WO2025166154A1 - Enzymatic engineering of extracellular vesicles - Google Patents

Abstract

The disclosure provides methods of functionalizing and/or labeling an extracellular vesicle via enzymatic glycan engineering. Also provided are extracellular vesicles that are functionalized and/or labeled according to methods of the disclosure.

Description

ENZYMATIC ENGINEERING OF EXTRACELLULAR VESICLES

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant Numbers R01 CA260132, R01 CA243445, and R35 GM131686, awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[0002] The present disclosure relates to enzymatic engineering methods for functionalizing and labeling extracellular vesicles and to extracellular vesicles prepared by such methods.

BACKGROUND

[0003] Extracellular vesicles (EVs), such as exosomes, are lipid bilayer-defined particles secreted by cells into the extracellular space. EVs can vary in size and composition, depending on the parent cells and culture conditions, as well as the tissue types and species. EVs can carry many of the materials of the parent cells, including mRNA, microRNA, proteins, glycans, and lipids; accordingly, EVs can act as useful messengers and substance transporters that mediate intercellular communications via molecular exchanges and interactions with recipient cells. EVs can enter target cells by several mechanisms, including endocytosis, membrane fusion, and binding to specific receptors. In general, EVs are stable particles that can penetrate through tissues, diffuse in the blood, and cross the blood-brain barrier. Furthermore, EVs can be highly compatible with the hosts of their parent cells. Therefore, EVs are generally well- suited for targeted drug delivery, immunotherapy, and other biomedical applications.

[0004] Like the exterior surface of a cell, the exterior surface of an EV generally includes glycoproteins and glycolipids, i.e. , proteins and lipids that are functionalized with oligosaccharides or glycans. In general, the glycan signatures of EVs are similar to those of the parent cells, though EVs and their parent cells can differ in certain specific glycans. For example, it has been observed that the glycans of plasma-derived EVs are different from glycans of the donor-matched whole plasma, especially in glycan nodes corresponding to that of chondroitin sulfate, dermatan sulfate, and type I and II keratan sulfates, which are enriched on EVs. The unique glycans of EVs associated with specific cells and diseases play a key role in various biological and pathological processes. As a result, the glycans on EVs are not only important biomarkers and targets for their biomedical application but also potentially useful molecular handles for EV engineering. [0005] EVs can be engineered to carry specific ligands, receptors, or other payloads, for instance to attain cell and tissue specificity that can be advantageous for practical use in biomedical applications. To this end, several methods have been developed for modifying EVs, including genetic and metabolic engineering methods, ligand labeling, and direct chemical functionalization. Genetic engineering of EVs can generally start with genetic modification of the parent cells to express specific target proteins or specific binding ligands. Metabolic engineering methods can take advantage of biosynthetic pathways of cells to incorporate unique functional groups into biomolecules within cells, and the resulting functionalized biomolecules can eventually be passed on to an EV. Ligand labeling can be carried out on isolated EVs via specific recognition of, and binding to, ligands on the EVs. Chemical functionalization can be carried out via incorporation of functionalized biomolecules onto an EV followed by chemical modification of the biomolecules. However, each of these modification methods generally has one or more limitations. For example, modification of EVs by genetic or metabolic engineering generally involves complex processes and can result in low-abundance and/or heterogeneous expression. Similarly, it can be complex and difficult to perform EV modification via ligand labeling. For chemical EV modification, specially functionalized groups can be required to enable chemoselective reaction on the EV surface; otherwise, chemical modifications can be nonspecific and can affect functional molecules besides the target molecules. Chemoselective modification can be realized by incorporating functionalized lipophilic molecules into the EV membrane, but this process can be difficult to control and it can affect the properties of the resulting EVs. Accordingly, EV modification methods that do not suffer these limitations are of interest to meet the demands of practical applications of EVs.

SUMMARY

[0006] Disclosed herein are methods of functionalizing and labeling a vesicle via enzymatic glycan engineering (EGE) and functionalized and/or labeled vesicles.

[0007] One aspect of the disclosure provides methods of functionalizing a vesicle, the methods comprising: providing a vesicle in the form of a sphere comprising an internal volume defined by a lipid bilayer having an interior facing hydrophilic surface and an exterior facing hydrophilic surface opposing the interior facing hydrophilic surface, wherein the vesicle has an external surface comprising the exterior facing hydrophilic surface of the lipid bilayer and the external surface comprises a first sugar residue connected thereto; and admixing the vesicle and an enzyme, to provide a functionalized vesicle comprising the vesicle functionalized at the first sugar residue. [0008] Another aspect of the disclosure provides a functionalized vesicle obtained by a method of the disclosure.

[0009] Another aspect of the disclosure provides a labeled vesicle, the labeled vesicle comprising a vesicle in the form of a sphere comprising an internal volume defined by a lipid bilayer having an interior facing hydrophilic surface and an exterior facing hydrophilic surface opposing the interior facing hydrophilic surface, wherein the vesicle has an external surface comprising the exterior facing hydrophilic surface of the lipid bilayer and the external surface comprises a first sugar residue connected thereto; wherein the vesicle comprises a labeling group connected to the external surface of the vesicle at the first sugar residue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows a generalized scheme of functionalizing an extracellular vesicle using a glycosyltransferase according to methods of the disclosure.

[0011] Figure 2 shows a generalized scheme of functionalizing an extracellular vesicle using a glycosyltransferase according to methods of the disclosure.

[0012] Figure 3 shows a generalized scheme of functionalizing an extracellular vesicle using a sugar oxidase according to methods of the disclosure.

[0013] Figure 4 shows fluorescence data for several samples of labeled exosomes of the disclosure and control samples.

[0014] Figures 5A-5D show fluorescence data for several samples of labeled exosomes of the disclosure and control samples.

[0015] Figure 6 shows optical density data for samples containing modified exosomes of the disclosure after various treatments and for control samples containing unmodified exosomes.

[0016] Figure 7 shows mean fluorescence intensity data for samples of HeLa cells incubated from 0 to 24 hr with exosomes of the disclosure that were functionalized and labeled via EGE.

DETAILED DESCRIPTION

[0017] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed methods and compositions pertain having the benefit of the teachings presented herein. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0018] “Comprising” as used herein means that various components, ingredients or steps can be conjointly employed in practicing the present disclosure. Accordingly, the term “comprising” encompasses the more restrictive terms “consisting essentially of” and “consisting of.” The present methods and compositions can comprise, consist essentially of, or consist of any of the required and optional steps and elements disclosed herein. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined herein.

[0020] All ranges set forth herein include all possible subsets of ranges and any combinations of such subset ranges. By default, ranges are inclusive of the stated endpoints, unless stated otherwise. Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also contemplated to be part of the disclosure.

[0021] It is expressly contemplated that for any number value described herein, e.g., as a parameter of the subject matter described or part of a range associated with the subject matter described, an alternative which forms part of the description is a functionally equivalent range surrounding the specific numerical value (e.g., for a dimension disclosed as “40 mm,” an alternative embodiment contemplated is “about 40 mm”).

[0022] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Definitions

[0023] As used herein, the term “alkyl” refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty two carbon atoms, or one to twenty carbon atoms, or one to ten carbon atoms. The term C_n means the alkyl group has “n” carbon atoms. For example, C4 alkyl refers to an alkyl group that has 4 carbon atoms. C1-7 alkyl and C1-C7 alkyl refer to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 7 carbon atoms), as well as all subgroups (e.g., 1-6, 2-7, 1-5, 3-6, 1 , 2, 3, 4, 5, 6, and 7 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), t-butyl (1 ,1 - dimethylethyl), 3,3-dimethylpentyl, and 2-ethylhexyl. Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group. For example, a “haloalkyl” is an alkyl group that is substituted with one or more halo, and can be perhalogenated (i.e., each hydrogen atom of the alkyl group is substituted with a halo atom).

[0024] As used herein, the term “alkylene” refers to a branched, straight chain, or cyclic hydrocarbon group having two radical centers derived by the removal of two hydrogen atoms from carbon atoms of a parent alkane. For example, an alkylene group can be -CH₂CH₂- or -CH₂-. The term C_n means the alkylene group has “n” carbon atoms. For example, C1-6 alkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range, as well as all subgroups, as previously described for “alkyl” groups. Unless otherwise indicated, an alkylene group can be an unsubstituted alkylene group or a substituted alkylene group.

[0025] As used herein, the term “alkyne” refers to a -C=CH group or a -C=C- group. As used herein, the term “azide” refers to a -N3 group (i.e., -N=N=N). As used herein, the term “amino” refers to a -NH₂ group or a -N(H)- group, wherein one or both hydrogens can be replaced with an alkyl, cycloalkyl, or aryl group. As used herein, the term “hydrazide” refers to a -N(H)-NH₂ group or a -N(H)-N(H)- group. As used herein, the term “carboxy” or “carboxyl” refers to a -C(=O)OH group and the term “carboxylate” refers to a -C(=O)O“ group.

[0026] As used herein, the term “heteroalkyl” is defined similarly as alkyl except that the straight chained and branched saturated hydrocarbon group contains one to five heteroatoms (e.g., 1-5, 1—4, 1-3, 1-2, 1 , 2, 3, 4, or 5 heteroatoms) independently selected from oxygen (O), nitrogen (N), and sulfur (S). In particular, the term “heteroalkyl” refers to a saturated hydrocarbon containing one to thirty carbon atoms and one to five heteroatoms. In general, in embodiments wherein the heteroalkyl is provided as a substituent, the heteroalkyl is bound through a carbon atom, e.g., a heteroalkyl is distinct from an alkoxy or amino group. Unless otherwise indicated, a heteroalkyl group can be an unsubstituted or a substituted heteroalkyl group.

