEK inhibitors such as ARRY 142886 from Array BioPharma, AZD6244 from AstraZeneca, PD181461 from Pfizer and leucovorin.

Additional therapeutic agents for co-use with the lectin-modified, cargo-loaded GVs as described herein are known in the art and/or disclosed in WP2018102397 and the references cited therein, the relevant dislcosures of each of which are incorporated by reference for the purposes or subject matter referenced herein.

General techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed,, 1989) Academic Press; Animal Cell Culture (R. 1. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P, Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & SJ. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»;

Immobilized Cells and Enzymes (1RL Press, ( 1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not. limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

Example 1: Identification of Lectins Capable of Binding to Gastrointestinal Tissues

Rodent (rats and mice) and human and non-human primate (NHP) fresh gastrointestinal (GI) tissues were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Human GI tissues were fixed using the same approach. 5 mm sections were rehydrated and incubated with various biotinylated lectins diluted in PBS and followed by incubation with AF647- streptavidin. Slides were imaged using EVOS and various regions of GI tract were scored from no lectin binding (-) to strong lectin binding (+++).

Lectin binding in rat and mouse intestine tissues are provided in Table 1 and lectin binding in human intestine tissues are provided in Table 2 below. Table 3 list common lectins for preclinical validation. Table 4 list the lectin binding to the brush border of villus enterocytes in the intestine segments of various species. A strong binding of lectins ECL and SBA to the brush border in the intestinal segments of the mouse was observed. A strong binding of lectins ECL. and UEA-1 to the brush border in the intestinal segments of rat, cynomolgus monkey and human was also observed.

Table 1. Lectin Binding in Rodent Intestine Tissues

Table 2, Lectin Binding in Human Intestine Tissues

Table 3, Common Lectins for Preclinical Validation

Table 4, Lectin Binding to Brush Border of Intestine Segments of Various Species

Example 2: Lectin Binding in Fresh Tissues

Mice were orally administered fluorescently labeled WGA (1 mg per dose or 0.125 mg per dose) in PBS solution. The animals were euthanized 2 hours post administration. Intestine tissues were isolated from the animals and imaged using an in vivo imaging system (IVIS) to examine lectin binding. The results show that the orally administered WGA was retained in duodenum and jejunum for at least 2 hours.

Further, fluorescently labeled polystyrene beads were modified with WGA, using 2 kDa 3.4 kDa, or 5 kDa PEG as a linker. WGA modified beads or unmodified beads (as a blank control) were administered into mouse duodenum. Fluorescence intensity of was monitored by IVIS for 3 hours. As shown in Figure 1, retention of WGA modified beads (having the various

PEG linkers) was observed in duodenum for at least 3 hours. The WGA modified beads using the 2 kDa PEG linker showed better retention as relative to the WGA modified beads using the

3.4 kDa or the 5 kDa PEG linker at various time points.

Example 3: Incorporation of Biotinylated Lectins into Streptavidin Functionalized GVs

This example provides a fusion-based approach for surface modification of GVs by lectins mediated by biotin-streptavidin interaction. More specifically, liposomes carrying surface lipid PEG-streptavidin are fused with GVs to load the lipid PEG-slreptavidin onto the surface of fused GVs. Biotinylated lectins are then attached to the surface of the fused GVs via biotin- streptavidin interaction. This process is illustrated in Figure 3A.

(i) Liposome composition and fusion conditions

One or more of the following components were used for preparing liposomes carrying surface streptavidin:

DOTAP - 18:1 TAP l,2-dioleoyl-3-trimethylammonium-propane (chloride salt) - Cholesterol

DOPC - 18:1 (A9-Cis) PC; l,2-dioleoyl-sn-glycero-3-phosphocholine

DSPE-mPEG2000 - 18:0 PEG2000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanoIamine- N - [ methox y ( poIyethylene glycol) -2000] ( ammonium salt)

RhDPPE - 16:0 Liss Rhod PE l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (lissamine rhodamine B sulfonyl) (ammonium salt)

DSPE-mPEG5000 - 18:0 PEG5000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000]

DSPE-PEG5000-azide - 18:0 azide PEG5000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[azido(polyethylene glycol)-5000] (ammonium salt)

- DSPE-PEG5000-biotin - 18:0 biotin PEG5000 PE; 1 „2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[biotin(polyethylene glycol)-5000]

- DMPE-PEG5000-Streptavidin - 14:0 Streptavidin PEG5000 PE; 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[streptavidin(polyethylene glycol)-5000]

DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-mPEG2k at 50 / 26.9 -27.8 / 20 / 0.7 - 1 / 1.5 - 2.1 mol % were used as a control. DOTAP/Cholesterol/DOPC/RhodamineDPPE/ DSPE-PEG2k/DMPE-PEG5k-STV at 50 / 26.6 - 27.5 / 20 / 0.7 - 1 / 1.5 -2.1 / 0.22 - 0.3 mol % were used for preparing streptavidin-carrying liposomes.

The lipid solutions were prepared in 0.1 um filtered absolute ethanol. The final lipid concentration was about 1 mg/ml. The aqueous phase was 0.1 um filtered 10 mM Citrate buffer pH-5. The liposomes were made using Dolomite microfluidics system with 2 pumps using compressed air supplied by an air compressor and 3 channel micromixer chip. The total flow rate was set at 1 ml/min and the flow rate ratio was 3/1 (0.75 ml/min for buffer and 0.25 ml/min for lipids). The first 0.25 ml of the formulation were discarded. The scale of the formulation was up to 10 ml. Each sample was dialyzed in 10 mM citrate buffer pH=5.5 in regenerated cellulose membranes Slide- A-Lyzer Dialysis Cassette G2 with 20,000 MWCO for 2 h at room temperature in order to removed ethanol. The samples were characterized using Zetasizer Ultra.

(ii) Preparation of streptavidin labeled hybrid G\^! /liposome particles 0.22 - 0.3 mg of DMPE-PEG5k-Streptavidin were dissolved in 10Oul of nuclease free water. The solution was mixed with 1 ml of dialyzed liposomes DOTAP/Cholesterol/DOPC/RhDPPE/DSPE-mPEG2k in 10 mM Citrate buffer pH=5.5. The final liposome composition was DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE- PEG2k/DMPE-PEG5k-STV at 50 / 26.6 - 27.5 / 20 / 0.7 - 1 / 1.5 -2.1 / 0.22 - 0.3 mol % at -1 - 6el3 particles/ml. The mixture was incubated at 40 C on a shaker for 2 h. The number of Streptavidin molecules per particle was 200 - 600. The DOTAP liposomal formulation in 10 mM citrate buffer at pH-5.5 as mixed with GVsin PBS for final GV concentration 3 - 6 e12 particles/ml. The ratio of DOTAP liposomes to GV was 2:1 - 10:1. The final buffer was 10 mM Citrate buffer pH - 5 - 5.5, 27 mM NaCl. Fusion was done for 3 h at 40 C. At the end the pH of the samples was increased to pH=8 by addition of 0.5 M NaOH. Aliquots were taken after the fusion for particle size and concentration measurements by NT A and MADLS measurements.

