Methods and compositions for targeted delivery through polymer vesicles

Abstract

The present disclosure relates to copolymers and polymeric vesicles for targeted delivery of biomolecules to living organisms. Exemplary copolymers include an initiator block, a extender block, and a linkage connecting the initiator block and the extender block. The initiator block comprises a glycan head configured to provide targeted delivery, and the extender block comprises a functional moiety configured to provide desired characteristics to the polymeric vesicle.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 458,102, filed April 8, 2023; U.S. Provisional Patent Application No. 63 / 587,231, filed October 2, 2023; and U.S. Provisional Patent Application No. 63 / 575,056, filed April 5, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure generally relates to compositions and methods associated with polymer nanocarriers, and more particularly, pharmaceutical formulations comprising polymer vesicles capable of selectively / targeting a payload to desired target regions in tissues or to cells. Background Technology

[0004] Delivery via nanotechnology has been widely used in scientific, industrial, and clinical applications. It has emerged as a promising drug delivery method, offering advantages including improved solubility and permeability of drug molecules. In a recent example, considering the generally unstable nature of mRNA molecules and their need for cryogenic storage (e.g., -70°C), mRNA vaccines developed for the COVID-19 virus utilize a special type of lipid nanoparticle (LNP) suitable for encapsulating and stabilizing mRNA molecules. Typical lipid nanoparticles are usually composed of several types of lipids. The ratio of these lipids needs fine-tuning, and the manufacturing cost of lipid nanoparticles can be high. Furthermore, lipid nanoparticles typically cannot selectively deliver mRNA molecules. Therefore, there is a need in this field for nanoparticles with simpler construction and more selective delivery capabilities. Summary of the Invention

[0005] In one aspect, this disclosure provides a copolymer for forming polymer vesicles. The copolymer comprises: an initiator block comprising a glycan head; and a propagator block further comprising a functional portion comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and a linker covalently connecting the initiator block and the propagator block, wherein the linker comprises a disulfide bond.

[0006] In one aspect, this disclosure provides a polymer vesicle. The polymer vesicle includes a membrane defining an internal space, wherein the membrane includes an exemplary copolymer of this disclosure.

[0007] In one aspect, this disclosure provides a pharmaceutical formulation comprising the polymer vesicles of this disclosure.

[0008] In one aspect, this disclosure provides a kit for preparing polymer vesicles. The kit comprises a first reagent and a second reagent, the first reagent comprising an initiator, wherein the initiator comprises a glycan head and an initiator linker portion, the second reagent comprising an elongator, wherein the elongator comprises a functional portion and an elongator linker portion, wherein the functional portion comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the initiator linker portion is configured to be coupled to the elongator linker portion via a disulfide bond.

[0009] In one aspect, this disclosure provides a method for targeted delivery of a payload to an individual. The method comprises administering to the individual an effective amount of a pharmaceutical formulation comprising polymeric vesicles, wherein the polymeric vesicles comprise a membrane encapsulating the payload, and wherein the membrane comprises a copolymer of this disclosure.

[0010] In one aspect, this disclosure provides a method for preventing or treating a disease in an individual, comprising administering to the individual an effective amount of a pharmaceutical preparation comprising polymeric vesicles, wherein the polymeric vesicles comprise a membrane, wherein the membrane comprises a polymeric component of any one of claims 1 to 34; and an effective load encapsulated within the membrane; and wherein the effective load is a therapeutic agent or a derivative therapeutic agent.

[0011] In one aspect, this disclosure provides a method for enhancing an adaptive immune response, comprising administering to an individual an effective amount of a pharmaceutical formulation comprising polymeric vesicles, wherein the polymeric vesicles comprise a membrane encapsulating an effective load, and wherein the membrane comprises a copolymer of this disclosure, wherein the effective load is an immunogenic or derived immunogenic biomolecule. Attached Figure Description

[0012] Figure 1A This diagram illustrates the exemplary synthesis of the exemplary copolymers of this disclosure. X and Y are both integers and are independently from 9 to 14.

[0013] Figure 1B The diagram illustrates the structures of some exemplary polymer vesicles comprising copolymers according to some embodiments of the present disclosure.

[0014] Figure 2A The results of an agarose gel electrophoresis assay are presented, demonstrating the encapsulation efficiency of several exemplary copolymers according to some embodiments of this disclosure. The payload in this efficacy validation assay was approximately 920 bp of GFP mRNA. The copolymers, with or without GFP mRNA, have a size greater than 10 kDa.

[0015] Figure 2B A graphic illustration is provided showing the mean fluorescence intensity of HEK293 cells after transfection with polymeric vesicles encapsulating GFP-mRNA according to some working embodiments of this disclosure.

[0016] Figure 3A The results of agarose gel electrophoresis assays demonstrate the encapsulation efficiency of several copolymers according to some working embodiments of this disclosure. The payload in this validation assay was a spike mRNA with a size of approximately 2550 bp. Exemplary copolymers with or without spike mRNA had a size greater than 10 kDa. The N / P ratio (positively charged polymer amine groups (N = nitrogen) to negatively charged nucleic acid phosphate (P) groups) for each experimental group is indicated by a number at each lane. The N / P ratios tested in this experiment were 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20.

[0017] Figure 3B Provides illustrations with CryoEM images and insets showing the particle size and zeta potential of mRNA-PNP (P1 / P5).

[0018] Figure 3C This image presents chemiluminescent images demonstrating spike protein expression mediated by spike mRNA-PNP (I1-P1 / P5) in HEK293T cells. The first lane shows protein expression from cells transfected with spike mRNA, and the second lane shows protein expression from cells transfected with spike mRNA-PNP (I1-P1 / P5). β-actin was used as the baseline expression in this experiment.