[0027] As used herein, the term “cycloalkyl” refers to an aliphatic monocyclic or polycyclic hydrocarbon ring containing three to twenty two carbon atoms, for example, three to twenty, three to fifteen carbon atoms, or three to ten carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 carbon atoms). The term C_n means the cycloalkyl group has “n” carbon atoms. For example, C5 cycloalkyl refers to a cycloalkyl group that has 5 carbon atoms in the ring. C5-8 cycloalkyl and Cs-Cs cycloalkyl refer to cycloalkyl groups having a number of carbon atoms encompassing the entire range (i.e., 5 to 8 carbon atoms), as well as all subgroups (e.g., 5-6, 6-7, 6-8, 7-8, 5-7, 5, 6, 7, and 8 carbon atoms). Nonlimiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be an unsubstituted cycloalkyl group or a substituted cycloalkyl group. The cycloalkyl groups described herein can be isolated or fused to another cycloalkyl group, a heterocycloalkyl group, an aryl group and/or a heteroaryl group.

[0028] As used herein, the term “heterocycle” refers to a monocyclic or polycyclic hydrocarbon ring in which from one to four carbon atoms are replaced with heteroatoms independently selected from oxygen, nitrogen, and sulfur. In particular, the term “heterocycle” refers to a ring containing a total of five to twenty atoms, for example five to fifteen atoms, five to twelve, or five to ten atoms, of which 1 , 2, 3, or 4 of those atoms are heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur, and the remaining atoms in the ring are carbon atoms. Nonlimiting examples of heterocycloalkyl rings include piperidine, pyrazolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, and morpholine. The heterocycloalkyl groups described herein can be isolated or fused to another heterocycloalkyl group, a cycloalkyl group, an aryl group, and/or a heteroaryl group. In some embodiments, the heterocycloalkyl groups described herein comprise one oxygen ring atom (e.g., oxiranyl, oxetanyl, tetrahydrofuranyl, and tetrahydropyranyl). The heterocycloalkyl can include one or more unsaturated bonds, but is not aromatic. Unless otherwise indicated, a heterocycloalkyl group can be an unsubstituted or a substituted heterocycloalkyl group. [0029] As used herein, the term “aryl” refers to monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) carbocyclic aromatic ring systems. The term C_n means the aryl ring has “n” carbon atoms. For example, Ce aryl refers to an aryl ring that has 6 carbon atoms in the ring. Examples of aryl groups include, but are not limited to, phenyl, methoxyphenyl, chlorophenyl, naphthyl, methylnaphthyl, fluoronaphthyl, tetrahydronaphthyl, phenanthrenyl, indanyl, indenyl, anthracenyl, tetracenyl, chrysenyl, triphenylenyl, pyrenyl, fluorenyl. Unless otherwise indicated, an aryl group can be an unsubstituted aryl group or a substituted aryl group. In particular, one to four carbon atoms of an aryl ring can be independently substituted with a group selected from, for example, halo, alkyl, alkenyl, OCF₃, NO2, CN, NC, OH, alkoxy, amino, CO2H, CO2alkyl, aryl, and heteroaryl. Other substituents are also contemplated, including Co-salkylene-halo, Co-salkylene-CN, Co-salkylene-NH2, Co-salkylene-OH, and Co-3alkylene-0-Ci-3alkyl.

[0030] As used herein, the term “heteroaryl” refers to a monocyclic or polycyclic aromatic ring system having five to twenty total ring atoms (e.g., a monocyclic aromatic ring with 5-12 total ring atoms), of which 1 , 2, 3, or 4 of those atoms are heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur, and the remaining atoms in the ring are carbon atoms. Unless otherwise indicated, a heteroaryl ring can be unsubstituted or substituted with one or more, and in particular one to four, substituents selected from, for example, halo, alkyl, alkenyl, OCF3, NO2, CN, NC, OH, alkoxy, amino, CO2H, CO2alkyl, aryl, and heteroaryl. In some cases, the heteroaryl ring is substituted with one or more of alkyl and alkoxy groups. Heteroaryl rings can be isolated (e.g., pyridyl) or fused to another heteroaryl group (e.g., purinyl), a cycloalkyl group (e.g., tetrahydroquinolinyl), a heterocycloalkyl group (e.g., dihydronaphthyridinyl), and/or an aryl group (e.g., benzothiazolyl and quinolyl). Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyridyl, pyrrolyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl. When a heteroaryl ring is fused to another heteroaryl group, then each ring can contain five to twenty total ring atoms and one to five heteroatoms in its aromatic ring.

[0031] As used herein, the term “substituted,” when used to modify a chemical functional group, refers to the replacement of at least one hydrogen radical on the functional group with a substituent. Substituents can include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycloalkyl, heterocycloalkenyl, ether, polyether, thioether, polythioether, aryl, heteroaryl, hydroxyl, oxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, ester, thioester, carboxy, cyano, nitro, amino, amido, acetamide, and halo (e.g., fluoro, chloro, bromo, or iodo). When a chemical functional group includes more than one substituent, the substituents can be bound to the same carbon atom or to two or more different carbon atoms.

Methods of the Disclosure

[0032] The disclosure provides methods of functionalizing a vesicle, the methods comprising (a) providing a vesicle in the form of a sphere comprising an internal volume, the internal volume defined by a lipid bilayer having an interior facing hydrophilic surface and an exterior facing hydrophilic surface opposing the interior facing hydrophilic surface, wherein the vesicle has an external surface comprising the exterior facing hydrophilic surface of the lipid bilayer and the external surface comprises a first sugar residue connected thereto; and (b) admixing the vesicle and an enzyme, to provide a functionalized vesicle comprising the vesicle functionalized at the first sugar residue. Methods of the disclosure can further include (c) admixing a functionalized vesicle with a labeling reagent to provide a labeled vesicle.

[0033] Admixing the vesicle and the enzyme can include mixing the vesicle and enzyme in water or an aqueous solvent. The admixing can be performed in the presence of one or more additional components, including but not limited to buffers, salts, and proteins. Admixing the vesicle and the enzyme can be carried out at a temperature at which the enzyme exhibits enzymatic activity. For instance, the admixing may be carried out at about 20 °C, or about 25 °C, or about 30 °C, or about 35 °C, or about 37 °C, or about 40 °C, or about 50 °C. Admixing the vesicle and the enzyme at about 37 °C is particularly contemplated.

[0034] Admixing the vesicle and the enzyme can be carried out for a time sufficient to functionalize a first sugar residue on the vesicle via an enzyme-driven reaction. For instance, the vesicle and the enzyme can be admixed for at least 30 minutes, or at least 1 hour, or at least 2 hours, or at least 3 hours.

[0035] In some aspects, admixing the vesicle and the enzyme further comprises admixing with a donor substrate. The donor substrate can be a sugar donor substrate. The donor substrate can include a sugar residue bound to a nucleotide, and the enzyme can be selected to transfer the sugar residue from the donor substrate to be attached to the first sugar residue.

[0036] Admixing a functionalized vesicle and a labeling reagent can include mixing the functionalized vesicle and labeling reagent in water or an aqueous solvent, optionally in the presence of one or more additional components including, but not limited to, buffers and salts. Time and temperature conditions for admixing a functionalized vesicle and a labeling reagent are not particularly limited and can be selected so as to connect the labeling reagent (and accordingly the labeling group) to the vesicle, for instance by covalently connecting the labeling reagent to the vesicle. For instance, the admixing can be carried out for at least 30 minutes, or at least 1 hour, or at least 2 hours, or at least 3 hours, and at a temperature of about 20 °C, or about 25 °C, or about 30 °C, or about 35 °C, or about 37 °C, or about 40 °C, or about 50 °C, or greater than about 50 °C. Connecting the labeling reagent to the vesicle via an azide-alkyne cycloaddition reaction is particularly contemplated.

[0037] Methods disclosed herein can be carried out on vesicles that are dissolved or dispersed in aqueous solution. Alternatively, methods disclosed herein can be carried out on vesicles that have been immobilized on a substrate. For instance, vesicles can be immobilized on a substrate (such as, for example, a glass slide or a plate) by modifying the substrate to facilitate binding of vesicles, followed by providing vesicles to the modified substrate, such that the modified substrate binds a plurality of the vesicles. Reagents and optionally other components can then be introduced to the substrate-bound vesicles to functionalize and/or label the vesicles according to methods disclosed herein. Without intending to be bound by theory, it is believed that immobilizing vesicles on a substrate can enable (i) functionalization and labeling of the vesicles using lower amounts of reagents (for instance, enzymes) that would otherwise be required to perform methods of the disclosure on vesicles dissolved or dispersed in solution, as well as (ii) faster and more convenient washing of the vesicles.

[0038] As a non-limiting example of immobilizing vesicles on a substrate, a substrate can be modified by attaching silica beads to the substrate, modifying the silica beads to facilitate binding of vesicles, and providing vesicles to the substrate, such that vesicles bind to the modified silica beads. Techniques for modifying the surface of a substrate and for modifying the surface of silica beads will be familiar to those of skill in the art.

[0039] Methods of the disclosure can be carried out within a microfluidic device, on plates, or in solution, as described herein.