( iii)Fluorescence labeling of biotinylated lectins

Biotinylated Griffonia (Bandeiraea) Simpli cifolia Lectin II (GSL 2), 2 mg, were dissolved in 1 ml of nuclease free water to give a solution of GSL2, 2 mg/ml in 10 mM HEPES pH=7.5, 150 mM NaCl, 0.1 mM CaC12, 0.08 % NaN3. 0.5 ml of 2 mg/ml were mixed with 0.5 ml of 10 mM phosphate buffer pH=8 for final concentration of 1 mg/ml or ~8.6 uM (using approximate MW-116 kDa). To the biotinylated GSL 2, 16 ul of 5 mM VivoTag645 were added for final dye concentration of 80.6 uM or 10 equivalents. The mixture was placed on a shaker at 37 C for 2 hours and then on a vortex at 4C for 16 h at 500 rpm. The reaction mixture was purified by filtration through hollow fiber filter mPES, 10 kDa cut off (MICROKROS 20CM 10K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8. The total volume of buffer used was 40 ml. The reaction mixture was concentrated down to about 1 ml. The concentration of the purified biotin-GSL2-VT645 was about 1 mg/ml as measured by BCA. Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II (GSL 2), 2 mg, were dissolved in 1 ml of nuclease free water to give a solution of GSL2, 2 mg/ml in 10 mM HEPES pH=7.5, 150 mM NaCl, 0.1 mM CaC12, 0.08 % NaN3. 0.5 ml of 2 mg/ml were mixed with 0.5 ml of 10 mM phosphate buffer pH-8 for final concentration of 1 mg/ml or -8.6 uM (using approximate MW~ 1 16 kDa). To the biotinylated GSL 2, 16 ul of 5 mM SulfoCyanine5.5 NHS ester were added for final dye concentration of 80.6 uM or 10 equivalents. The mixture was placed on a shaker at 37 C for 2 hours and then on a vortex at. 4C for 16 h at 500 rpm. The reaction mixture was purified by filtration through hollow fiber filter mPES, 10 kDa cut off (MICROKROS 20CM 10K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8. The total volume of buffer used was 40 ml. The reaction mixture was concentrated down to about 1.25 ml. The concentration of the purified biotin-GSL2-SulfoCy5.5 was about 0.7 mg/ml as measured by BCA. Biotinylated Erythrina Cristagalli Lectin (ECL), 5 mg, were dissolved in 1 ml of nuclease free water to give a solution of GSL2, 2 mg/ml in 10 mM HEPES pH-7.5, 150 mM NaCl, 0.1 mM CaC12, 0.08 % NaN3. 0.2 ml of 5 mg/ml were mixed with 0.8 ml of 10 mM phosphate buffer pH=8 for final concentration of 1 mg/ml or -16.6 uM (using approximate MW-60 kDa). To the biotinylated ECL, 16 ul of 5 mM SulfoCyanine5.5 NHS ester were added for final dye concentration of 80.6 uM or 5 equivalents. The mixture was placed on a shaker at 37 C for 2 hours and then on a vortex at 4C for 16 h at 500 rpm. The reaction mixture was purified by filtration through hollow fiber filter mPES, 10 kDa cut off (MICROKROS 20CM 10K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8. The total volume of buffer used was 40 ml. The reaction mixture was concentrated down to about 1 .25 ml. The concentration of the purified biotin- ECL-SulfoCy5.5 was about 0.7 mg/ml as measured by BCA.

The samples from the reaction mixture and purified lectin were placed in 96 well half- area plate (clear, UV clear) with a final volume of 60 ul. The absorbance was measured using M5 well-plate reader: absorbance 210-750 nm. Figure 4A and Figure 4B show fluorescent labeling of lectins.

(iv) Streptavidin / Biotin-GSL2' loading onio hybrid GV/DOTAP liposomes

Biotin-GSL 2 labeled with VivoTag645 or SulfoCyanine5.5 in 10 mM phosphate buffer pH=8 was mixed with GlcNAc in water and incubated at 37C on a shaker for 1 h. The final concentration of GSL2 was 0.5 -1 mg/ml and of GlcNAc was 50-200 mM.

GV./DOTAP2k 5kSTV (DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE- PEG2k/DMPE-PEG5k-STV at 50 / 26.6 - 27.5 / 20 / 0.7 - 1 / 1.5 -2.1 / 0.22 - 0.3 mol %) at -1 - 6el3 particles/ml were mixed with biotin-GSL2-dye/GlcNAc. The final concentration was 0.19 - 0.28 mg/ml GSL2 and 30-100 mM GlcNAc. As a control the same amounts were mixed using GV/DOTAP 2k (no streptavidin). The 2 mixtures were left for 2 h at room temperature on a vortex at 500 rpm and then at 4C overnight. The number of Streptavidin molecules per particle was 200 - 600. The number of GSL2 molecules per particle was 150-250.

The samples were purified by filtration through hollow fiber filter mPES, 500 kDa cut off (MICROKROS 20CM 500K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8 with 20-50 mM GlcNAc. The total volume of buffer used was 30 ml for each mixture. The reaction mixture was concentrated down to about 1 ml. The number of GSL2 molecules per particle retained after purification was 50-150.

The samples from the reaction mixtures and purified lectin loaded GV/DOTAP particles were placed in 96 well half-area plate (clear, UV clear) with a final volume of 60 ul. The absorbance and fluorescence was measured using M5 well-plate reader: absorbance 210-750 nm and fluorescence with excitation at 540 mn, cut off at 550 nm, emission at 560 to 850 nm, gain at 300, 50 lamp flashes and a second fluorescence measurement with excitation at either 610 mn, cut off at 635 nm, emission at 635 to 850 nm (for VivoTag645) or 640 mn, cut off at 665 nm, emission at 665 to 850 nm (for SulfoCyanine5.5), gain at 500, 50 lamp flashes.

Figures 5A-5C show surface lectin modification of hybrid GV/DOTAP liposomes via streptavidin/biotin interaction. The particle concentration is about -foe 12 particles/ml. The reaction was performed in the presence of 50 mM GlcNAc (pre-incubated with 100 mM). About 300 - 550 STV molecules were present per hybrid particles. .About 50-120 GSL2 molecules were present on the surface of each particle (equi valent to about 20 - 72 % of GSL.2 put into the reaction). No GSL2 is retained in EV/DOTAP2k (which has no surface streptavidin).

(v) Streptavidin / Biotin- ECL loading onto hybrid GV/DOTAP liposomes

Biotin-ECL labeled with VivoTag645 or SulfoCyanine5.5 in 10 mM phosphate buffer pH-8 was mixed with Galactose or Lactose in water and incubated at 37C on a shaker for 1 h. The final concentration of ECL was 0.25 -1 mg/ml and of Galactose or Lactose was 50-200 mM.

GV/DOTAP2k 5kSTV (DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE- PEG2k/DMPE-PEG5k-STV at 50 / 26.6 - 27.5 / 20 / 0.7 - 1 / 1.5 -2.1 / 0.22 - 0.3 mol %) at ~1 - 6el 3 particles/ml were mixed with biotin-ECL-dye/sugar. The final concentration was 0.14 - 0.18 mg/ml GSL2 and 30-100 mM Galactose or Lactose. As a control the same amounts were mixed using GV/DOTAP 2k (no streptavidin). The 2 mixtures were left for 2 h at room temperature on a vortex at 500 rpm and then at 4C overnight. The number of Streptavidin molecules per particle was 200 - 600. The number of ECL molecules per particle was 250 - 350.