[0019] Figure 4 This image illustrates the colocalization of lysosomes and mRNA-PNPs in cells studied over four hours using confocal fluorescence imaging. Images were collected at 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours, respectively. mRNA-PNPs were FITC-labeled for detection.

[0020] Figure 5 This illustration shows the cellular uptake of fluorescence signals in BMDCs, B cells, and T cells treated with either Siglec-2-targeted spike mRNA-PNP (I1-P1 / P4-FITC / P5) or spike mRNA-PNP (I2-P1 / P4-FITC / P5) one hour later. Data were analyzed using flow cytometry.

[0021] Figure 6 This illustration shows the cellular uptake of fluorescence signals by BMDCs, B cells, and T cells treated one hour after treatment with either DC-SIGN-targeted spike mRNA-PNP (I1-P1 / P4-FITC / P5) or spike mRNA-PNP (I5-P1 / P4-FITC / P5). Data were analyzed using flow cytometry.

[0022] Figure 7Illustrations are provided showing the binding analysis of DC-SIGN, MMR, MINCLE, Dectin-2 and Langerin (0.625 μg / mL) with polymer vesicles according to some embodiments of the present disclosure at pH 7.4.

[0023] Figure 8A A schematic diagram illustrating the design of an animal immunization experiment is shown.

[0024] Figure 8B Provides a graph showing the serum spike-specific IgG titers induced by immunization with I1-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA-PNP, and LNP-mRNA (control), measured on day 28 post-immunization.

[0025] Figure 8C A graph is provided showing the neutralizing titers (ID50) of I1-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA-PNP, and LNP-mRNA (control) measured on day 28 post-immunization. The neutralizing titer was calculated as the reciprocal of the serum dilution that caused a 50% reduction in RLU compared to the virus control well, after subtracting background RLU. ID50 values ​​are plotted on the graph with the standard error of the mean.

[0026] Figure 9 A graphic illustration of FACS data for C2C12 muscle cell uptake of polymer vesicles of this disclosure (at 0 and 1 hours after treatment) and polymer vesicles without targeted glycans (at 1 hour after treatment). Detailed Implementation

[0027] The delivery of nanoparticles has been widely used in various applications. Besides lipid nanoparticles (LNPs), which are considered the most common type of nanoparticle, another type of nanoparticle, polymer vesicles, is gaining increasing attention in industrial and clinical applications. Polymer vesicles (i.e., polymer-based nanoparticles, polymer vesicles, or polymer nanoparticles (PNPs)) are shells self-assembled from amphiphilic block copolymers. These amphiphilic block copolymers are macromolecules containing at least one hydrophobic polymer block and at least one hydrophilic polymer block. When hydrated, these amphiphilic block copolymers self-assemble into a shell such that the hydrophobic blocks tend to bind together to minimize direct exposure to water and form the inner surface of the shell, while the hydrophilic blocks face outwards, forming the outer surface of the shell. The hydrophobic core of these water-soluble polymer vesicles provides an environment for dissolving additional hydrophobic molecules. Therefore, these water-soluble polymer vesicles can act as carrier polymers for encapsulating hydrophobic molecules within the polymer vesicle. Furthermore, the self-assembly of amphiphilic block polymers occurs in the absence of stabilizers, which would otherwise provide colloidal stability and prevent aggregation.

[0028] Polymer vesicles offer numerous advantages, such as high storage stability, ease of manufacturing, convenient surface modification, high biocompatibility, and controlled release mechanisms. However, industry still lacks polymer vesicles for effectively delivering drug molecules to specific regions of the body. This is likely because conventional polymer vesicles do not effectively encapsulate the payload (e.g., biomolecules administered to achieve therapeutic effects) and are generally unstable in serum. Furthermore, conventional polymer vesicles are not capable of selective delivery.

[0029] Polymer vesicles

[0030] Therefore, one aspect of this disclosure provides a polymer vesicle. The polymer vesicles of this disclosure are designed to provide selective delivery (or targeted delivery) and good encapsulation efficiency, particularly for nucleic acid-type payloads. Exemplary polymer vesicles comprise a membrane defining an internal space configured to encapsulate or carry a payload. The membrane of the polymer vesicles of this disclosure comprises a copolymer comprising an initiator block, an elongator block, and a linker, wherein the linker covalently connects the initiator block and the elongator block, and the linker comprises a disulfide bond. The initiator block comprises a glycan head, and the elongator block comprises a functional portion comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof.

[0031] Without being bound by theory, the disulfide bond (i.e., disulfide linkage) was chosen because of its biodegradability in the intracellular environment and / or thiol-mediated uptake. Therefore, the disulfide bond facilitates the uptake of the polymer vesicles of this disclosure and their subsequent degradation to release the encapsulated payload via, for example, intracellular glutathione-mediated cleavage.

[0032] In some embodiments, the copolymer may comprise more than one initiator block and more than one grower block. For example, the copolymer may comprise an initiator block, a first grower block, and a second grower block, wherein the initiator block and the first grower block, as well as the first grower block and the second grower block, are all linked by bonding.

[0033] Initiator Block

[0034] In some embodiments, the initiator block includes a glycan head configured to provide selective delivery. For this purpose, the glycan head may have a targeting portion that acts as a ligand for a target (e.g., a receptor on a target cell). In some embodiments, the targeting portion may be a terminal portion of the glycan head to obtain a better chance of interacting with and binding to the target. However, this disclosure is not limited to the configuration described above. In some embodiments, the target cell for selective delivery is an antigen-presenting cell (APC, such as dendritic cells). In some embodiments, the target cell may be other types of immune cells. In still other embodiments, the target may be any biological cell to which the payload is designed to interact.