[0040] The first sugar residue can be a sugar residue that is native to the vesicle. Alternatively, a vesicle can be modified to include a particular first sugar residue on the vesicle surface and the new first sugar residue can be a target for functionalization.

[0041] The disclosure also provides a functionalized vesicle or labeled vesicle prepared according to a method of the disclosure.

[0042] Providing a functionalized vesicle can include functionalizing a first sugar residue on the external surface of a vesicle via reaction with a sugar oxidase to install a reactive group on the first sugar residue. The sugar oxidase can comprise glucose oxidase, galactose oxidase, or a combination thereof. The reaction with the sugar oxidase can convert a hydroxy group or hydroxymethyl group on the sugar residue to an aldehyde group, and the resulting aldehyde group can be further reacted to install a labeling group, such as biotin, a fluorophore, or a drug, on the vesicle.

[0043] Providing a functionalized vesicle can include functionalizing a first sugar residue on the external surface of a vesicle via reaction with a glycosyltransferase and a glycosyl donor to attach a second sugar residue to the first sugar residue. The second sugar residue can include a substituent that is susceptible to further chemical modifications, enabling further targeted functionalization or labeling of the vesicle.

Extracellular Vesicles

[0044] Vesicles that can be functionalized and/or labeled according to methods of the disclosure can include, but are not limited to, extracellular vesicles (EVs). Nonlimiting examples of EVs include, but are not limited to, exosomes, microvesicles, apoptotic bodies, stressomes, autophagic extracellular vesicles, and combinations thereof.

[0045] Exosomes are particularly contemplated as EVs that can be functionalized and/or labeled according to methods of the disclosure. Exosomes can contain cargo including, but not limited to, RNA, DNA, and/or proteins. In particular, methods of the disclosure can be used to functionalize exosomes from various cell sources, including but not limited to exosomes derived from A549, PC3, or COLO-1 cells, or serum or other body fluids, or combinations thereof.

[0046] In general, a vesicle according to the disclosure or a vesicle prepared according to the method of the disclosure is a lipid bilayer-defined vesicle. The vesicle is generally spherical and comprises an internal volume, the internal volume defined by a lipid bilayer having an interior facing hydrophilic surface and an exterior facing hydrophilic surface opposing the interior facing hydrophilic surface, wherein the vesicle has an external surface comprising the exterior facing hydrophilic surface of the lipid bilayer. Figure 1 includes a representation of a cross-sectional view of a vesicle, indicating a lipid bilayer-based structure, interior and exterior facing hydrophilic surfaces, and an internal volume defined by the lipid bilayer. Figure 1 also depicts a sugar residue at the external surface of the vesicle. Sugar residues at the external surface of the vesicle can be, for instance, components of glycolipids or glycoproteins; as a non-limiting example, Figure 1 depicts sugar residues of a glycoprotein. [0047] The external surface of an EV such as an exosome generally includes glycoproteins and glycolipids, i.e., proteins and lipids that are functionalized with oligosaccharides or glycans. As such, the external surface of an EV can include sugar residues, as components of oligosaccharides, polysaccharides, glycans, or other surface-bound components of the EV. Sugar residues at the external surface of an EV can be targets for reactions, including enzyme- driven reactions according to methods of the disclosure. Such reactions can introduce nonnative substituents or functional groups to the EV.

[0048] An EV can include on its external surface a first sugar residue that can be functionalized upon admixing with an enzyme according to methods of the disclosure. The first sugar residue is not particularly limited. The first sugar residue can be a monosaccharide residue. Residues of galactose, N-acetylgalactosamine, glucose, and N-acetylglucosamine are particularly contemplated as the first sugar residue. The first sugar residue can be a nonreducing sugar residue. The external surface can include a non-reducing end of the first sugar residue.

Functionalization of Extracellular Vesicles

[0049] As described above, existing methods of modifying the external surface of an EV can, disadvantageously, exhibit high complexity, low selectivity, and/or low yield. Methods disclosed herein can take advantage of the inherent selectivity of enzyme-driven reactions to introduce functional groups connected to the external surface of an EV. The introduced functional groups can then serve as molecular handles for further functionalization steps, such as to install a chemoselective group (e.g., biotin), a fluorescent tag, or a therapeutic agent (e.g., a drug) intended for targeted delivery.

[0050] Functionalizing a first sugar residue of the external surface of an EV can include transforming a group on the sugar residue (for instance, a hydroxy group or hydroxymethyl group) to form a different functional group, such as a functional group that is more reactive than the hydroxy groups and/or hydroxymethyl groups on the native sugar residue (e.g., an aldehyde). The functional group that is more reactive than the hydroxy and/or hydroxymethyl group on the native sugar residue can then be further functionalized by additional reactions at the more reactive functional group. For example, a hydroxymethyl group (-CH₂OH) on a saccharide ring can be oxidized to an aldehyde group, which can be further reacted to install additional groups on the first sugar residue and/or covalently attach a second sugar residue to the first sugar residue Enzymatic Glycan Engineering

[0051] The disclosure provides methods of functionalizing a vesicle, such as an EV, via enzymatic glycan engineering. In general, an EV can be modified by an enzyme-driven reaction to functionalize one or more sugar residues on the external surface of the EV. The enzyme- driven reaction can modify a sugar residue on the external surface of an EV to include a nonnative functional group. For instance, the enzyme-driven reaction can convert a hydroxymethyl group on a sugar residue to an aldehyde group. As another example, the enzyme-driven reaction can modify a sugar residue on the external surface of an EV to include a second sugar residue that includes a non-native functional group. The non-native functional group on the second sugar residue is not particularly limited; in some aspects the non-native functional group on the second sugar residue can be an azide group or an alkyne group. Azide and alkyne groups are susceptible to cycloaddition reactions (often referred to as “click” reactions) whereby an azide group and an alkyne group are reacted, optionally in the presence of a catalyst, to form a 1 ,2,3-triazole.

Glycosyltransferase

[0052] The enzyme used in methods of the disclosure can be a glycosyltransferase. In general, glycosyltransferases can catalyze formation of glycosidic linkages. In particular, glycosyltransferases can catalyze transfer of a sugar residue from a glycosyl donor (i.e. , a donor substrate) to an acceptor moiety. The donor substrate used in methods of the disclosure that include a glycosyltransferase-driven reaction can include a nucleotide sugar, i.e., a sugar residue bound to a nucleotide, providing transfer of the sugar residue to an EV.

[0053] Thus, methods according to the disclosure that comprise a glycosyltransferase-driven reaction can further include admixing with the glycosyltransferase a donor substrate that provides a second sugar residue to an EV. The donor substrate can comprise a second sugar residue bound to a nucleotide, and a glycosyltransferase-driven reaction transfers the second sugar residue to the vesicle at the first sugar residue.

[0054] Figure 1 shows a generalized, non-limiting scheme of functionalizing an extracellular vesicle (EV) using a glycosyltransferase. Initially, the EV (upper left) includes a natural sugar residue (the ‘first sugar residue’ of the methods disclosed herein) at the external surface of the EV. The EV is admixed with a glycosyltransferase and a donor substrate comprising an unnatural (i.e., non-naturally-occurring) sugar bound to a nucleotide; the resulting functionalized vesicle (upper right) includes the unnatural sugar bound to the vesicle at the first sugar residue. Subsequently, the functionalized vesicle is admixed with a labeling reagent including a molecular label (“labeling group”) that can react with a functional group on the unnatural sugar; the resulting labeled vesicle includes the labeling group bound to the vesicle through the natural sugar residue on the EV.

[0055] Non-limiting examples of glycosyltransferases include, but are not limited to, a(2,3)sialyltransferase, a(2,6)sialyltransferase, a(2,8)sialyltransferase, a(1 ,2)fucosyltransferase, a(1 ,3)fucosyltransferase, a(1 ,6)fucosyltransferase, [3(1 ,2)galactosyltransferase, [3(1 ,3)galactosyltransferase, [3(1 ,4)galactosyltransferase, [3(1 ,6)galactosyltransferase, [3(1 ,2)galactosaminosyltransferase, [3(1 ,3)galactosaminosyltransferase,

[3(1 ,4)galactosaminosyltransferase, [3(1 ,6)galactosaminosyltransferase, or a combination of any of the foregoing. Sialyltransferases, including a(2,3)sialyltransferases, a(2,6)sialyltransferases, and a(2,8)sialyltransferases, are particularly contemplated.

[0056] Sources of glycosyltransferases for use in methods of the disclosure include, but are not limited to, bacteria. For instance, suitable bacterial sources of glycosyltransferases include, but are not limited to, Photobacterium leiognathi, Photobacterium phosphoreum, Photobacterium damselae, Vibrio sp., Pasteurella multocida, Pasteurella dagmatis, Haemophilus ducreyi, Bibersteinia trehalosi, Campylobacter jejuni, Neisseria meningitidis, Neisseria gonorrheae, Haemophilus influenzae, Streptococcus agalactiae, and Escherichia coli.

[0057] CSTII, or CST2, is a bacterial sialyltransferase (Sia-T) that can catalyze a2-3 and a2- 8-sialylations. Its a2-8 sialylation activity is generally low; its primary activity is a2-3-sialylation. It has been shown that CSTII can accept sialyl donor substrates that include artificial derivatives of N-acetylneuraminic acid. CSTII-mediated modification of EVs to install an artificial Neu5Ac derivative containing an azide group are described herein.