The samples were purified by filtration through hollow' fiber filter mPES, 500 kDa cut off (MICROKROS 20CM 500K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8 with 20-60 mM Galactose or Lactose. The total volume of buffer used was 30 ml for each mixture. The reaction mixture was concentrated down to about 1 ml. The number of ECL molecules per particle retained after purification was 60-150. The samples from the reaction mixtures and purified lectin loaded GV/DOTAP particles were placed in 96 well half- area plate (clear, UV clear) with a final volume of 60 ul. The absorbance and fluorescence was measured using M5 well-plate reader: absorbance 210-750 nm and fluorescence with excitation at 540 mu, cut off at 550 nm, emission at 560 to 850 nm, gain at 300, 50 lamp flashes and a second fluorescence measurement with excitation at either 610 nm, cut off at 635 nm, emission at 635 to 850 nm (for VivoTag645) or 640 mn, cut off at 665 nm, emission at 665 to 850 nm (for SulfoCyanine5.5), gain at 500, 50 lamp flashes.

Figures 6A-6C show surface lectin modification of hybrid GV/DOTAP liposomes via streptavidin/biotin interaction. The particle concentration is about ~6el2 particles/ml. The reaction was performed in the presence of 30 mM galactose (pre-incubated with 100 mM). About 300 - 550 STV molecules were present per hybrid particles. About 75 ECL molecules were present on the surface of each particle (equivalent to about 20% of ECL put into the reaction). No ECL is retained in EV/DOTAP2k (which has no surface streptavidin).

(vi) Characterization of Streptavidin functionalized liposomes and hybrid GV/DOTAP liposomes

Sizes of the particles were measured by nanoparticle tracking analysis (NTA) as follows. The samples of GV were diluted 100,000x and the samples from fused GV/DOTAP liposomes were diluted l(),()()()-20,000x in 0.1 um filtered lx PBS pH-7.4 for NTA measurement of particle size and concentration. Each sample was injected into the Malvern Nanosight NS300 from Malvern Pananalytical using a 1 ml syringe and a syringe pump set at flow⁷ rate 30. The samples were recorded for 4x30s using camera level 14 and analyzed using level 5 setting.

The size, concentration and charge of the samples was measured using Zetasizer U ltra form Malvern Pananalytical. Samples were placed as is in a small volume fluorescence cuvette (Sigma- Aldrich). The volume used was 50 ul of each sample without any further dilution. The size and concentration of the samples was measured using MADLS method.

The charge of the particles was measured in a cuvette for measuring zeta potential from Malvern Pananalytical. The cuvette has filled with 1 ml of 100 ul liposomes sample diluted in 900 ul 10 mM Citrate buffer pH-5, 10 mM NaCl. Each sample was measured in triplicate and an average of the values was taken. As shown in Figure 7A, a clear shift in liposome size was observed upon addition of

DMPE-PEG5k-STV. No significant difference in GV size was observed after fusion of liposomes with or without DMEP-PEG5k-STV. Figure 7B

In sum, the results of this Example show that up to 550 molecules of Streptavidin- PEG5k-DMPE were loaded onto hybrid GV/Liposome particles. Further, streptavidin-biotin interaction successfully allowed for significant loading of biotinylated lectins onto hybrid GV/Liposome particles. Up to 120 lectin molecules per hybrid GV/Liposome particle were loaded. Moreover, STV-Biotin lectin loading method successfully allowed for quick screening of multiple lectins for retention in the GI.

Example 4: Incorporation of Lipid Modified Lectins into Liposomes for Fusion with GV or Post Fusion Directly into Hybrid GV/Liposomes

This example describes exemplary approaches for incorporating lipid functionalized lectins into liposomes or hybrid GVs. These exemplary approaches are illustrated in Figures 3B and 3C.

(i) Liposome composition and fusion conditions One or more of the following components can be used in thi s example:

DOTAP - 18:1 TAP l,2-dioleoyl-3-trimethylammonium-propane (chloride salt)

Cholesterol

DO PC - 18:1 (A9-Cis) PC; l,2-dioleoyl-sn-glycero-3-phosphocholine DSPE-mPEG2000 - 18:0 PEG2000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)

DSPE-mPEG5000 - 18:0 PEG5000 PE; 1, 2-di stearoyl -sn-glycero-3- phosphoethanolamine-N-[rnethoxy(polyethylene glycol)-5000] - DSPE-PEG5000-azide - 18:0 azide PEG5000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[azido(polyethylene glycol)-5000] (ammonium salt)

DSPE-PEG5000"biotin - 18:0 biotin PEG5000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N~[biotin(polyethylene glycol)-5000]

- DMPE-PEG50()()-Streptavidin - 14:0 Streptavidin PEG5000 PE; 1 ,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[streptavidin(polyethylene glycol)-5000] A lipid solution comprising DOTAP/Cholesterol/DOPC/RhodarnineDPPE/DSPE- mPEG2k at 50 / 26.9 -27.8 / 20 / 0.7 - 1 / 1.5 - 2.1 mol % at ~1 6el3 particles/ml was prepared as follows. The lipid solutions were prepared in 0.1 um filtered absolute ethanol. The final lipid concentration was about 1 mg/ml. The aqueous phase was 0.1 um filtered 10 mM Citrate buffer pH=5. The liposomes were made using Dolomite microfluidics system with 2 pumps using compressed air supplied by an air compressor and 3 channel micromixer chip. The total flow rate was set at 1 ml/min and the flow rate ratio was 3/1 (0.75 ml/min for buffer and 0.25 ml/min for lipids). The first 0.25 ml of the formulation were discarded. The scale of the formulation was up to 10 ml. Each sample was dialyzed in 10 mM citrate buffer pH-5.5 in regenerated cellulose membranes Slide- A-Lyzer Dialysis Cassette G2 with 20,000 MWCO for 2 h at room temperature in order to removed ethanol. The samples were characterized using Zetasizer Ultra.

The DOTAP liposomal formulation in 10 mM citrate buffer at pH-5.5 as mixed with GVs in PBS for final GV concentration 3 - 6 e!2 particles/ml. The ratio of DOTAP liposomes to GV was 2:1 - 10:1. The final buffer was 10 mM Citrate buffer pH = 5 - 5.5, 27 mM NaCl.

Fusion was done for 3 h at 40 °C. At the end the pH of the samples was increased to pH=8 by addition of 0.5 M NaOH. Aliquots were taken after the fusion for particle size and concentration measurements by NTA and MADLS measurements.

(ii) Charactsrization of hybrid GV/DOTAP liposomes Sizes of the particles were measured by nanoparticle tracking analysis (NTA) as follows.

The samples of GV were diluted 100,000x and the samples from fused GV/DOTAP liposomes were diluted 10, 000-20, OOOx in 0.1 um filtered lx PBS pH=7.4 for NTA measurement of particle size and concentration. Each sample was injected into the Malvern Nanosight NS300 from Malvern Pananalytical using a 1 ml syringe and a syringe pump set at flow' rate 30. The samples were recorded for 4x30s using camera level 14 and analyzed using level 5 setting.

The size, concentration and charge of the samples was measured using Zetasizer Ultra form Malvern Pananalytical. Samples were placed as is in a small volume fluorescence cuvette (Sigma - Aldrich). The volume used was 50 ul of each sample without any further dilution. The size and concentration of the samples was measured using MADLS method.