[0035] In some embodiments, the initiator block is configured to bind to lectin receptors, such as Siglec-1 (sialic acid adhesin), Siglec-2, Siglec-5 / E, and DC-SIGN, with a certain affinity, thereby exhibiting better uptake of specific types of APCs. In some embodiments, the glycan head of the initiator block contains a mannosidase, which may be a terminal mannose configured to bind to DC-SIGN on dendritic cells. In some embodiments, the glycan head contains a sialoside. In some embodiments, for targeting Siglec-1, the glycan head may contain 9-N-(4H-thieno[3,2-c]chromene-2-carbamoyl)-Neu5Ac-α2,3-Gal-GlcNAc. In some embodiments, for targeting Siglec-2, the glycan head may contain 9-biphenylNeu5Ac-α2,6-Gal. In some embodiments, the glycan head may contain Neu5Ac-α2,3-Gal-GlcNAc for targeting Siglec-5 / E.

[0036] Binding occurs under acidic conditions. In some embodiments, the binding between the initiator block and the target is Ca... 2+Relatedly, calcium coordination may decrease at low pH, leading to lower binding affinity. Therefore, to provide better binding affinity under acidic conditions, the initiator block glycan head may contain an aryl group. Without wishing to be bound by any theory, the aryl group may participate in CH-π and hydrophobic interactions, thereby enhancing binding under acidic conditions. The aryl group may be unsubstituted benzene or benzene substituted with a halide or alkyl halide (e.g., CF3). In some embodiments, the aryl group is coupled to the targeting moiety. For example, the initiator block glycan head may contain an O-arylmannoside.

[0037] Structural configuration of the glycan head. In some embodiments, the glycan head may be a linear or branched structure. In some embodiments, the glycan head may have multiple targeting portions, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting portions. The multiple targeting portions may be arranged linearly, branched, or star-shaped. For example, the glycan head of the initiator block may contain monomannoside, dimannoside, or trimannoside, and when the glycan head contains trimannoside, the trimannoside may be in a linear form or a branched structure, such as α-1,3-α-1,6-trimannoside. In some embodiments, it has been noted that in some cases, the branched configuration (e.g., the trimannoside glycan head) exhibits superior binding affinity to its target receptor.

[0038] Initiator spacer group. In some embodiments, the initiator block further comprises an initiator spacer group. The initiator spacer group is configured to provide structural flexibility to the glycan head and / or hydrophobicity to the entire copolymer to facilitate the assembly of polymer vesicles. Flexibility allows the glycan head to move during the interaction between the initiator block and the target, where theoretical constraints are not desired, thereby promoting bonding therebetween.

[0039] Preferred spacer groups are biocompatible. In some embodiments, the initiator spacer group comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof. For example, the spacer group may be a polyethylene glycol (PEG) moiety formed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 30, 36, 40, 48, 50, 55, 60, 65 or 72 (OCH2CH2) subunits, or any range defined by the aforementioned endpoints, such as 2 to 72, 2 to 60, 2 to 48, 2 to 36, 2 to 24, 2 to 18, 2 to 15, 2 to 10, 4 to 72, 4 to 60, 4 to 48, 4 to 36, 4 to 24, 4 to 18, 4 to 15, 4 to 10, 8 to 72, 8 to 60, 8 to 48, 8 to 36, 8 to 24, 8 to 18, 8 to 15 or 8 to 10 (OCH2CH2) subunits. In some embodiments, the PEG portion may be a linear, branched, or star-shaped structure.

[0040] In some embodiments, the spacer group is a saturated carbon portion, which may be a lipid tail attached to the glycan head. In some embodiments, the saturated carbohydrate contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or any range of carbons defined by the aforementioned endpoints, such as 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, or 6 to 8 carbons.

[0041] Binding affinity. In some embodiments, the binding affinity between the initiator block's glycan head and the target can be determined by the dissociation constant (K). D (Definition). In some embodiments, K at pH 7.4 D The values ​​can be 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3250, 3500, 3750, 4000, or 4250. 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750 or 8000 nM, or any range defined by the aforementioned endpoints, such as 5 to 8000, 5 to 7000, 5 to 6000, 5 to 5000, 5 to 4000, 5 to 3000, 5 to 2500, 5 to 2000, 5 to 1500, 5 to 1250, 5 to 1000, 5 to 900, 5 to 800, 5 to 700, 5 to 600, 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 150, 5 to 100, 5 to 75, 5 to 50, 5 to 30, 5 to 20, 10 to 8000, 10 to 7000, 10 to 6000, 10 to 5000, 10 to 4000 10 to 3000, 10 to 2500, 10 to 2000, 10 to 1500, 10 to 1250, 10 to 1000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 150, 10 to 100, 10 to 75, 10 to 50, 10 to 30 or 10 to 20 nM.

[0042] In some other embodiments, K at pH 5 D It can be 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 1750, or 2000 nM, or any range defined by the aforementioned endpoints, such as 1 to 2000, 1 to 1500, 1 to 1000, 1 to 900, 1 to 800, 1 to 750, 1 to 700, 1 to 650, 1 to 600, 1 to 550, 1 to 500, 1 to 450. 1 to 400, 1 to 350, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 75, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 or to 5, 5 to 2000, 5 to 1500, 5 to 1000, 5 to 900, 5 to 800, 5 to 750, 5 to 700, 5 to 650, 5 to 600, 5 to 550, 5 to 500, 5 to 450, 5 to 400, 5 to 350, 5 to 300, 5 to 250, 5 to 200, 5 to 150, 5 to 100, 5 to 75, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10 nM.