Glycosyl Donors / Donor Substrates

[0058] The donor substrate for glycosyltransferase-driven reactions can include a sugar residue (i.e., a second sugar residue) bound to a nucleotide. The second sugar residue can include, but is not limited to, a residue of fucose, galactose, neuraminic acid, N- acetylneuraminic acid, galactosamine, or N-acetylgalactosamine. Residues of sialic acids, including but not limited to neuraminic acid (Neu), N-acetylneuraminic acid (Neu5Ac), and derivatives thereof, are particularly contemplated.

Neu Neu5Ac

[0059] The second sugar residue can comprise a non-native functional group. The nonnative functional group can be selected to impart reactivity to the vesicle comprising the second sugar residue. While the non-native functional group is not particularly limited, azide and alkyne groups are particularly contemplated, as vesicles modified with these functional groups can be further functionalized using well-known azide-alkyne cycloaddition reactions (often referred to as “click” reactions). The non-native functional group can include a functional group that enables selective binding (such as biotin), a fluorophore, or a drug.

[0060] The nucleotide comprising the donor substrate is not particularly limited. The nucleotide can comprise cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, thymidine monophosphate, thymidine diphosphate, thymidine triphosphate, guanosine monophosphate, guanosine diphosphate, or guanosine triphosphate. Cytidine monophosphate is particularly contemplated. , wherein Ac is acetyl and O-Nu indicates a bond between an oxygen of the second sugar residue and the nucleotide (Nu), and wherein R comprises an azide group, an alkyne group, biotin, a fluorophore, or a drug. Non-limiting a bond or R³ is selected from O-, Ci-6alkylene-, (Ci-6alkylene)O-, O(Ci-6alkylene)-,

NH(Ci_₆alkylene)O- NH-, (Ci-₆alkylene)NH- NH(Ci_₆alkylene)-, O(Ci-₆alkylene)NH-

NH(Ci_₆alkylene)NH- NHC(=O)(Ci-₆alkylene), OC(=O)(Ci_₆alkylene)- O(CH₂CH₂O)_n-,

O(CH₂CH₂O)_n(Ci-6alkylene)O-, O(CH₂CH₂O)n(Ci-₆alkylene)NH-, NH(CH₂CH₂O)_n(Ci-6alkylene)O-, and NH(CH₂CH₂O)n(Ci-6alkylene)NH- wherein each n is independently 1 , 2, 3, 4, 5, or 6. R can comprise biotin-R³, fluorophore-R³, or drug-R³, where R³ is defined as above.

[0062] The donor substrate can comprise 9-azido-9-deoxy-N-acetylneuraminic acid-cytidine monophosphate (9-N₃-Neu5Ac-CMP, 1 ). Enzyme-driven reactions involving 1 can transfer a 9- N₃-Neu5Ac residue (2) to the 3-0-positions of galactose (Gal) or N-acetylgalactosamine (GalNAc) residues in glycans on the EV surface, providing azide groups on the resulting EV that can serve as molecular handles for further reactions.

[0063] Figure 2 shows a generalized scheme of functionalizing a first sugar residue on the external surface of an EV using a glycosyltransferase (GT) to install a second, modified sugar residue covalently bound to the first sugar residue. In Figure 2, the sialyl donor substrate is shown as the modified sugar residue containing an R group (which can be, for instance, a functional group such as an azide group or alkyne group) attached to a nucleotide (Nu) at a phosphate group (P) on the nucleotide. Sugar Oxidase

[0064] The enzyme used in methods of the disclosure can comprise a sugar oxidase, such as galactose oxidase, glucose oxidase, or a combination thereof. The enzyme can comprise galactose oxidase. The enzyme can comprise glucose oxidase. In general, sugar oxidases can catalyze oxidation of a specific sugar or sugar residue, such as a hydroxymethyl group on galactose or a galactose residue oxidized to an aldehyde group or a hydroxy group on glucose or a glucose residue oxidized to a ketone group.

[0065] The method can comprise admixing an EV and galactose oxidase to oxidize a hydroxymethyl group on a galactose residue on the external surface of an EV to an aldehyde group. The aldehyde group can be subjected to further reactions to attach a labeling group to the EV. Figure 3 shows a generalized, non-limiting scheme of functionalizing an EV using galactose oxidase. In the figure, a sugar residue (for instance, a galactose residue) on the surface of the EV is modified to contain an aldehyde group, and the aldehyde group is functionalized with biotin hydrazide to provide a biotin-labeled vesicle.

Labeled Vesicles

[0066] The disclosure further provides labeled vesicles and methods of introducing a labeling group to a vesicle that has been functionalized via enzymatic glycan engineering, to provide a labeled vesicle. The methods of the disclosure can further include (c) admixing the functionalized vesicle with a labeling reagent to provide a labeled vesicle. Optionally, step (c) can further comprise admixing, with the functionalized vesicle and the labeling reagent, a reducing agent.

[0067] The labeled vesicle can comprise a vesicle in the form of a sphere comprising an internal volume defined by a lipid bilayer having an interior facing hydrophilic surface and an exterior facing hydrophilic surface opposing the interior facing hydrophilic surface, wherein the vesicle has an external surface comprising the exterior facing hydrophilic surface of the lipid bilayer and the external surface comprises a first sugar residue connected thereto, wherein the vesicle comprises a labeling group selected from biotin, fluorophores, and drugs, wherein the labeling group is connected to the external surface of the vesicle at the first sugar residue. The vesicle can be any vesicle disclosed herein. The first sugar residue can be any sugar residue disclosed herein.

[0068] The labeling group can be selected to impart useful properties to the vesicle, including but not limited to selective binding, fluorescence, and targeted delivery. [0069] The labeling reagent can comprise a linker group bound to a labeling group, and admixing the functionalized vesicle with the labeling reagent can connect the linker group to the external surface of the vesicle. Accordingly, extracellular vesicles of the disclosure, and extracellular vesicles functionalized according to methods of the disclosure, can include a labeling group connected to the external surface of the vesicle. The external surface of the vesicle can comprise a first sugar residue connected thereto, and the labeling group can be connected to the external surface of the vesicle at the first sugar residue. The labeling group can be connected to the external surface of the vesicle through covalent bonds, ionic bonds, van der Waals interactions, or a combination of any of the foregoing. Vesicles in which the labeling group is connected to the external surface of the vesicle through covalent bonds are particularly contemplated.

[0070] As used herein, “linker group” refers to a component of a labeling reagent or to chemical group(s) coupled to a labeling group that can link the labeling group of a labeled vesicle to the external surface of the vesicle. The linker group present in a labeled vesicle can be different from the linker group present in the labeling reagent as a result of reactions involved in linking the labeling reagent to the vesicle. As a non-limiting example, a linker group comprising an azide or alkyne group can be attached to a functionalized vesicle comprising an alkyne or azide group via an azide-alkyne cycloaddition reaction to form a substituted 1 ,2,3- triazole group, such that the linker group present in the labeled vesicle comprises a substituted 1 ,2,3-triazole group and the labeled vesicle comprises the labeling group connected to the external surface of the vesicle through the substituted 1 ,2,3-triazole group .

[0071] The labeling group can comprise biotin, a fluorophore, a drug, or a combination thereof. The labeling group can comprise biotin. The selective binding of biotin to avidin, streptavidin, and related derivatives is well-known. Vesicles labeled with biotin can selectively bind to substrates or other species containing avidin or streptavidin.

[0072] The labeling group can be a fluorophore. The fluorophore is not particularly limited; suitable fluorophores include, but are not limited to, fluorescein, cyanine dyes, and derivatives thereof. Vesicles labeled with a fluorophore can be tracked and/or quantified in subsequent manipulations using fluorescence or fluorescent imaging techniques.

[0073] The labeling group can be a drug. Use of vesicles labeled with a drug for targeted delivery applications is contemplated. As used herein, “drug” includes pharmaceutically active agents and therapeutic agents, including, but not limited to, small-molecule and large-molecule diagnosis reagents and anti-cancer drugs. [0074] The linker group can comprise a first terminus and a second terminus, wherein the second terminus is bound to the labeling group and the second terminus comprises a group capable of reacting with the vesicle surface to link the labeling group to the vesicle. In some aspects, the first terminus of the linker group comprises an alkyne group, an azide group, a carboxyl group, an amine group, or a hydrazide group. In some aspects the first terminus comprises an amine group, a hydrazine group, or a hydrazide group and the linker group comprises -HN-R¹-, =N-R¹-, -HN-NH-R¹- or =N-NH-R¹-, wherein R¹ comprises the second terminus and R¹ is a bond or R¹ is selected from O, O-ealkylene, O(Ci-ealkylene), (Ci_₆alkylene)O, (CH₂CH₂O)_n, O(CH₂CH₂O)_n, (Ci-₆alkylene)NH, (CH₂CH₂O)_nCH₂CH₂NH, and O(CH₂CH₂O)_nCH₂CH₂NH, wherein each n is independently 1 , 2, 3, 4, 5, or 6. For example, in some aspects, the linker group can be -HN-, =N-, =N-NH-, or -HN-NH-, wherein the first terminus is bound to the first sugar residue and the second terminus is bound to the labeling group. For example, the schematic in Figure 3 indicates a labeled vesicle including biotin bound to a hydrazide group and the hydrazide group bound to a sugar residue on the surface of the vesicle. For aspects in which the linker group is -HN- or =N-, the first terminus and second terminus are the same amine group. For aspects in which the linker group is =N-NH- or -HN- NH-, the first terminus and the second terminus are the same hydrazide group.