The charge of the particles was measured in a cuvette for measuring zeta potential from Malvern Pananalytical. 'The cuvette has filled with 1 ml of 100 ul liposomes sample diluted in 900 ul 10 mM Citrate buffer pH-5, 10 mM NaCl. Each sample was measured in triplicate and an average of the values was taken. The results are provided in Figures 8A and 8B, and in Table 5 below.

Table 5. Characterization of Hybrid GV/DOTAP Liposomes

(Hi) Functionalizing lectins with DSPE-PEG5k

Griffonia (Bandeiraea) Simplicifolia Lectin II (GSL2) 1 - 2 mg/ml or 8.85 - 17.7 uM (using MW 113 kDa) was dissolved in 10 mM phosphate buffer pH=8. To the solution of GSL2 6 equivalents of VivoTag645 NHS ester or SulfoCy5.5 NHS ester in DMSO were added, followed by 15 - 50 equivalents of DBCO-STP ester in DMSO. The mixture was placed on a vortex at 500 rpm at room temperature for 2 hours and then at 4C for 16 h. The yield of the reaction was between 30-60%.

Erythrina Cristagalli Lectin (ECL) 1 - 2 mg/ml or 18.5 - 37 uM (using MW 54 kDa) was dissolved in 10 mM phosphate buffer pH=8. To the solution of ECL 4 equivalents of VivoTag645 NHS ester or SulfoCy5.5 NHS ester in DMSO were added, followed by 10 - 30 equivalents of DBCO-STP ester in DMSO. The mixture was placed on a vortex at 500 rpm at room temperature for 2 hours and then at 4C for 16 h. The yield of the reaction was between 30- 60%. Lectin-DBCO-dye at 0.7-2 mg/ml in 10 mM Phosphate buffer pH=8 was mixed with 5 -

10 equivalents of DSPE-PEG5k-N3 in ethanol. The mixture was placed on a shaker at 500 rpm at 37C for 6 hours and then at 4C overnight. The yield of the reaction was between 20 - 100%.

.After each reaction the reaction mixtures were purified by filtration through hollow fiber filter mPES, 10 kDa cut off (MICROKROS 20CM 10K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8. The total volume of buffer used was 30-40 ml. The reaction mixture was concentrated down to about 1 ml with final concentration of 0.7-2 mg/ml as measured by BCA.

The samples from the reaction mixture and purified lectin were placed in 96 well half- area plate (clear, UV clear) with a final volume of 60 ul. The absorbance was measured using M5 well-plate reader: absorbance 210-750 nm.

As shown in Figure 9, DBCO functionalized ECL reacts fully with 10 eq DSPE-PEG5k- azide and DBCO functionalized GSL2 reaction with 10 eq DSPE-PEG5k-azide gives 50-70% yield.

(iv) Direct loading of Lectins: Addition of Lectin-5k-DSPE to Liposomes followed by fusion with GV

DOTAP2k liposomes (DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-

PEG2k/DMPE-PEG5k-STV at 50 / 26.9 - 27.8 / 20 / 0.7 - 1 / 1.5 -2.1 mol %) in 10 mM citrate buffer at pH 5.5 (~1 - 6el3 particles/ml) were mixed with ECL-dye-PEG5k-DSPE (0.5-1 mg/ml). The final concentration of ECL was 0.1 - 0.3 mg/ml. The mixture was incubated at 37C for Ih.

The DOTAP liposomal formulation containing ECL-dye-PEG5k-DSPE in 10 mM citrate buffer at pH-5.5 was mixed with GalcNAc for 30 min at 37C. GVs in lx PBS pH-7.4 were added for final GV concentration 3 - 6 el2 particles/ml and sugar concentration of 25 - 100 mM. with GV in PBS for final GV concentration 3 - 6 el2 particles/ml. The ratio of liposomes to GV was 2:1 - 10:1 . The amount of ECL-dye-PEG5k-DSPE per particle was 200 - 400 molecules/particle. The final buffer was 10 mM Citrate buffer pH = 5 - 5.5, 27 mM NaCl .

Fusion was done for 3 h at 40 C.

The ECL-PEG5k-DSPE -GV/DOTAP2k sample was purified by filtration through hollow fiber filter mPES, 500 kDa cut off (MICROKROS 20CM 500K MPES 0.5MM MLL X

FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8 with 60 mM of lactose. The total volume of buffer used was 30 ml. The reaction mixture was concentrated down to about 1 ml. The lectin retained was about 30-50 % or 80 - 200 molecules per particle.

The samples from the reaction mixtures and purified lectin loaded GV/DOTAP particles were placed in 96 well half-area plate (clear, UV clear) with a final volume of 60 ul. The absorbance and fluorescence was measured using M5 well-plate reader: absorbance 210- /50 nm and fluorescence with excitation at 540 mn, cut off at 550 nm, emission at 560 to 850 nm, gain at 300, 50 lamp flashes and a second fluorescence measurement with excitation at either 610 mn, cut off at 635 nm, emission at 635 to 850 nm (for VivoTag645) or 640 mn, cut off at 665 nm, emission at 665 to 850 nm (for SulfoCyanine5.5), gain at 500, 50 lamp flashes.

As shown in Figure 10A, -140 ECL-dye-PEG5k-DSPE/particle of lectin retained (~44% of ECL, put into the reaction). The reaction was performed in the presence of 60 mM Lactose (pre-incubated with 120 mM). Figures 10B and 10C show fluorescence intensities of lectin- loaded liposome/GV hybrid particles.

(v) Direct loading of Lectins: Addition of Leclin-5k-DSPE to hybrid GV/DOTAP liposomes DOTAP2k liposomes (DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE- PEG2k/DMPE-PEG5k-STV at 50 / 26.9 - 27.8 / 20 / 0.7 - 1 / 1.5 -2.1 mol %) in 10 mM citrate buffer at pH 5.5 (-1 - 6el3 particles/ml) were mixed with ECL-dye-PEG5k-DSPE (0.5- 1 mg/ml). The final concentration of ECL was 0.1 - 0.3 mg/ml. The mixture was incubated at 37C for Ih.

The DOTAP liposomal formulation containing ECL-dye-PEG5k-DSPE in 10 mM citrate buffer at pH=5.5 was mixed with galactose or lactose for 30 min at 37C. GVs in lx PBS pH-7.4 were added for final GV concentration 3 - 6 el2 particles/ml and sugar concentration of 25 - 100 mM. The ratio of liposomes to GV was 2:1 - 10: 1 . The amount of ECL-dye-PEG5k-DSPE per particle was 200 - 400 molecules/particle. The final buffer was 10 mM Citrate buffer pH = 5 - 5.5, 27 mM NaCl. Fusion was done for 3 h at 40 C.

The ECL-PEG5k-DSPE -GV/DOTAP2k sample was purified by filtration through hollow fiber filter mPES. 500 kDa cut off (MICROKROS 20CM 500K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8 with 60 mM of lactose. The total volume of buffer used was 30 ml. The reaction mixture was concentrated down to about 1 ml. The lectin retained was about 30 - 50 % or 80 - 200 molecules per particle.