[0043] Example. In some embodiments, the initiator block of the glycan head contains 9 BPC Neu5Ac-conjugated N-glycan heads (e.g., IB2), Neu5Ac-conjugated N-glycans (e.g., IB3), 9T CCN eu5Ac-conjugated N-glycans (e.g., IB4) or combinations thereof. In some embodiments, the glycan head comprises at least one of the following structures (note that "IB" stands for initiator block, which describes an initiator conjugated in the copolymer of this disclosure. However, for the sake of brevity, "IB" may be used interchangeably with "I" to represent an initiator):

[0044]

[0045] Solid circles represent mannosides, hollow circles represent galactose, solid squares represent GlcNAc, and rhombuses represent Neu5Ac.

[0046] Growth agent block

[0047] In some embodiments, the exemplary polymer vesicles of this disclosure exhibit desired properties, such as effective payload encapsulation, reduced serum protein adsorption, enhanced membrane fusion, and effective payload release after uptake. The growth agent blocks of this disclosure provide at least one desired property. To this end, the growth agent blocks of this disclosure include a functional portion comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof.

[0048] In some embodiments, the copolymers of this disclosure comprise a single grower block that provides at least one desired property. In some embodiments, the copolymers of this disclosure comprise a plurality of grower blocks, each providing at least one desired property.

[0049] Effective payload encapsulation. In some embodiments, the polymer vesicles of this disclosure are designed to carry nucleic acid-type payloads, such as mRNA or DNA molecules. In such embodiments, the growth block of the copolymer of the polymer vesicle may contain guanidine groups. In some embodiments, the growth block contains 1, 2, 3, 4, 5, or more guanidine groups. Where it is not desirable to be bound by theory, multiple guanidine groups in the growth block provide a stronger salt bridge between the guanidine salt group of the copolymer and the phosphate ester group of the nucleic acid molecule (e.g., mRNA). In some embodiments, the growth block contains three guanidine groups.

[0050] Reduced serum protein adsorption and enhanced membrane fusion. As mentioned above, one of the common drawbacks of polymeric vesicles is their instability in serum due to serum protein adsorption. To reduce serum protein adsorption and enhance serum stability, the copolymers of this disclosure may contain zwitterions, which also enhance the fusion of the polymeric vesicle membrane with target cells. The zwitterions of this disclosure may be molecules / parts having an overall zero charge by carrying the same number of positively and negatively charged functional groups at pH 4.5 to 7.5. In some embodiments, the zwitterions of this disclosure have an isoelectric point between pH 4.5 and 7.5. The zwitterions may be (but are not limited to) phosphocholine (CP), sulfothetin, phosphonium sulfonate, or psilocybin. In some embodiments, the zwitterions of this disclosure contain an alkyl phosphate betaine group comprising a phosphate ester group and an amine group, respectively providing a negative charge and a positive charge.

[0051] Effective payload release after uptake. Another desirable characteristic of polymeric vesicles is the ability to effectively release the encapsulated / encapsulated payload after uptake. Release of the encapsulated / encapsulated payload occurs in the lysosomes of the target cell, resulting from escape from the endosome / lysosomal pathway or degradation of the polymeric vesicle. In some embodiments, the zwitterionic extensional block can facilitate endosome escape. However, in some embodiments, the extensional block may further comprise an alkyl chain, which also contributes to endosome escape.

[0052] On the other hand, in some embodiments, to promote lysosomal degradation, the growth agent block of this disclosure may include a diethylenetriamine moiety. The terminal amine residue of the diethylenetriamine moiety may also be used for additional functionalization. Alternatively, the growth agent block of this disclosure may include ethylenediamine, 1-(2-aminoethyl)piperazine, and / or tris(2-aminoethyl)amine.

[0053] Growth agent spacer group. In some embodiments, the growth agent block of this disclosure includes a growth agent spacer group that is primarily configured to provide the copolymer with the hydrophobicity required for assembly into polymer vesicles. In some embodiments, the growth agent spacer group includes a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0054] In some embodiments, the spacer group may be a polyethylene glycol (PEG) moiety formed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 30, 36, 40, 48, 50, 55, 60, 65, or 72 (OCH2CH2) subunits, or any range defined by the aforementioned endpoints, such as 2 to 72, 2 to 60, 2 to 48, 2 to 36, 2 to 24, 2 to 18, 2 to 15, 2 to 10, 4 to 72, 4 to 60, 4 to 48, 4 to 36, 4 to 24, 4 to 18, 4 to 15, 4 to 10, 8 to 72, 8 to 60, 8 to 48, 8 to 36, 8 to 24, 8 to 18, 8 to 15, or 8 to 10 (OCH2CH2) subunits. In some embodiments, the PEG moiety may be linear, branched, or star-shaped.

[0055] In some embodiments, the saturated carbon portion may be a lipid tail extending from the growth agent block. In some embodiments, the saturated carbohydrate comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or any range of carbons defined by the aforementioned endpoints, such as 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, or 6 to 8 carbons. In some embodiments, the grower spacer group comprises a monocarboxylic acid amide moiety (e.g., thioctinamide moiety) or other biocompatible structure that provides a saturated carbohydrate and functional groups for conjugation.

[0056] Example. In some embodiments, the grower block of this disclosure comprises at least one of the following structures (note that "PB" represents a grower block, which describes a grower conjugated in the copolymer of this disclosure. However, for the sake of brevity, "PB" may be used interchangeably with "P," which represents a grower):

[0057]

[0058] Copolymer extender blocks. In some embodiments, the copolymer comprises a plurality of extender blocks and a plurality of links, wherein each extender block is connected to at least one other extender block or initiator block via one of the links. Where it is not desired to be bound by theory, this disclosure contemplates the advantage of having two or more extenders in the structure of the copolymer. Each of the two or more extenders provides at least one of the following desired properties: effective load encapsulation, reduced serum protein adsorption, enhanced membrane fusion, and effective load release after uptake. In some embodiments, each of the two or more extenders differs in structure and / or the desired properties it provides, thereby forming a copolymer in the form of a heteropolymer.