[0075] The second terminus can include O-, Ci-6alkylene-, O(Ci-ealkylene)-, (Ci_₆alkylene)O-, (CH₂CH₂O)_n-, O(CH₂CH₂O)_n-, (Ci_₆alkylene)NH-, (CH₂CH₂O)_nCH₂CH₂NH-, or O(CH₂CH₂O)_nCH₂CH₂NH-, wherein each n is independently 1 , 2, 3, 4, 5, or 6.

[0076] The labeled vesicle can include the labeling group connected to a sugar residue at the external surface of the vesicle through an amine group, a hydrazine group, or a hydrazide group or a reaction product thereof.

[0077] The labeled vesicle can include a second sugar residue connected to the first sugar residue, wherein the labeling group is connected to the second sugar residue through the linker. In some aspects, the second sugar residue is a non-naturally-occurring sugar residue. Non- naturally-occurring sugar residues can include sugar residues that have been modified to introduce a functional group, including, but not limited to, an azide group or an alkyne group. [0078] The linker group as present in the labeling reagent can be HC=C-R²-, N3-R²-, HO2C- , p s a bond or is selected from O, Ci-6alkylene, O(Ci-6alkylene), (CH2CH2O)_n, O(CH2CH2O)_n, (Ci_₆alkylene)NH, (CH2CH₂O)nCH2CH₂NH, and O(CH₂CH2O)nCH2CH₂NH, wherein each n is independently 1 , 2, 3, 4, 5, or 6.

[0079] As described herein, methods of making a functionalized or labeled vesicle according to the disclosure can include using click reactions, such as azide-alkyne cycloaddition reactions, to attach functional groups to the vesicle. Azide-alkyne cycloaddition reactions generally provide a 1 ,2,3-triazole group; accordingly, vesicles according to the disclosure can comprise a 1 ,2,3-triazole group as part of the linker group connecting the labeling group to the external surface of the vesicle. The linker group can comprise , wherein R⁴ is selected from Ci-ealkylene, (Ci-6alkylene)O(Ci-6alkylene), (CH2CH2O)_n(Ci-6alkylene), (Ci-6alkylene)NH(Ci-6alkylene), or (CH2CH₂O)n(Ci-6alkylene), and wherein R⁵ and R⁶ are independently selected from O, (O-ealkylene), O(Ci-ealkylene)O, (CH₂CH₂O)n, O(CH₂CH₂O)_n, (Ci-₆alkylene)NH, (CH₂CH₂O)_nCH₂CH₂NH, O(Ci_₆alkylene)NH, and

R⁵ R⁶ R⁵ R⁶

O(CH2CH2O)nCH2CH₂NH. The linker group can comprise , wherein R⁴ is selected from Ci-6alkylene, (Ci-6alkylene)O(Ci-6alkylene), (CH2CH₂O)_n(Ci-6alkylene), (Ci-6alkylene)NH(Ci-6alkylene), or (CH2CH2O)_n(Ci-6alkylene), and wherein R⁵ and R⁶, together with the carbon atoms to which they are attached, form a carbocycle or heterocycle. [0080] The linker group comprising a labeled vesicle can comprise wherein R⁷ is selected from

Ci-6alkylene, (Ci-6alkylene)O(Ci-6alkylene), (CH₂CH₂O)_n(Ci-6alkylerie), (Ci-6alkylene)NH(Ci-6alkylene), and (CH₂CH₂O)_n(Ci-6alkylene), wherein each n is independently 1 , 2, 3, 4, 5, or 6.

[0081] Labeling reagents comprising an amine group, a hydrazine group, or a hydrazide group are particularly suitable for reacting with a first sugar reside that has been modified according to methods of the disclosure to contain an aldehyde group, for instance as shown in Figure 3. The labeling reagent can comprise an amine group, a hydrazine group, or a hydrazide group directly bound to a labeling group. The labeling reagent can comprise biotin hydrazide.

Microfluidic Devices and Plates

[0082] Functionalization of EVs in solution can be limited by low recovery rates associated with conventional solution-based methods of handling and purifying EVs. Attaching EVs to a plate to perform modification can address limitations of conventional solution-based methods. Methods of the disclosure were carried out on surface-bound vesicles on a plate. Plates for carrying out methods of the disclosure can be any made from any suitable material, as will be known to persons of skill in the art.

[0083] The use of microfluidic technology can also address limitations of solution-based processing. Manipulating surface-bound EVs via microfluidic techniques can enable improved yields compared to processes for manipulating solution-phase EVs, due in part to the higher surface-to-volume ratio inherent to such surface techniques.

[0084] Methods of the disclosure were carried out on surface-bound vesicles within a microfluidic device or chip. The microfluidic chips included reaction chambers comprising silica beads immobilized on a glass surface; the silica beads were functionalized to enable immobilization of vesicles on the bead surface. In general, the microfluidic chip comprised a polydimethylsiloxane (PDMS) pneumatic layer and a PDMS fluidic layer on a glass substrate. The pneumatic channels, actuated by a solenoid controller, enabled control of flow through the fluidic channels. Each chip contained eight parallel reaction chambers, each chamber containing silica nanobeads (~0.5 pm diameter) immobilized on the glass substrate in a herringbone pattern, enabling eight simultaneous reactions. Methods used to bind EV’s to the surface of a microfluidic device or chip are described herein.

EXAMPLES

Materials and Methods

[0085] Lyophilized exosomes/EVs from A549 cells were purchased from Galen Laboratory Supplies (USA). Annexin V and 10x Annexin binding buffer were obtained from BD Biosciences (USA). Absolute ethanol (200% proof), CaCL, 3-mercaptopropyl-trimethoxysilane (3-MPS), N- (y-maleimidobutyryloxy)succinimide ester (GMBS), 3-aminopropyl-triethoxysilane (APTES), and glutaraldehyde were obtained from Fisher Scientific (USA). Anti-CD81 , biotinylated anti-CD81 , biotinylated anti-CD63, and biotinylated anti-CD9 monoclonal antibodies were obtained from Ancell (USA). Biotinylated Sambucus Nigra lectin (SNA) and Maackia Amurensis lectin II (MAL II) were ordered from Vectorlabs (USA). Phosphate buffered saline (PBS, 1 x) was obtained from ThermoFisher Scientific (USA). Bovine serum albumin (BSA) and MgCl2 were obtained from Sigma-Aldrich (USA). Campylobacter jejuni o-2,3/8-sialyltransferase (CST-II) was obtained from Chemily, LLC (USA). a2-3,6,8-Neuraminidase was obtained from New England Biolabs (USA). Streptavidin p-D-galactosidase (S|3G) was purchased from Invitrogen (USA), and fluorescein di-p-D-galactopyranoside (FDG) was obtained from Life Technologies (USA). Aqueous suspension of 0.5 pm-diameter silica beads was obtained from Bangs Laboratories (USA).

[0086] Fluorescence measurements were performed according to standard techniques using a commercial fluorescence spectrometer or imager, including a microscope with a camera. Specific conditions (for instance, excitation or emission wavelength) for characterizing fluorophore-labeled vesicles can be selected to optimize fluorescence measurements based on the properties of the fluorophore, as will be familiar to those of skill in the art.

[0087] Optical density (OD) measurements can be performed according to standard techniques. OD measurements described herein were performed using a biotinylated additive and alkaline phosphatase-streptavidin conjugate (AP-Strep) via colorimetric measurement of AP-catalyzed p-nitrophenylphosphate (PNPP) hydrolysis, determining OD at 405 nm. A BioTek Cytation 1 plate reader or equivalent can be used for performing OD measurements.

Microfluidic Chip Fabrication

[0088] Microfluidic devices comprised a PDMS pneumatic layer, a PDMS fluidic layer, and a glass substrate functionalized with silica beads packed in a herringbone pattern. The molds of the microfluidic device were fabricated using SU-8 photolithography with 50 pm height channels for both molds of the pneumatic layer and the fluidic layer. The PDMS layers were made using standard soft-lithography techniques. In general, a glass slide was cleaned with piranha solution for 15 min, and silica beads (500 nm diameter) were patterned for 10 layers on the glass slide in a herringbone pattern using a nano-printer (FUJIFILM Dimatix). Silica beads were functionalized with mercapto (-SH) groups by treating the glass slide having the nano-patterned silica beads with 3-MPS (5 wt.% solution in ethanol) on a dancer for 1 h, followed by heating at 80 °C for 30 min. Finally, the PDMS slab was aligned to the nano-pattern and attached to the glass slide to provide a microfluidic device with reaction chambers containing the functionalized silica beads.

Example 1 : Modification of microfluidic chips with Annexin V

[0089] EV’s were immobilized on a chip surface by binding to Annexin V, or A5. Annexin V is a non-glycosylated adhesion protein that binds to phosphatidylserine, a phospholipid that can be present on the surface of EVs. Unless stated otherwise, Annexin V was attached to nanopatterned silica beads comprising a microfluidic chip by functionalizing the silica beads with 3- MPS, treating the mercapto-functionalized silica beads with GMBS, and treating the GMBS- treated silica beads with Annexin V. In general, following 3-MPS treatment, each chip channel was rinsed with ethanol and flushed with water, then GMBS aqueous solution (0.28 mg/mL) was pumped through the channel for 30 min. The channel was washed twice with 1 x PBS buffer. Subsequently, Annexin V in 1 x PBS (100 pg/mL) was introduced into the channel and the chip was held at 4 °C overnight, followed by washing twice with 1 xPBS buffer, to remove excess unbound Annexin V.