The results show that 24 % of GSL2 is retained in EV/DOTAP2k and 34 % of ECL is retained in EV/DOTAP2k (accounted for dilution). Figure 11 A. When lectins were added after fusion (6el2 particles/ml), 0.23 mg/ml GSL2 or 170 molecules/particle before purification and 40 molecules/particle retained after purification were observed. Further, 0.18 mg/ml ECL or 230 molecules/particle before purification and about 80 molecules/particle retained after purification were observed. Figures 11B and 11C. The amount of ECL retained is the same regardless of whether it was added to the particles before fusion or after fusion with GV.

In summary, results from this Example demonstrated that lectins can be chemically functionalized with a controlled number of lipophilic moiety-PEGx molecules (x - 2000 - 5000 Da) per lectin. Up to 10 lipid-PEG5k molecules per lectin were placed. Further, up to 140 lectin- PEGx-lipid molecules were loaded per hybrid GV/Liposome particle. This was achieved by adding the lipophilic-PEGx-Lectin to the cationic liposomes before fusion with GVs or to fused hybrid GV/Liposome particles.

Example 5: Formulation of Azide Modified lipids for Surface Modification of Liposomes or GV-Liposome Hybrid Particles

This example describes an approach for surface loading of lectins onto GVs using azide functionalized liposomes or liposome-GV hybrid particles. An exemplary process is illustrated in Figure 3D.

(i) Liposome compositions and fusion conditions

Lipid components used in this example include:

DOTAP - 18:1 TAP l,2-dioIeoyl-3- trimethylammonium -propane (chloride salt) Cholesterol

DOPC - 18:1 (A9-Cis) PC; l,2-dioleoyl-sn-glycero-3-phosphocholine

DSPE-mPEG2000 - 18:0 PEG2000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[rnethoxy(polyethylene glycol)-2000] (ammonium salt)

RhDPPE - 16:0 Liss Rhod PE l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-(lissamine rhodamine B sulfonyl) (ammonium salt)

- DSPE-mPEG5000 - 18:0 PEG5000 PE; 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000]

- DSPE-PEG5000-biotin - 18:0 biotin PEG5000 PE; l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[biotin(polyethylene glycol)-5000]

DMPE-PEG5000-Slreptavidin - 14:0 Streptavidin PEG5000 PE; 1 ,2-dimyristoyl-sn- gfycero-3-phosphoethanolamine”N"[streptavidin(polyethylene glycol)-5()()0]

The following liposome compositions are used in this example: DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-mPEG2k @ 50 / 26.9 -27.8 / 20 / 0.7 - 1 / 1.5 - 2.1 mol %

- DOTAP/Cholesterol/DOPC/RliodamineDPPE/DSPE-mPEG5k @ 50 / 26.9 -27.8 / 20 / 0.7 - 1 / 1.5 - 2.1 mol % - DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-mPEG2k/DSPE-mPEG5k@ 50 / 26.9 / 20 / 1 / 1.4 / 0.7 mol % - DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-PEG2k/DS PE-PEG5k-N3 @ 50

/ 26.9 - 27.3 / 20 / 0.7 - 1 / 0.7 - 1 .4 / 0.7 - 1.3 mol% - DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-PEG2k/DSPE- PEG5kN3/DSPE-PEG5k-Biotin @ 50 / 26.9 / 20 / 1 / 1 / 0.7 / 0.4 mol% The lipid solutions were prepared In 0.1 um filtered absolute ethanol. The final lipid concentration was about 1 mg/ml. The aqueous phase was 0.1 um filtered 10 mM Citrate buffer pH=5. The liposomes were made using Dolomite microfluidics system with 2 pumps using compressed air supplied by an air compressor and 3 channel micromixer chip. The total flow rate was set at 1 ml/min and the flow rate ratio was 3/1 (0.75 ml/min for buffer and 0.25 ml/min for lipids). The first 0.25 ml of the formulation were discarded. The scale of the formulation was up to 10 ml.

Each sample was dialyzed in 10 mM citrate buffer pH-5.5 in regenerated cellulose membranes Slide- A-Lyzer Dialysis Cassette G2 with 20,000 MWCO for 2 h at room temperature in order to removed ethanol. The samples were characterized using Zetasizer Ultra. The DOTAP liposomal formulation in 10 mM citrate buffer at pH-5.5 as mixed with GVs in PBS for final GV concentration 3 - 6 e!2 particles/ml. The ratio of DOTAP liposomes to GV was 2: 1 - 10: 1. The final buffer was 10 mM Citrate buffer pH = 5 - 5.5, 27 mM NaCl .

Fusion was done for 3 h at 40 C. At the end the pH of the samples was increased to pH-8 by addition of 0.5 M NaOH. Aliquots were taken after the fusion for particle size and concentration measurements by NTA and MADLS measurements.

(it) Azide-alkyne cycloaddition reaction of GSL2-DBCO with hybrid GVs

GSL2-DBCO-dye (VivoTag645 or SulfoCy5.5) was incubated with GlcNAc for 30 min - Ih at 37C. Hybrid GV/DOTAP2k 5kN3 particles (DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-PEG2k/DSPE-PEG5k-N3 at 50 / 26.9 - 27.3 / 20 / 0.7 - 1 / 0.7 - 1.4 / 0.7 - 1.3 mol%) were mixed with GSL2-DBCO-dye (VivoTag645 or SulfoCy5.5) and GlcNAc. The final concentration of lectin was 0.1 - 0.3 mg/ml GSL2 and 25 - 100 mM GlcNAc. As a control the same amounts were mixed using GV/DOTAP 2k (DOTAP/Cholesterol/IX)PC/RhodamineDPPE/DSPE-mPEG2k @ 50 / 26.9 -27.8 / 20 / 0.7 - 1 / 1.5 - 2.1 mol %). The 2 mixtures were left for 10 h at 37C and then 4C overnight on a shaker at 500 rpm.

The samples were then purified by filtration through hollow fiber filter mPES, 500 kDa cut off (MICROKROS 20CM 500K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8 with 25 - 50 mM GlcNAc. The total volume of buffer used was 30 ml for each mixture. The reaction mixture was concentrated down to about 1 ml. The lectin retained was about 20 - 65 % or 50 - 100 molecules per particle.

As shown in Figures 12A-12C, about 65 % of GSL2 is retained in EV/DOTAP2k5kN3 and about 50 % of GSL2 is retained in EV/DOTAP2k. After purification, the average level of GSL.2 attachment to the liposome/GV hybrid particles is about 65 molecules per particle. (iii) Azide-alkyne cycloaddition reaction of ECL-DBCO with hybrid GVs ECL-DBCO-dye (VivoTag645 or SulfoCy5.5) was incubated with lactose for 30 min - Ih at 37C. Hybrid GV/DOTAP2k 5kN3 particles

(DOT AP/Cholesterol/DOPC/RhodamineDPPE/DSPE-PEG2k/DS PE-PEG5k-N3 @ 50 / 26.9 - 27.3 / 20 / 0.7 - 1 / 0.7 - 1.4 / 0.7 - 1.3 mol% ) were mixed with ECL-DBCO-dye (VivoTag645 or SulfoCy5.5) and lactose. The final concentration of lectin was 0.1 - 0.2 mg/ml ECL and 25 - 100 mM lactose. As a control the same amounts were mixed using GV./DOTAP 2k (DOTAP/Cholesterol/DOPC/RhodamineDPPE/DSPE-mPEG2k @ 50 / 26.9 -27.8 / 20 / 0.7 - 1 / 1.5 - 2.1 mol %). The 2 mixtures were left for 10 h at 37C and then 4C overnight on a shaker at 500 rprn.