[0059] In some embodiments, the heteropolymer comprises any two or more growth agent blocks PB1, PB2, PB3, PB4, and PB5. In some embodiments, the copolymer comprises at least two growth agent blocks, said at least two growth agent blocks being (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, (4) PB2 and PB5, or (5) PB1, PB4, and PB5, wherein the two or more growth agents in the copolymer can be represented by formula numbers of the two or more growth agents separated by the symbol " / ". For example, the two growth agent blocks PB1 and PB5 in the copolymer can be represented as PB1 / PB5. However, the order and amount of growth agent blocks in the copolymers mentioned are not limited by their naming convention. Without wishing to be bound by theory, this disclosure finds that copolymers of PB1 / PB4, PB2 / PB4, PB1 / PB5, and PB2 / PB5 exhibit superior intracellular delivery due to efficient membrane fusion and effective load release.

[0060] In some embodiments, heteropolymers having two or more different types of extender blocks can be configured with any of the initiators I2, I3, I4, I5, I6, I7, I8, I9, and I10. The combination of initiator and extender can be indicated by formula numbers of the initiator and extender separated by a hyphen “-”. For example, a copolymer containing initiator I5 and extender P5 can be represented as I5-P5. However, the order and amount of initiator blocks and / or extender blocks in the copolymers mentioned are not limited by their naming convention. In some embodiments, the copolymer of a heteropolymer having two or more different types of growers conjugated with an initiator of this disclosure may be selected from the group consisting of: I5-P1 / P5, I5-P1 / P4, I5-P2 / P5, I5-P2 / P4, I5-P1 / P4 / P5, I6-P1 / P5, I6-P1 / P4, I6-P2 / P5, I6-P2 / P4, I6-P1 / P4 / P5, I7-P1 / P5, I7-P1 / P4, I7- P2 / P5, I7-P2 / P4, I7-P1 / P4 / P5, I8-P1 / P5, I8-P1 / P4, I8-P2 / P5, I8-P2 / P4, I8-P1 / P4 / P5, I9-P1 / P5, I9-P 1 / P4, I9-P2 / P5, I9-P2 / P4, I9-P1 / P4 / P5, I10-P1 / P5, I10-P1 / P4, I10-P2 / P5, I10-P2 / P4 and I10-P1 / P4 / P5.

[0061] In some embodiments, the heteropolymer comprises a plurality of first growth agent blocks and a plurality of second growth agent blocks. In some embodiments, the number of first growth agent blocks is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, or any range defined by the aforementioned endpoints, such as 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 4, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 9 to 20, 9 to 18, 9 to 16, 9 to 14, 9 to 12, 12 to 20, 12 to 18, 12 to 16. In some embodiments, the number of second growth agent blocks is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, or any range defined by the aforementioned endpoints, such as 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 4, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 9 to 20, 9 to 18, 9 to 16, 9 to 14, 9 to 12, 12 to 20, 12 to 18, 12 to 16. In some embodiments, mass spectrometry analysis may be used to determine the number of the first growth agent block and / or the number of the second growth agent block, but is not limited thereto.

[0062] Polymer vesicles comprising the copolymers disclosed herein

[0063] The polymer vesicles of this disclosure comprise membranes containing copolymers of this disclosure. In some embodiments, the membrane comprises a plurality of copolymers of this disclosure assembled into a membrane, wherein the promoter blocks are coupled to each other via hydrophobic interactions. The initiator block of each copolymer of the polymer vesicle extends from the membrane and is exposed to the surrounding environment.

[0064] In some embodiments, the copolymer of this disclosure constitutes at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the polymer vesicle membrane, or any range defined by the aforementioned endpoints, such as 50% to 99%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 60% to 99%, 60% to 99%. 0% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 60% to 70%, 60% to 65%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 50% to 75%, 80% to 99%, 80% to 95%, 80% to 90%, 80% to 85%, 90% to 99%, or 90% to 95%.

[0065] In some embodiments, the membrane comprises at least two different types of copolymers of the present disclosure. In some embodiments, the membrane comprises a copolymer comprising at least two different types of growth agent blocks of the present disclosure. For example, the membrane comprises a copolymer comprising a first growth agent block and a second growth agent block, wherein the molecular ratio of the first growth agent block and the second growth agent block in the membrane is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 15, 20, or 25, or any range defined by the aforementioned endpoints, such as 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2.5, 1 to 2, 1 to 1.5, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2 to 2.5.

[0066] In some embodiments, the polymer vesicles disclosed herein may be dendritic cell-targeting vaccines (DCTVs) used to specifically target and deliver a payload to dendritic cells, thereby achieving and / or improving the immunogenicity of the vaccine.

[0067] Size of the polymer vesicles. In some embodiments, the diameter of the polymer vesicles disclosed herein is 0.001, 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 micrometers, or any range defined by the aforementioned endpoints, such as 0.001 to 5, 0.001 to 4, 0.001 to 3, 0.001 to 2, 0.001 to 1, 0.001 to 0.8, 0.001 to 0.6, 0.001 to 0.4, 0.001 to 0.2, 0.001 to 0.1, 0.001 to 0. 0.05, 0.001 to 0.01, 0.001 to 0.005, 0.05 to 5, 0.05 to 4, 0.05 to 3, 0.05 to 2, 0.05 to 1, 0.05 to 0.8, 0.05 to 0.6, 0.05 to 0.4, 0.05 to 0.2, 0.05 to 0.1, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 0.5 to 0.8, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 micrometers. The size of the polymer vesicles can be determined using (but not limited to) dynamic light scattering (DLS). In some embodiments, the polydispersity index (PDI) of the polymer vesicles disclosed herein is about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1, or any range defined by the foregoing endpoints, such as 0.01 to 1, 0.01 to 0.9, 0.01 to 0.8, or 0. 01 to 0.7, 0.01 to 0.6, 0.01 to 0.5, 0.01 to 0.4, 0.01 to 0.3, 0.01 to 0.2, 0.01 to 0.1, 0.01 to 0.05, 0.1 to 1, 0.1 to 0.9, 0.1 to 0.8, 0.1 to 0.7, 0.1 to 0.6, 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3 or 0.1 to 0.2.