[0090] In general, the binding of Annexin V to phosphatidylserine is calcium-dependent. Consequently, EVs bound on chips captured by Annexin V could be readily released (for instance, after functionalizing or labeling the EVs using enzymatic glycan engineering) by depleting Ca²⁺ from the chip environment, for instance by introducing a chelant such as ethylenediaminetetraacetic acid (EDTA) to sequester Ca²⁺. Example 2: On-chip enzymatic glycan engineering and further functionalization of exosomes

[0091] A microfluidic chip prepared and functionalized with Annexin V as described above was blocked with BSA by treatment for 1 hr in a 3% BSA solution in 1 x PBS buffer. Then, 10 pL of 0.05 mg/mL A549 exosomes in the capture and wash buffer (i.e. , 1 xAnnexin binding buffer containing 1% BSA and 10 mM CaCy was pumped into the channel within 1 h. A solution of 1 (9-N₃-Neu5Ac-CMP; 3.51 mg/mL) and CST-II (3.3 U/mL) containing 3.35 mM MgCL, 20 mM CaCl2, and 1% BSA in 1 x Annexin binding buffer (5 pL) was introduced to the channel at 37 °C for 1 h, to functionalize the exosomes with azide-modified sugar residues. The channel was washed with 30 pL of the capture and wash buffer. DBCO-biotin (25 pM, 3 pL) in the capture and wash buffer was pumped into the channel at room temperature for 1 h, and the channel was washed with 30 pL of the capture and wash buffer. The channel was then flushed with SpG (20 ng/mL) in the working buffer (1 xAnnexin V binding buffer containing 10 mM CaCL, 2 mM MgCl2, and 1% BSA) at a flow rate of 10 pL/h for 15 min, and washed with 30 pL of the capture and wash buffer. FDG (500 pM) was introduced into the channel by vacuum suction. The chip was held at room temperature for 30 min, and the image of each channel was taken and analyzed using a fluorescent microscope.

[0092] Enzymatic glycan engineering and additional modification of COLO-1 and PC3 cell exosomes were performed according to similar methods. Fluorescence characterization of labeled A549, COLO-1 , and PC3 exosomes (i.e., exosomes derived from A549, COLO-1 , or PC3 cells) and of a negative control sample (i.e., no exosomes) are shown in Figure 5D.

Example 3

[0093] Subsequent experiments assessed whether Annexin V could bind EV’s to a substrate without substantially interfering with affecting enzymatic reactions. Annexin V was immobilized to silica beads on chips by the 3-MPS/GMBS cross-linking approach as described in Example 1 . One channel of the microfluidic device was treated with A549-derived EVs, CSTII, and 1 , and finally with the fluorescent probe by the same protocol as mentioned above. Other channels were treated with the fluorescent probe following treatment with only 1 , or 1 + CSTII (no EVs), or 1 + EVs (no enzyme). As shown in Figure 4, fluorescence signal intensity of the (EVs + 1 + CSTII) treatment samples was significantly higher than that of all other groups lacking the EV, the enzyme, or both. The fluorescence data indicate that EVs were effectively captured by the Annexin V-modified chip and that Annexin V did not significantly participate in or interfere with the enzyme-driven reaction. Example 4

[0094] To validate that treating EVs via enzymatic glycan engineering methods and by treating with 1/CSTII and labeling with biotin-DBCO and a fluorophore did lead to fluorophore- labeled EV’s, and that labels were indeed attached to glycans, functionalized EVs treated with 1 and CSTII as described in Example 2 were treated on-chip with a2-3,6,8-neuraminidase or peptide N-glycosidase (PNGase). a2-3,6,8-Neuraminidase is a selective sialidase which hydrolyzes the glycosidic linkages of natural Neu5Ac but does not hydrolyze glycoside linkages of unnatural derivatives of Neu5Ac (such as 1). PNGase F is a hydrolase that catalyzes the cleavage of N-linked glycans. The azide-tagged EVs were incubated with a2-3,6,8- neuraminidase or PNGase F, respectively, before being treated with DBCO-biotin, S G, and FDG as described in Example 2. Fluorescence data on the resulting EV’s indicate that a2- 3,6,8-neuraminidase treatment did not cause a significant change in fluorescence intensity (Figure 5B; ‘Sialidase treatment’ refers to treatment with a2-3,6,8-neuraminidase), though the release of natural Neu5Ac was detected in the flow-through solution, confirming the neuraminidase activity. However, PNGase F treatment resulted in a >30% decrease of the fluorescent signals of the glycoengineered EVs vs. a positive control (‘PC’ in Figure 5B; a functionalized EV treated with 1 and CSTII but not treated with sialidase or PNGase F), demonstrating that at least a portion of the fluorescent labels were N-glycan-linked. The remaining labels should be linked to N-glycans with fucosylated core structure, O-glycans, and glycolipids, which are resistant to PNGase F. Figure 5B also shows fluorescence data for a negative control sample (‘NC).

[0095] Enzymatic glycan engineering and additional modification were also performed on exosomes derived from HeLa, HEK293 (renal), MDA-MB-231 , MDA-MB-468 and SK-BR-3 (breast), and SK-MEL-28 (skin) cell lines, according to similar methods. As shown in Figure 6, the increased OD values of exosomes treated via EGE and bound on a surface (‘EGE exosome’; second bar in each set) compared to OD values for native exosomes (‘native exosome’; first bar in each set) demonstrate successful binding of exosomes via EGE. Slight discrepancies in OD values for exosomes from these cell lines may indicate that exosomes of different origins can differ in their surface glycan profiles.

[0096] To further study the glycan profiles of various exosomes, biotinylated exosomes from different cell lines were incubated with PNGase F, followed by treatment with AP-Strep/PNPP and colorimetric analysis. Results are shown in Figure 6 (“EGE/PNGase exosomes”; third bar in each set). PNGase F treatment of exosomes from HeLa, MDA-MB-231 , and SK-MEL-28 cells did not result in a significant decrease in OD value, while PNGase F treatment of exosomes from HEK293, MDA-MB-468, and SK-BR-3 cells resulted in decreases in OD value to almost background levels. Without intending to be bound by theory, the results suggest that there are variations in the glycan profile of exosomes from these cells and that tagged glycans on some exosomes are more resistant to PNGase than tagged glycans on other exosomes. The results further suggest that EGE of HEK293, MDA-MB-468, and SK-BR-3 exosomes can occur mainly at their N-glycans, while EGE of HeLa, MDA-MB-231 , and SK-MEL-28 exosomes can occur mainly at their glycolipids, O-glycans, and PNGase F-resistant N-glycans.

[0097] The above-described biotinylated exosomes were also incubated with EDTA, followed by treatment with AP-Strep/PNPP and colorimetric analysis. Decreased OD values following treatment with EDTA to sequester Ca²⁺ and disrupt binding of Annexin V to the surface (Figure 6, “EGE exosome/EDTA”; fourth bar in each set) indicates release of bound exosomes from the surface, further demonstrating on-plate labeling of exosomes by EGE. Figure 6 also indicates that some SK-MEL-28-derived exosomes remained on the plate following EDTA treatment, possibly indicating interaction of these exosomes with the plate through molecules other than phosphatidyl serine and Annexin V.

Example 5: On-chip vs. off-chip EGE

[0098] Efficiencies of on-chip and off-chip EGE of EVs were investigated. The quantities and concentrations of A549 EVs and all reagents for off-chip EGE were the same as those utilized for on-chip EGE as described in Example 2. After A549 EVs were treated with 1 and CSTII in solution, the reaction mixture was diluted with the capture solution, and then introduced to a microfluidic chip for EV capture. Subsequent EV labeling and fluorescence measurement steps were performed as described in Example 2. Fluorescence results, shown in Figure 50, demonstrated that off-chip enzymatic engineering of EVs was efficient and facilitated subsequent labeling. (The left and right bars in each pair in Figure 50 are fluorescence data for a negative control and the A549 EV’s, respectively.) .However, the results also suggested that off-chip EGE may produce less consistent results (i.e., larger error bars associated with repeated measurements) and slightly less efficient labeling compared to on-chip EGE.

Example 6: Cellular uptake of exosomes modified via EGE

[0099] Uptake of EGE-treated exosomes by cells was demonstrated using fluorescencebased detection methods. Initially, exosomes were bound to a plate, functionalized via EGE with a fluorescent label having strong excitation at 488 nm (i.e., A488 exosomes), released from the plate, and collected, as described in Example 4. The labeled exosomes were incubated with HeLa cells for 10 hr, washed extensively, and analyzed via fluorescence imaging. The resulting cells exhibited strong fluorescence upon 488 nm excitation compared to HeLa cells treated with unlabeled exosomes or HeLa cells treated with PBS buffer, indicating uptake of the A488 exosomes by the cells.

[0100] Kinetics of cellular uptake of exosomes were assessed by incubating A488 exosomes with HeLa cells as described above for varying times, up to 24 hr, measuring mean fluorescence intensities from the resulting cells via flow cytometry. Results are shown in Figure 7. Figure 7 indicates that uptake of the labeled exosomes increased after 8 hr of incubation and exhibited a plateau after 24 hr of incubation.