The samples were then purified by filtration through hollow' fiber filter mPES, 500 kDa cut off (MICROKROS 20CM 500K MPES 0.5MM MLL X FLL 1/PK) using 2x 10 ml syringes. The buffer used was 10 mM phosphate buffer pH-8 with 25 - 50 mM Lactose. The total volume of buffer used was 30 ml for each mixture. The reaction mixture was concentrated down to about 1 ml. The lectin retained was about 25 - 95 % or 50 - 200 molecules per particle.

As shown in Figures 13A-13C, about 94 % of ECL is retained in EV7DOTAP2k5kN3 and about 55 % of ECL is retained in EV/DOTAP2k. After purification, the average level of ECL. attachment to the liposome/GV hybrid particles is about 188 molecules per particle.

In summary, this Example demonstrates that lectins can be chemically functionalized with a controlled number of DBCO groups that are reactive in strain-promoted azide-alkyne cycloaddition. DBCO functionalized lectins can react with lipophilic moiety-PEGx-azide molecules (x = 2000 - 5000 Da) incorporated into cationic liposomes, GVs, or fused hybrid GV/Liposome particles. Increasing the amount of DBCO molecules per lectin leads to higher retention in hybrid GV/Liposome particles with some non-specific binding. Up to 200 lectin- DBCO molecules were successfully loaded per hybrid GV/Liposome particle. Azide-alkyne cycloaddition reaction of DBCO-Lectins and azide-PEGx functionalized hybrid GV/Liposomes is compatible with various cargos and cargo loading/encapsulation strategies.

Example 6: Validation of Surface Modification of GVs

The GSL2 samples were pulled down using N-Acetyl-D-glucosamine-agarose beads. The ECL samples were pulled-down using Galactose-Separopore-4B CL (epoxy-coupled). 60 ul of sugar beads were centrifuged at 1500 g for 5 mln. The supernatant was removed and the beads were washed with 100 ul of 10 mM phosphate buffer pH=8. The beads were centrifuged at 1500 g for 5 min and the supernatant was removed.

30 ul of the lectin samples were diluted with 120 ul of 10 mM phosphate. 80 ul of each of the diluted lectin samples were added to the dry beads. The samples were left incubating at 37C for 30 min on a shaker at 850 rpm. The beads were centrifuged at 1500 g for 5 min and the supernatant was removed. The fluorescence of the supernatant (SN) and the initial diluted lectin solutions was measured. The retention of the lectins by the sugar-beads was calculated by the ratio of SN/initial sample.

Retention was calculated by measuring fluorescence in the stock solution and removed supernatant. The levels of lectin retention are provided in Table 6 below.

Table 6. Levels of Lectin Retention

The degree of surface functionalization was evaluated using sulfoCy5.5-DBCO as illustrated in Figure 14A. As shown in Figure 14B, about 2000:1 Toc-PEG2k-N3:EV was observed in reaction and about 1000:1 Toc-PEG2k-N3:EV was observed after purification. In other words, about 51% of the dyes were retained.

Example 7: Preparation of Lectinized AAV Loaded GV

GVs (4.34 E+13, 462 uL ), AAV1 particles carrying transgenes encoding a transgene (e.g., a luciferase reporter protein) (2.60 E+13, 770 uL) were initially mixed at the equal particles concentration to achieve the final concentration of 1.00E+13 particles/mL. PEG (MW400, 600 uL) was added to make the total concentration of 30% v/v. 1XPBS buffer (168 uL) was added to make up the total volume to 2000 uL. It was then put on Microfluidizer LV1 at 20,000 psi with single pass. The collected formulation was then purified by using hollow fiber membrane (500kD mPES 0.5mm) to remove PEG.

200 uL of each lectins solution at 1 mg/mL was added to 1 mL of AAV- loaded GV prepared as described above. It was then put on tumbler at 4 °C for 1 h. The resultant formulation was then used for further analysis without further purification.

The resultant latinized, AAV- Loaded GV particles were characterized to determine their sizes, polydispersity index (EDI), particle concentrations, and zeta potential (ZP) using conventional methods for measuring physical characteristics of particles. Briefly, samples for analysis were prepared by diluting particle-containing solutions ten times in O.lx PBS. Size, particle concentrations and zeta potentials were measured by Malvern Zetasizer Ultra. The results are provided in Table 7. Table 7. Characterization of Letinized AAV Loaded GV

Example 8: Lectin- Targeted Delivery of Transgenes to Intestinal Ceils for Producing Reporter Proteins Lectinized and AAV-loaded GVparticles were given to mice via direct duodenum injection. The particles investigated in this example are loaded with AAV1 particles carrying a transgene encoding NanoLuc luciferase as a reporter protein and lectin SBA or ECL, GV/AAV1/SBA particles or GV/AAV1/ECL particles. Plasma samples were collected from the treated mice on Days 2, 4, and 10 post administration and subject to analysis by the NanoGio® Plasma NanoIuc® assay .

The procedure to perform the NanoGio® Plasma Nanoluc® assay is as follows. The NanoGio® assay buffer (-20 °C) was thawed to room temperature. The mouse plasma samples (- 80 °C) was thawed on ice. 40 uL of IX PBS (Room Temp) was added to each well of a 96-well, flat bottomed white plate. 10 uL of individual mouse plasma was added to each of three wells (triplicates) of the assay plate. NanoGio® substrate was added to NanoGio® Assay Buffer (1:50), e.g., 200 uL substrate to 9.8 mL assay buffer. After vortex, the NanoGio® substrate solution was transferred to a reagent reservoir and add 50 uL to each well of the assay plate. The plate was incubated for 3 minutes at RT on an orbital shaker then transfer the plate to the SpectraMAX plate reader. The plate configuration was set using the software. The Luminescence Endpoint program was run on SpectraMax. The samples were transferred from the white assay plate to a 96-well, flat-bottomed black assay plate. The black plate was placed in the IVIS and image using Living Image Software on an open-filter bioluminescent well-plate program. The luminescent signal was quantitated using the Living Image Software.

On Day 2 post administration of OSO-0037 (GV/AAV1/SBA), NanoLuc® was detected in plasma with mean total flux values of 43,358 + 59,759 photons/sec (p/s, mean + standard deviation) in 5/5 animals assayed. On Day 2 post administration of 080-0038 (GV/AAV1/ECL), NanoLuc® was detected in plasma with mean total flux values of 17,860 ± 9463 p/s in 3/3 animals assayed. On Day 2 post administration of DS-0634 (GV/AAV1 particles with no lectin), NanoLuc® was detected in plasma with mean total flux values of 51,842 ± 30,126 p/s in 4/4 animals assayed. Total flux values statistics for Day 2 post administration is as shown in Figure 16A. On Day 4 post administration of OSO-0038, NanoLuc® was detected in plasma with mean total flux values of 17,080 ± 12,381 p/s in 4/4 animals assayed. On Day 4 post administration of DS-0634, NanoLuc® was detected in plasma with mean total flux values of 155,182 ± 167,813 p/s in 5/5 animals assayed. Samples collected from animals treated with OSO-0037 (SBA) were not of sufficient quality to be assayed. Total flux values statistics for Day 4 post administration is as shown in Figure 16B.