[0068] Zeta potential and molecular weight. Without being bound by theory, the zeta potential and molecular weight of polymeric vesicles may influence cellular uptake. In some embodiments, the polymeric vesicles of this disclosure comprise about -50, -40, -30, -20, -15, -10, -5, 0, +5, +10, +15, +20, +30, +40, or +50, or any range defined by the aforementioned endpoints, such as -50 to +50, -50 to +40, -50 to +30, -50 to +20, -50 to +15, -50 to +10, -50 to +5, -50 to -5, -50 to -10, -50 to -15, -50 to -20, -20 to +50. ζ potentials of -20 to +40, -20 to +30, -20 to +20, -20 to +15, -20 to +10, -20 to +5, -20 to -5, -20 to -10, -20 to -15, -15 to +50, -15 to +40, -15 to +30, -15 to +20, -15 to +15, -15 to +10, -15 to +5, -15 to -5, -15 to -10, +5 to +50, +5 to +40, +5 to +30, +5 to +20, +5 to +15, or +5 to +10. In some embodiments, the polymer vesicles of this disclosure comprise about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50 kDa, or any range defined by the aforementioned endpoints, such as 1 to 50 kDa, 1 to 40 kDa, 1 to 30 kDa, 1 to 20 kDa, 1 to 15 kDa, 1 to 10 kDa, 1 to 5 kDa, 2 to 50 kDa, 2 to 40 kDa, 2 to 30 kDa, 2 to 20 kDa, 2 to 15 kDa, 2 to 10 kDa, 2 to 5 kDa. Molecular weights of 5 to 50 kDa, 5 to 40 kDa, 5 to 30 kDa, 5 to 20 kDa, 5 to 15 kDa, 5 to 10 kDa, 8 to 50 kDa, 8 to 45 kDa, 8 to 40 kDa, 8 to 35 kDa, 8 to 30 kDa, 8 to 25 kDa, 8 to 20 kDa, 8 to 15 kDa, 8 to 10 kDa, 12 to 50 kDa, 12 to 45 kDa, 12 to 35 kDa, 12 to 25 kDa, 12 to 15 kDa, 25 to 50 kDa, 25 to 40 kDa, or 25 to 30 kDa.

[0069] Payload. In some embodiments, the membrane of the polymer vesicle defines an internal space configured to encapsulate or carry a payload. As described herein, "encapsulating a payload," "encapsulated within an internal space," or similar descriptions refer to a situation where the payload is held, closed, or surrounded by the membrane of the polymer vesicle. The payload may move freely within the internal space or be covalently or non-covalently attached to the membrane. Encapsulation may be substantial, complete, or partial, and does not preclude the possibility that a portion of the payload may be exposed to the external environment of the polymer vesicle. In partially encapsulated embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the payload is held, closed, or surrounded by the membrane of the polymer vesicle. In some embodiments, the payload may be a biomolecule, such as a nucleic acid, compound, peptide, protein, glycan head, or a combination thereof.

[0070] In some embodiments, the payload is ribonucleic acid (RNA, such as mRNA) or deoxyribonucleic acid (DNA, such as double-stranded or single-stranded DNA), which can encode polypeptides or proteins in vivo after being delivered to target cells using the polymeric vesicles of this disclosure. Nucleic acids, such as the mRNA molecules used in this disclosure, can be prepared from a reference nucleic acid by in vitro transcription. In vitro transcription can be performed as described in PCT patent publication WO2014 / 152027, filed March 13, 2014, which is incorporated herein by reference in its entirety.

[0071] In some embodiments, the polypeptide or protein is immunogenic (e.g., antigenic) to the organism to which the polymer vesicles are administered. In such embodiments, the polymer vesicles of this disclosure are used to encapsulate and carry immunogenic proteins or nucleic acids, such as mRNA molecules in RNA vaccines, configured to encode immunogenic proteins in vivo. Immunogenic proteins may be proteins of pathogens derived from viruses (e.g., SARS-CoV-2, influenza, respiratory syncytial virus (RSV), EBV, dengue fever (DENGUE), VZV, HIV, ZIKA, or NIPAH), bacteria, or fungi. In some embodiments, the immunogenic protein may be a viral spike protein. In some embodiments, the spike protein may be of coronavirus (CoV) origin (e.g., SARS-CoV, MERS-CoV, and SARS-CoV-2). In some embodiments, examples of coronaviruses (CoV) described herein include (but are not limited to) α-SARS-CoV2, β-SARS-CoV2, γ-SARS-CoV2, δ-SARS-CoV2, o-SARS-CoV2, and variants thereof.