Example 7: Attachment of exosomes to a plate, and enzymatic functionalization of surfacebound exosomes

[0101] 200 pL of a buffer solution containing A5 (2 pL of a 0.5 mg/mL stock solution) was added to each well of a 96-well plate. The plate was incubated at 4 °C overnight, then at 37 °C for 1 h, and washed 3 times with PBST (phosphate-buffered saline with Tween™) buffer. 300 pL of a blocking solution (3% BSA in 1 x PBS) was added to each well, and the plate was incubated at room temperature for 45 min, followed by washing 3 times with PBST. 1 .0 pg of exosomes (10 pL of 0.1 mg/mL stock solution) was then added to each well, followed by A5 binding buffer (1x) to bring the volume to 200 pL. The plate was incubated at 37 °C for 2 h with slow shaking (100 rpm) and then washed 3 times with PBST.

[0102] Following the washing step, a solution comprising 1 (9-N₃-Neu5Ac-CMP) (1 .67 mg/mL; 5.0 pL of a 0.1 g/mL stock solution) and Pd-2,6-ST (an a(2,6)sialyltransferase from Photobacterium damsel; 3.33 pg/mL; 2 pL of a 0.5 mg/mL stock solution) in 300 pL of an enzymatic reaction buffer was added to each well of the plate. The plate was incubated at 37 °C for 1 h with swirling at 100 rpm and washed 3 times. DBCO-biotin (200 pL, 100 pM in Tris buffer, pH 7.5, from 0.4 pL of a 50 mM stock solution) was added to each well, and the plate was incubated at room temperature for 1 .5 h with swirling at 100 rpm, then washed 3 times, to provide functionalized surface-bound exosomes. Surface-bound exosomes were characterized and analyzed as described herein.

Example 8: Recovery of exosomes

[0103] Functionalized surface-bound exosomes from Example 7 were released from the plate as follows. 200 pL of Tris buffer (pH 8.0) containing 10 mM EDTA (2 pL from a 1 M stock solution in Tris buffer) was added to each well, and the plate was incubated at 37 °C for 1 .5 h while swirling at 100 rpm. The supernatant from each well containing released exosomes was collected, and the released exosomes were washed 3 times with PBS and characterized.

Comparative Example 1 : Antibody-based labeling of exosomes

[0104] To further assess the efficiency of EV labeling methods of the disclosure, enzymatic glycan engineering-based labeling was compared to conventional antibody-based methods of EV labeling. A549 EVs were immobilized on a microfluidic chip using Annexin V, as described in Example 1 . In one channel, bound EV’s were modified via EGE as described in Example 2 to provide fluorescein-labeled EV’s. In another channel, samples of bound EVs in other channels were treated with a mixture of biotinylated anti-CD81 , CD63, and CD9 antibodies, then reacted with DBCO-biotin and labeled with fluorescein using S^G-streptavidin and FDG, as described in Example 2. Figure 5A shows fluorescence intensity data of both samples; also included in Figure 5A are fluorescence intensity data of negative controls (i.e., no exosome; the left bar in each pair). The fluorescence intensity of the EGE-labeled EVs was >2 times greater than that of antibody-labeled EVs, indicating that the EGE-based method for EV labeling was more efficient than conventional antibody-based labeling methods.

[0105] EGE-based labeling also provided improved detection limits compared to antibodybased labeling. A plate coated with exosomes at concentrations ranging from 1 .25 to 5.00 pg/mL and labeled via EGE exhibited greater OD signals compared to a negative control, while plates coated with exosomes at concentrations of 2.50, 3.75, and 5.00 pg/mL and treated with biotinylated anti-CD63, CD81 , and CD9 antibodies exhibited no greater OD signals compared to a negative control.

Comparative Example 2: On-chip quantitation of exosomes

[0106] A glass slide was patterned with silica nanobeads and incubated with 5% 3-MPS in ethanol for 1 h. After being washed and air-dried, the glass slide was heated at 80 °C on a hotplate for 30 min. The PDMS slab was aligned on the glass slide. Aqueous GMBS solution (0.28 mg/mL) was flushed through each channel for 30 min. After each channel was washed with 30 pL of 1 x PBS, anti-CD-81 antibody (100 pg/mL) in 1 x PBS was introduced into each channel and incubated at 4 °C overnight. The channel was washed with 30 pL of 1 x PBS and blocked with 3% BSA in 1 x PBS for 1 h. Exosomes in 10 pL of 1 x PBS containing 1% BSA was pumped through each channel for 1 h. A solution of biotinylated anti-CD81 (10 pg/mL), antiCD63 (10 pg/mL), and anti-CD9 (10 pg/mL) antibodies in 1 x PBS (4 pL) containing 1% BSA was introduced to each channel for 30 min. Then, each channel was washed with 30 pL of 1 x PBS containing 1 % BSA and 0.05% Tween® 20. The channel was flushed with SpG in 1 x PBS (20 ng/mL) containing 1% BSA and 2 mM MgCl2 for 15 min. The channel was washed with 30 pL of 1 x PBS containing 1% BSA and 0.05% Tween® 20. FDG (500 pM) in 1 x PBS containing 1% BSA and 2 mM MgCh was introduced into the channel by vacuum suction. The chip was held at room temperature for 30 min, and the fluorescence image of each channel was taken and analyzed with a fluorescent microscope.

Comparative Example 3: On-chip sialic acid quantitation of antibody-bound exosomes

[0107] A glass slide was patterned with silica nanobeads and incubated with 5% APTES in 95% ethanol for 30 min. After being washed and air-dried, the glass slide was heated at 80 °C on a hotplate for 2 h. The PDMS slab was aligned on the glass slide. Aqueous glutaraldehyde solution (2.5%) was pumped through each channel for 2 h. Each channel was then washed with 30 pL of 1 x PBS. Anti-CD-81 antibody in 1 x PBS (100 pg/mL) was introduced to each channel and incubated at 4 °C overnight. The channels were washed with 30 pL of 1 x PBS and blocked with 5% BSA in 1 x PBS for 1 h. Exosomes dissolved in 10 pL of 1 xPBS containing 1% BSA were pumped through each channel for 1 h. Then, a solution of biotinylated SNA (10 pg/mL) and MAL-II (10 pg/mL) in 1 x PBS buffer (4 pL) containing 1 % BSA was introduced to each channel for 30 min. Each channel was washed with 30 pL of 1 x PBS containing 1% BSA and 0.05% Tween® 20 and flushed with SpG (20 ng/mL) in 1 x PBS containing 1% BSA and 2 mM MgCh for 15 min. After the channel was washed with 30 pL of 1 x PBS containing 1 % BSA and 0.05% Tween® 20, FDG (500 pM) in 1 x PBS containing 1% BSA and 2 mM MgCl2 was introduced to the channel by vacuum suction. The chip was held at rt for 30 min, and the fluorescence image of each channel was taken and analyzed with a fluorescent microscope.

Claims

What is claimed is:

1 . A method of functionalizing a vesicle, comprising:

(a) providing a vesicle in the form of a sphere comprising an internal volume, the internal volume defined by a lipid bilayer having an interior facing hydrophilic surface and an exterior facing hydrophilic surface opposing the interior facing hydrophilic surface, wherein the vesicle has an external surface comprising the exterior facing hydrophilic surface of the lipid bilayer and the external surface comprises a first sugar residue connected thereto; and

(b) admixing the vesicle and an enzyme, to provide a functionalized vesicle comprising the vesicle functionalized at the first sugar residue.

2. The method of claim 1 , wherein the vesicle is an extracellular vesicle selected from exosomes, microvesicles, apoptotic bodies, stressomes, and autophagic extracellular vesicles.

3. The method of claim 2, wherein the extracellular vesicle is an exosome.

4. The method of any one of the preceding claims, wherein the first sugar residue is selected from residues of galactose, N-acetylgalactosamine, glucose, N- acetylglucosamine, and derivatives thereof.

5. The method of any one of the preceding claims, wherein the enzyme comprises a sugar oxidase.

6. The method of claim 5, wherein the sugar oxidase is selected from galactose oxidase, glucose oxidase, and combinations thereof.

7. The method of claim 6, wherein the sugar oxidase is galactose oxidase.

8. The method of any one of claims 5 to 7, further comprising:

(c) admixing the functionalized vesicle with a labeling reagent to provide a labeled vesicle.

9. The method of claim 8, wherein the labeling reagent comprises a linker group covalently bound to a labeling group, wherein the linker group comprises a first terminus and a second terminus, wherein the second terminus is covalently bound to the labeling group.

10. The method of claim 9, wherein the labeling group is selected from biotin, a fluorophore, a drug, and combinations thereof.

11 . The method of claim 9, wherein the first terminus comprises an amine group, a hydrazine group, or a hydrazide group.

12. The method of any one of claims 9 to 1 1 , wherein the second terminus comprises O-, Ci-6alkylene-, O(Ci-ealkylene)-, (Ci-6alkylene)O-, (CH2CH₂O)n-, O(CH₂CH₂O)n-, (Ci-₆alkylene)NH-, (CH₂CH₂O)nCH₂CH₂NH-, or O(CH₂CH₂O)_nCH₂CH₂NH-, wherein each n is independently 1 , 2, 3, 4, 5, or 6.

13. The method of any one of claims 9 to 12, wherein the first terminus is bound directly to the second terminus.

14. The method of claim 13, wherein the linker group comprises H₂N-R¹ or H₂NNH-R¹, wherein

R¹ comprises the second terminus, and R¹ is a bond or R¹ is selected from O-, Ci-6alkylene-, O(Ci-ealkylene)-, (Ci-ealkylene)O-, (CH₂CH₂O)_n-, O(CH₂CH₂O)_n-, (Ci_₆alkylene)NH-, (CH₂CH₂O)_nCH₂CH₂NH-, and O(CH₂CH₂O)_nCH₂CH₂NH-, wherein each n is independently 1 , 2, 3, 4, 5, or 6.