On Day 10 post administration of OSO-0037, NanoLuc® was detected in plasma with mean total flux values of 28,940 ± 17,542 p/s in 5/5 animals assayed. On Day 10 post administration of OSO-0038, NanoLuc® was detected in plasma with mean total flux values of 56,092 + 78,856 p/s in 4/4 animals assayed. On Day 10 post administration of DS-0634 (no Lectin), NanoLuc® was detected in plasma with mean total flux values of 240,267 + 242,254 p/s in 5/5 animals assayed. Total flux values statistics for Day 10 post administration is as shown in Figure 16C.

Background mean luminescence values were 3,321 ± 2,226 p/s. All three treatment groups showed some individual animals with detectable NanoLuc® in plasma on Days 2, 4, and 10 post administration. By Day 10 post administration all animals treated with OSO-0037

(SBA), OSO-0038 (ECL), and DS-0634 (no lectin) showed detectable NanoLuc® in plasma. Descriptive statistics are shown in Tables 8-10 below. A chart showing total Flux from Day 0 to Day 10 in plasma samples of the treated animals is provided in Figure 17A.

Table 8. Day 2 Statistics

Table 9. Day 4 Statistics

Table 10. Day 10 Statistics

In some instances, the AAV1 particles loaded to GVs also carry a second transgene encoding EPO. The weights of spleens obtained on Day 10 post administration were analyzed and the results are shown in Figure 17B.

Example 9: Staining of Mouse Intestinal Tissue with Biotinylated Lectins

Pre -biotinylated lectins ECL, Jacalin, ConA, and LEL were purchased from Vector Laboratories. Serial sections of mouse duodenal tissue were exposed to solutions of 4 micrograms/mL of each biotinylated lectin, then stained with streptavidin-Cy5 and DAPI, and imaged using a slide scanner.

As shown in Figures 18A-18D, different lectins showed different staining patterns in mouse duodenal tissue, indicating different binding affinity and/or specificity to different duodenal tissues and/or cells. All of the four tested lectins, ECL, Jacalin, ConA, and LEL, stained the apical brush border, which is the target for intestinal cargo delivery mediated by GVs, and Jacaline showed the strongest signal. ECL was found to have high binding affinity to goblet cells relative to other types of duodenal cells. Figure 18A. On the other hand, Jacalin appeal's to have a broad binding specificity to duodenal tissues and/or cells relative to ECL. Figure 18B. LEL showed a similar albeit weaker binding pattern as Jacalin. Figure 18D.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature seiwing the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements): etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of' or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one. A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

What Is Claimed Is:

1. A modified glycocalyx vesicle (GV), comprising a lipid membrane, to which one or more lectins are attached, wherein the one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract.

2. The modified glycocalyx vesicle of claim 1, further comprising proteins associated with the lipid membrane, optionally wherein the proteins are transmembrane proteins or glycoproteins.

3. The modified glycocalyx vesicle of claim 1 or claim 2, wherein the lectin is embedded in the lipids in the lipid membrane, and/or attached to one or more of the proteins associated with the lipid membrane.

4. The modified glycocalyx vesicle of any one of claims 1-3, wherein the GI tract is a human GI tract.

5. The modified glycocalyx vesicle of any one of claims 1-4, wherein the compartment of the GI tract is duodenum, upper jejunum, lower jejunum, ileum, cecum, colon. or rectum

6. The modified glycocalyx vesicle of any one of claims 1-5, wherein the one or more lectins have substantially low binding activity to the modified glycocalyx vesicle.

The modified glycocalyx vesicle of any one of claims 1-6, wherein the one or more lectins are selected from the group consisting of ECL, SBA, GSL2, UEA, PNA, GSL1,

WGA, PH AL and DBA, optionally wherein the lectin is ECL and/or UEA1

8. The modified glycocalyx vesicle of any one of claims 1-7, wherein the one or more lectins are attached to the modified glycocalyx vesicle via a receptor-ligand pair.

9. The modified glycocalyx vesicle of claim 8, wherein the receptor-ligand pair is biotin-streptavidin, or nitrilotriacetic acid-His tag.

10. The modified glycocalyx vesicle of any one of claims 1-9, wherein the lipid membrane of the modified glycocalyx vesicle comprises phospholipids, cholesterol, and/or tocopherol, which is conjugated to polyethylene glycol (PEG) chains, and wherein the one or more lectins form a covalent bond to a functional moiety linked to the PEG chain.

11 . The modified glycocalyx vesicle of claim 10, wherein the functional moiety is a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANC).

12. The modified glycocalyx vesicle of claim 10 or claim 11, wherein the PEG chains have a molecular weight ranging from about 1 -10 kDa, optionally ranging from about 2-5 kDa.

13. The modified glycocalyx vesicle of any one of claims 1-12, wherein the modified glycocalyx vesicle has modified surface glycocalyx as compared with the wild-type counterpart to prevent interaction of the modified glycocalyx vesicle to the one or more lectins, to mucus, or both.

14. The modified glycocalyx vesicle of claim 13, wherein the modified glycocalyx comprises removal of surface sialic acid residues, change of sugar content of glycocalyx, or a combination thereof.

15. The modified glycocalyx vesicle of any one of claims 1-14, wherein the modified glycocalyx vesicle is an extracellular vesicle (EV).

16. The modified glycocalyx vesicle of any one of claims 1-15, wherein the modified glycocalyx vesicle is loaded with a cargo.

17. The modified glycocalyx vesicle of claim 16, wherein the cargo is a therapeutic agent or a diagnostic agent. 18. The modified glycocalyx vesicle of claim 16 or claim 17, wherein the cargo is a peptide, a protein, a nucleic acid, a polysaccharide, a small molecule, or a particle comprising a nucleic acid, which optionally is a viral particle.

19. A pharmaceutical composition, comprising a modified glycocalyx vesicle of any one of claims 1-18 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, further comprising an inhibitory sugar selected from the group consisting of chitotriose, galactose, N-acetylgalactosamine, and lactose.

21. The pharmaceutical composition of claim 20, wherein the lectin attached to the modified glycocalyx vesicle is ECL and the inhibitory sugar is galactose, N-acetylgalactosamine, or lactose.

22. The pharmaceutical composition of any one of claims 19-21, wherein the pharmaceutical composition is formulated for oral administration.

23. A method for making lectin-displaying glycocalyx vesicles (GVs), the method comprising:

(i) contacting GVs with lipid nanoparticles carrying one or more lectins to allow' for fusion of the GVs and the lipid nanoparticles, thereby forming hybrid vesicles displaying the one or more lectins, and

(ii) collecting the fused vesicles produced in step (1); wherein the one or more lectins bind enterocytes, Tuft cells. Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract. 24. The method of claim 23, wherein the lipid nanoparticles carrying the one or more lectins are prepared by a process comprising:

(a) providing lipid nanoparticles comprising a lipid conjugated to a first PEG chain, which is further conjugated to a member of a receptor- ligand pair;

(b) providing one or more lectins, which are conjugated to the other member of the receptor- ligand pair: and

(c) contacting the lipid nanoparticles in (a) with the one or more lectins in (b) under conditions allowing for interaction between the members of the receptor-ligand pair, thereby producing the lipid nanoparticles carrying the one or more lectins. 25. The method of claim 24, wherein the other member of the receptor- ligand pair is conjugated to the one or more lectins via a second PEG chain.