[0072] In some embodiments, the payload is a nucleic acid, which may be a polynucleotide having an open reading frame configured to encode a polypeptide or protein in vivo. Such polynucleotides may be 5' capped, the 5' cap being generated during in vitro transcription using the following chemical RNA cap analogues: 3”-O-Me-m7G(5)ppp(5')G[ARCA cap], G(5)ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, or m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Post-transcriptional 5'-capping of the modified polynucleotide can be performed using a vaccinia virus capping enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 0 can be generated using a vaccinia virus capping enzyme and a 2'-O methyltransferase. The Cap 1 structure is used to generate m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 structure can be generated from the Cap 1 structure, followed by 2'-O-methylation of the 5'-last nucleotide using a 2'-O-methyltransferase. The Cap 3 structure can be generated from the Cap 2 structure, followed by 2'-O-methylation of the 5'-last nucleotide using a 2'-O-methyltransferase. The enzyme can be derived from recombinant sources. After transfection into mammalian cells, the modified polynucleotide exhibits stability for 12 to 18 hours, or greater than 18 hours, such as 24, 36, 48, 60, 72, or greater than 72 hours.

[0073] In some embodiments, nucleic acids may be modified. In some embodiments, nucleic acids may have several (more than one) modifications that are the same as or different from each other. In some embodiments, nucleic acids contain one, two or more (optionally, different) nucleoside or nucleotide modifications in a specific region. In some embodiments, modified nucleic acids (e.g., modified mRNA polynucleotides) exhibit reduced degradation in cells or organisms relative to unmodified nucleic acids. In some embodiments, modified nucleic acids may exhibit reduced immunogenicity (e.g., reduced innate response) in organisms.

[0074] In some embodiments, the modification may include chemical modifications. In some embodiments, the modification may be naturally occurring, non-natural, or both. Some exemplary modifications applicable to this disclosure include, but are not limited to, modifications of sugars, nucleobases, nucleoside linkages (e.g., linkages to phosphate esters, phosphodiester linkages, or phosphodiester backbones), or combinations thereof. In some embodiments, the nucleic acid (e.g., RNA) used as the payload of this disclosure may be codon-optimized. For example, the nucleic acid may be modified to enhance its G / C content. The G / C content of a nucleic acid can affect its stability. Nucleic acids with increased amounts of guanine (G) and / or cytosine (C) residues may be functionally more stable than nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. For example, WO2002 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modifications in the translation region. Due to the degeneracy of the genetic code, the modification works by replacing existing codons with those that contribute to higher RNA stability without altering the resulting amino acids.

[0075] In some embodiments, the nucleic acid may further comprise a sequence encoding a signal peptide. The signal peptide may comprise three regions: (1) an N-terminal region of varying lengths, which typically contains positively charged amino acids, (2) a hydrophobic region, and (3) a short C-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across the membrane. The signal peptide is not typically responsible for the final destination of the mature protein, but is not limited thereto in this disclosure. The signal peptide is typically cleaved from the precursor protein by a resident ER signal peptidase. It may remain unclewd and act as a membrane anchor. In some embodiments, the signal peptide may be programmed to fuse with a polypeptide or protein encoded by the payload at its C-terminus or N-terminus.

[0076] In some embodiments, the payload may be a therapeutic or preventive agent for treating or preventing diseases such as cancer or infectious diseases. For example, the payload may be an antiviral agent, including (but not limited to) ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, remdesivir (GS-5734) and favipiravir (T-705), interferon, adefovir, tenofovir, acyclovir, brivudin, cidofovir, fomivirsen, foscarnet, ganciclovir, amantadine, rimantadine, zanamivir, remdesivir, molnupiravir, and nematvir / ritonavir. In other instances, the payload may be an anticancer agent. In some embodiments, the payload is a nucleic acid configured to encode a therapeutic or preventative agent.

[0077] In some embodiments, the N / P ratio (positively charged amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups) of the polymer vesicles encapsulating nucleic acids is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40. 45 or 50, or any range defined by the aforementioned endpoints (including or excluding the endpoints), such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 8 to 40, 8 to 20, 8 to 12, 9 to 50, 9 to 30, or 9 to 15. In another embodiment, the nanoparticle / mRNA (N / P) ratio of the polymeric vesicles encapsulating mRNA is about 10 or about 20.

[0078] In some embodiments where the effective load of the polymer vesicles is nucleic acid configured to encode a peptide or protein in a target cell, after being taken up by the target cell, the polymer vesicles are configured to encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 copies of the peptide or protein in vivo, or any range defined by the aforementioned endpoints (including or excluding the endpoints), such as 1 to 50, 1 to 40, 1 to 5 ... 30, 1 to 20, 1 to 10, 1 to 5, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 5 to 8, 4 to 50, 4 to 45, 4 to 35, 4 to 25, 4 to 15, 4 to 9, 4 to 6, 7 to 50, 7 to 45, 7 to 35, 7 to 25, 7 to 15, or 7 to 9 copies. In some embodiments, after being taken up by target cells, the polymeric vesicles are configured to continuously and in real-time encode peptides or proteins in vivo until the nucleic acid (i.e., the payload) is inactivated in vivo.

[0079] Composition / Formulation

[0080] One aspect of this disclosure relates to a composition (i.e., a formulation) comprising the polymeric vesicles of this disclosure. The polymeric vesicles of the composition may encapsulate a payload and are configured to deliver the payload to a target region of a organism. The payload may, as described herein, comprise nucleic acids, compounds, peptides, proteins, glycan heads, or combinations thereof. In some embodiments, the payload may be an immunogenic protein or a nucleic acid configured to encode an immunogenic protein in vivo. In some embodiments, the formulation further comprises pharmaceutically acceptable excipients, adjuvants, or combinations thereof. In some embodiments, the composition is a pharmaceutical composition or a pharmaceutical formulation.