15. The method of any of claims 8 to 14, wherein step (c) further comprises admixing, with the functionalized vesicle and the labeling reagent, a reducing agent.

16. The method of any one of claims 9 to 15, wherein the labeled vesicle comprises the labeling group connected to the external surface of the vesicle at the first sugar residue.

17. The method of any one of claims 1 to 4, wherein the enzyme comprises a glycosyltransferase.

18. The method of claim 17, wherein the glycosyltransferase is selected from a(2,3)sialyltransferases, a(2,6)sialyltransferases, a(2,8)sialyltransferases, a(1 ,2)fucosyltransferases, a(1 ,3)fucosyltransferases, a(1 ,6)fucosyltransferases, 3(1 ,2)galactosyltransferases, 3(1 ,3)galactosyltransferases, 3(1 ,4)galactosyltransferases, 3(1 ,6)galactosyltransferases, 3(1 ,2)galactosaminosyltransferases, 3(1 ,3)galactosaminosyltransferases, 3(1 ,4)galactosaminosyltransferases, 3(1 ,6)galactosaminosyltransferases, and combinations of any of the foregoing.

19. The method of claim 17 or claim 18, wherein the glycosyltransferase comprises a a(2,3)sialyltransferase or a a(2,6)sialyltransferase.

20. The method of any one of claims 17 to 19, wherein step (b) further comprises admixing, with the vesicle and enzyme, a donor substrate, wherein the donor substrate comprises a second sugar residue and a nucleotide bound to the second sugar residue.

21 . The method of claim 20, wherein the nucleotide is selected from cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, thymidine monophosphate, thymidine diphosphate, thymidine triphosphate, guanosine monophosphate, guanosine diphosphate, and guanosine triphosphate.

22. The method of claim 20 or claim 21 , wherein the second sugar residue is selected from residues of fucose, galactose, neuraminic acid, N-acetylneuraminic acid, galactosamine, and N-acetylgalactosamine.

23. The method of any one of claims 20 to 22, wherein the second sugar residue comprises an azide group, an alkyne group, biotin, a fluorophore, or a drug.

24. The method of any one of claims 20 to 23, wherein the donor substrate wherein Ac is acetyl and O-Nu indicates a bond between the second sugar residue and the nucleotide (Nu), and wherein R comprises an azide group, an alkyne group, biotin, a fluorophore, or a drug.

25. The method of claim 24, wherein R comprises N3-R³-, HC=C-R³-, biotin- wherein R³ is a bond or R³ is selected from O, Ci-6alkylene, (Ci-6alkylene)O, O(Ci_₆alkylene), NH(Ci_₆alkylene)O, NH, (Ci_₆alkylene)NH, NH(Ci_₆alkylene), O(Ci_₆alkylene)NH, NH(Ci_₆alkylene)NH, NHC(=O)(Ci_₆alkylene), OC(=O)(Ci_₆alkylene), O(CH₂CH₂O)n, O(CH₂CH₂O)n(Ci-₆alkylene)O, O(CH₂CH₂O)_n(Ci_₆alkylene)NH, NH(CH₂CH₂O)_n(Ci-6alkylene)O, and NH(CH₂CH₂O)_n(Ci-6alkylene)NH, wherein each n is independently 1 , 2, 3, 4, 5, or 6.

26. The method of claim 24 or claim 25, wherein the donor substrate comprises 9-azido-9-deoxy-N-acetylneuraminic acid-cytidine monophosphate.

27. The method of any one of claims 20 to 26, wherein the functionalized vesicle includes the second sugar residue covalently bound to the first sugar residue.

28. The method of any one of claims 17 to 27, further comprising:

(c) admixing the functionalized vesicle with a labeling reagent to provide a labeled vesicle.

29. The method of claim 28, wherein the labeling reagent comprises a linker group covalently bound to a labeling group, wherein the linker group is linear and comprises a first terminus and a second terminus, wherein the second terminus is covalently bound to the labeling group.

30. The method of claim 29, wherein the labeling group is selected from biotin, fluorophores, and drugs.

31 . The method of claim 29 or claim 30, wherein the first terminus of the linker group comprises an alkyne group, an azide group, a carboxyl group, an amine group, a hydrazine group, or a hydrazide group.

32. The method of any one of claims 29 to 31 , wherein the linker group

R² is a bond or

R² comprises the second terminus and R² is selected from O, Ci-ealkylene, O(Ci_₆alkylene), (CH₂CH₂O)_n, O(CH₂CH₂O)_n, (Ci_₆alkylene)NH, (CH₂CH₂O)_nCH₂CH₂NH, and O(CH₂CH₂O)nCH₂CH₂NH, wherein each n is independently 1 , 2, 3, 4, 5, or 6.

33. The method of any one of claims 29 to 32, wherein the labeled vesicle comprises the labeling group connected to the external surface of the vesicle at the first sugar residue.

34. The method of claim 33, wherein the labeled vesicle comprises the labeling group connected to the external surface of the vesicle through a substituted 1 ,2,3- triazole group.

35. A vesicle prepared according to the method of any one of claims 1 to 34.

36. A labeled vesicle, comprising: a vesicle in the form of a sphere comprising an internal volume defined by a lipid bilayer having an interior facing hydrophilic surface and an exterior facing hydrophilic surface opposing the interior facing hydrophilic surface, wherein the vesicle has an external surface comprising the exterior facing hydrophilic surface of the lipid bilayer and the external surface comprises a first sugar residue connected thereto; wherein the vesicle comprises a labeling group selected from biotin, fluorophores, and drugs, wherein the labeling group is connected to the external surface of the vesicle at the first sugar residue.

37. The labeled vesicle of claim 36, wherein the vesicle is an extracellular vesicle selected from exosomes, microvesicles, apoptotic bodies, stressomes, and autophagic extracellular vesicles.

38. The labeled vesicle of claim 37, wherein the extracellular vesicle is an exosome.

39. The labeled vesicle of any one of claims 36 to 38, wherein the first sugar residue is selected from residues of galactose, N-acetylgalactosamine, glucose, N- acetylglucosamine, and derivatives thereof.

40. The labeled vesicle of any one of claims 36 to 39, wherein the labeling group is connected to the first sugar residue through a linker group comprising a first terminus and a second terminus, wherein the second terminus is covalently bound to the labeling group.

41 . The labeled vesicle of claim 40, wherein the first terminus is covalently bound to the first sugar residue.

42. The labeled vesicle of claim 40 or claim 41 , wherein the first terminus comprises an amine group or a hydrazide group.

43. The labeled vesicle of claim 42, wherein the linker group comprises -HN- R¹- or -HNNH-R¹-, wherein

R¹ comprises the second terminus, and R¹ is a bond or R¹ is selected from O, Ci-ealkylene, O(Ci-ealkylene), (Ci-6alkylene)O, (CH₂CH₂O)_n, O(CH₂CH₂O)_n, (Ci-ealkylene)NH, (CH₂CH₂O)_nCH₂CH₂NH, and O(CH₂CH₂O)_nCH₂CH₂NH, wherein each n is independently 1 , 2, 3, 4, 5, or 6.

44. The labeled vesicle of claim 40, wherein the vesicle comprises a second sugar residue connected to the first sugar residue and wherein the first terminus is covalently bound to the second sugar residue.

45. The labeled vesicle of claim 44, wherein the second sugar residue is selected from residues of fucose, galactose, neuraminic acid, N-acetylneuraminic acid, galactosamine, N-acetylgalactosamine, and derivatives thereof.

46. The labeled vesicle of claim 44 or claim 45, wherein the second sugar residue is a residue of a non-naturally-occurring sugar.

47. The labeled vesicle of any one of claims 44 to 46, wherein the linker group comprises a substituted triazole group.

48. The labeled vesicle of claim 47, wherein the linker group comprises wherein R⁴ is selected from Ci-6alkylene, (Ci-6alkylene)O(Ci-6alkylene), (CH2CH2O)_n(Ci-6alkylene), (Ci-6alkylene)NH(Ci-6alkylene), or (CH2CH2O)_n(Ci-6alkylene), and wherein R⁵ and R⁶ are independently selected from O, (Ci-ealkylene), O(Ci-6alkylene)O, (CH₂CH₂O)n, O(CH₂CH₂O)n, (Ci-₆alkylene)NH, (CH2CH₂O)nCH₂CH2NH, O(Ci_₆alkylene)NH, and O(CH₂CH2O)nCH2CH₂NH.

49. The labeled vesicle of claim 47, wherein the linker group comprises wherein R⁴ is selected from Ci-6alkylene, (Ci-6alkylene)O(Ci-6alkylene), (CH2CH₂O)n(Ci-6alkylene), (Ci-6alkylene)NH(Ci-6alkylene), or (CH2CH₂O)_n(Ci-6alkylene), and wherein R⁵ and R⁶, together with the carbon atoms to which they are attached, form a carbocycle or heterocycle.

50. The labeled vesicle of claim 49, wherein the linker group comprises wherein R⁷ is selected from Ci-6alkylene, (Ci-6alkylene)O(Ci-6alkylene), (CH2CH2O)_n(Ci-6alkylene), (Ci-6alkylene)NH(Ci-6alkylene), and (CH2CH2O)_n(Ci-6alkylene), wherein each n is independently 1 , 2, 3, 4, 5, or 6.