26. The method of any one of claims 23-25, wherein the receptor-ligand pair is biotin- streptavidin or nitrilotriacetic acid- His tag.

27. 'The method of claim 26, wherein the lipid nanoparticles carrying the one or more lectins are prepared by a process comprising:

(a) providing lipid nanoparticles comprising a lipid conjugated to a first PEG moiety, which is further conjugated to a first functional moiety; (b) providing one or more lectins, which is conjugated to a functional agent reactive to the functional moiety; and (c) contacting the lipid nanoparticles in (a) with the one or more lectins in (b) under conditions allowing for reaction between the first functional moiety on the lipid nanopaiticles and the functional agent conjugated to the one or more lectins, thereby producing the lipid nanoparticles carrying the one or more lectins.

28. The method of claim 27, wherein the first functional moiety is a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANG).

29. The method of claim 27 or claim 28, wherein the functional agent conjugated to the one or more lectins is a PEG chain, which comprises a second functional moiety that is reactive to the first functional moiety.

30. The method of claim 29, wherein the lipid nanoparticles carrying the one or more lectins are prepared by a process comprising: contacting lipid nanopaiticles with a lipid-lectin conjugate under conditions allowing for incorporation of the lipid-lectin conjugate into the lipid nanoparticles, thereby producing the lipid nanoparticles carrying the one or more lectins.

31. The method of claim 30, wherein the lipid-lectin conjugate comprises a PEG chain, which connects the lipid and the lectin. 32. A method for making lectin-displaying glycocalyx vesicles (GV s), the method comprising:

(1) incubating hybrid GVs with one or more lectins to allow for attachment of the one or more lectins onto the hybrid GVs, thereby producing lectin-displaying GVs; and

(ii) collecting the lectin-displaying GVs produced in step (i): wherein the one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract.

33. The method of claim 32, further comprising, prior to step (i), fusing lipid nanoparticles with GVs to form the hybrid GVs, wherein the lipid nanoparticles comprise a lipid conjugated to a first PEG chain, which is further conjugated to a member of a receptor-ligand pair; wherein the one or more lectins are conjugated to the other member of the receptor-ligand pair; and wherein the one or more lectins are displayed on the surface of the hybrid GVs via the interaction between the members of the receptor-ligand pair.

34. The method of claim 33, wherein the one or more lectins are conjugated to the other member of the receptor-ligand pair via a second PEG chain.

35. The method of claim 33 or claim 34. wherein the receptor- ligand pair is biotin- streptavidin or nitrilotriacetic acid-His tag.

36. The method of claim 35, further comprising, prior to step (i), fusing lipid nanoparticles with GV s to form the hybrid GVs, wherein the lipid nanoparticle comprises a lipid conjugated to a first PEG chain, which is further conjugated to a first functional moiety; wherein the one or more lectins are conjugated to a functional agent, which is reactive to the first functional moiety; and wherein the one or more lectins are displayed on the surface of the hybrid GVs via the reaction between the functional moiety on the hybrid glycocalyx vesicle and the functional agent linked to the one or more lectins.

37. 'The method of claim 36, wherein the first functional moiety is a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nilrone cycloaddition ( SP ANC) .

38. The method of claim 36 or claim 37, wherein the functional agent conjugated to the one or more lectins is a PEG chain, which comprises a second functional moiety that is reactive to the first functional moiety.

39. The method of claim 32, wherein the one or more lectins are conjugated to a lipid; and wherein the lipid-conjugated one or more lectins are incorporated into the hybrid GVs in step (i).

40. The method of claim 39, wherein the one or more lectins are conjugated to a lipid via a PEG chain.

41 . A method for making lectin-displaying glycocalyx vesicles (GVs), the method comprising (i) incubating GVs with one or more lectins to allow for attachment of the one or more lectins onto the GVs, thereby producing lectin-displaying GVs; and

(ii) collecting the lectin-displaying GVs produced in step (i); wherein the one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer's patches at a compartment of a gastrointestinal (GI) tract.

42. The method of claim 42, wherein the GVs are conjugated to a member of a receptor-ligand pair; wherein the one or more lectins are conjugated to the other member of the receptor- ligand pair; and wherein the one or more lectins are displayed on the surface of the GVs via the interaction between the members of the receptor-ligand pair.

43. The method of claim 42, wherein the glycocalyx vesicles are conjugated to the member of the receptor-ligand pair via a first PEG chain, which optionally is conjugated to a lipid of the glycocalyx vesicles. 44. The method of claim 43, wherein the one or more lectins are conjugated to the other member of the receptor-ligand pair via a second PEG chain.

45. The method of claim 42, wherein the one or more lectins form a fusion polypeptide(s) with the other member of the receptor-ligand pair.

46. The method of claim 44 or claim 45, wherein the receptor-ligand pair is biotin- streptavidin, which optionally is monovalent, or nitrilotriacetic acid-His tag.

47. The method of claim 46, wherein the GVs are conjugated to a first functional moiety; wherein the one or more lectins are conjugated to a functional agent, which is reactive to the first functional moiety; and wherein the one or more lectins are displayed on the surface of the GVs via the reaction between the functional moiety on the glycocalyx vesicle and the functional agent linked to the one or more lectins. 48. The method of claim 47, wherein the first functional moiety is conjugated to the glycocalyx vesicles via a first PEG chain, which optionally is conjugated to a lipid of the glycocalyx vesicles .

49. The method of claim 48, wherein the first functional moiety is a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I) -catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANC). 50. The method of claim 48 or claim 49, wherein the functional agent conjugated to the one or more lectins is a PEG chain, which comprises a second functional moiety that is reactive to the first functional moiety.

51. The method of claim 50, wherein the one or more lectins are conjugated to a lipid; and wherein the lipid-conjugated one or more lectins are incorporated into the GVs in step (i).

52. The method of claim 51, wherein the one or more lectins are conjugated to a lipid via a PEG chain.

53. The method of any one of claims 24-52, further comprising treating the GVs or the hybrid GVs with sialidase, a glycosylation enzyme, a glycosyltransferase enzyme, or a combination thereof to modify surface glycocalyx of the GVs or the hybrid GVs.

54. The method of any one of claims 24-53, further comprising treating the lectin- displaying GVs with sialidase, a glycosylation enzyme, a glycosyltransferase enzyme, or a combination thereof to modify surface glycocalyx of the glycocalyx vesicle.

55. The method of any one of claims 24-54, wherein any of the PEG chains has a molecular weight of about 1 kDa to 10 kDa, optionally about 2 kDa to 5 kDa.

56. The method of any one of claims 24-53, wherein the one or more lectins are selected from the group consisting of ECL, SBA, GSL2, UEA, PNA, GSL1, WGA, PHAL and DBA, optionally wherein the lectin is ECL and/or UEA1.

57. The method of any one of claims 24-56, wherein the glycocalyx vesicle is an extracellular vesicle (GV).

58. A method for delivering a cargo to the gastrointestinal (Gl) tract in a subject, the method comprising administering to the subject orally an effective amount of a pharmaceutical composition set forth in any one of claims 20-23, wherein the modified glycocalyx vesicle in the pharmaceutical composition comprise a cargo.