[0081] In some embodiments, the composition comprises 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95% (w / w) of encapsulated or unencapsulated polymeric vesicles of the present disclosure, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints) 0.01% to 95% (w / w), 0.01% to 90% (w / w), 0.01% to 80% (w / w), 0.01% to 70% (w / w), etc. w / w), 0.01% to 60% (w / w), 0.01% to 50% (w / w), 0.01% to 40% (w / w), 0.01% to 30% (w / w), 0.01% to 20% (w / w), 0.01% to 10% (w / w), 0.01% to 5% (w / w), 0.01% to 1% (w / w), 0.01% to 0.1% (w / w), 0.1% to 95% (w / w), 0.1% to 90% (w / w), 0.1% to 80% (w / w), 0.1% Up to 70% (w / w), 0.1% to 60% (w / w), 0.1% to 50% (w / w), 0.1% to 40% (w / w), 0.1% to 30% (w / w), 0.1% to 20% (w / w), 0.1% to 10% (w / w), 0.1% to 5% (w / w), 0.1% to 1% (w / w), 1% to 95% (w / w), 1% to 90% (w / w), 1% to 80% (w / w), 1% to 70% (w / w), 1% to 60% (w / w), 1% to 50% (w / w) % (w / w), 1% to 40% (w / w), 1% to 30% (w / w), 1% to 20% (w / w), 1% to 10% (w / w), 1% to 5% (w / w), 5% to 95% (w / w), 5% to 90% (w / w), 5% to 80% (w / w), 5% to 70% (w / w), 5% to 60% (w / w), 5% to 50% (w / w), 5% to 40% (w / w), 5% to 30% (w / w), 5% to 20% (w / w), or 5% to 10% (w / w). The remaining percentage of the composition may be excipients as described herein.

[0082] In some embodiments, the composition is an mRNA vaccine, wherein polymeric vesicle encapsulation is configured to encode mRNA encoding an immunogenic protein in vivo. The immunogenic protein may be a viral spike protein or other antigenic molecules of a pathogen. In some embodiments, the compositions of the present invention may be a COVID-19 mRNA vaccine.

[0083] The exemplary COVID-19 mRNA vaccines described herein can be designed based on mRNA technology to remove the glycan shielding of coronavirus (e.g., SARS-CoV-2) spike proteins, thereby better exposing the conserved regions of the spike protein. Compared to unmodified mRNA, mRNA vaccines for coronavirus spike proteins have the deletion of glycosylation sites in the receptor-binding domain (RBD) or subunit 2 (S2) domain to expose highly conserved antigenic determinants and elicit antibody and CD8 T cell responses, providing broader protection against α, β, γ, δ, O, and various variants. The vaccine may be a low sugar universal vaccine (LSUV) as described in WO2022 / 221835 (wherein the mRNA contains sequences selected from SEQ ID NO 1-52), WO2022 / 221837A2 (wherein the mRNA contains sequences selected from SEQ ID NO 1-21), and US20200046826A1 (wherein the mRNA contains sequences selected from SEQ ID NO 1-20), which are incorporated herein by reference in their entirety.

[0084] In some embodiments, the compositions of the present invention are configured for treating or preventing diseases (e.g., cancer). In such embodiments, the payload carried by the polymer vesicles may be a therapeutic agent, a preventative agent, or a nucleic acid configured to encode a therapeutic or preventative agent in vivo. For example, the composition may be a personalized cancer vaccine (e.g., melanoma), a KRAS vaccine (KRAS-driven), or a checkpoint vaccine (e.g., PD-1, PDL-1 related).

[0085] In some embodiments, the compositions of the present invention may be administered together with another composition (e.g., a vaccine or a drug). Examples of the other composition may be (but are not limited to) influenza (flu) vaccines, adenovirus vaccines, anthrax vaccines, cholera vaccines, diphtheria vaccines, hepatitis A or B vaccines, HPV vaccines, measles vaccines, mumps vaccines, smallpox vaccines, rotavirus vaccines, tuberculosis vaccines, pneumococcal vaccines, and Haemophilus influenzae type b vaccines.

[0086] Combination composition

[0087] In some embodiments, the composition may be a combination composition (e.g., a combination vaccine) comprising a first polymeric vesicle encapsulating a first payload and a second polymeric vesicle encapsulating a second payload. The first and second polymeric vesicles may be in the form of polymeric vesicles described herein, but differ from each other in terms of the structure or properties of their copolymers. For example, the first and second polymeric vesicles may differ in size, the copolymer in which they form a film, the payload encapsulated within the polymeric vesicles, or combinations thereof.

[0088] For example, the first polymer vesicle contains a glycan head configured to bind DC-SIGN, while the second polymer vesicle contains a glycan head configured to bind Siglec-1. In another example, the first polymer vesicle contains a glycan configured to target antigen-presenting cells, while the second polymer vesicle contains a glycan configured to target cancer cells.

[0089] In some embodiments, the first payload and the second payload are different from each other. For example, the first payload may be a protein or peptide, while the second payload may be a nucleic acid. In some embodiments, both the first and second payloads may be mRNA molecules, but encoding different proteins. For example, the first payload may be mRNA configured to encode the spike protein of δ-SARS-CoV2, while the second payload may be mRNA configured to encode the spike protein of o-SARS-CoV2.

[0090] Additional components of the composition

[0091] In some embodiments, the compositions disclosed herein may further comprise adjuvants and / or inactive substances, such as pharmaceutically acceptable excipients. In some embodiments, the adjuvant may be (but is not limited to) C34, glucose-C34, 7DW8-5, C17, C23, C30, α-galactosidylceramide (α-GalCer), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts), squalene, MF59, QS-21, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), CpG1018 (Dynavax), or combinations thereof.

[0092] In some embodiments, pharmaceutically acceptable excipients may comprise solvents, dispersion media, diluents, dispersions, suspending agents, surfactants, isotonics, thickeners or emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, or mixtures thereof. Various excipients used to formulate pharmaceutical compositions and techniques used to prepare compositions are known in the art (see *Remington: The Science and Practice of Pharmacy*, 22nd edition, edited by Allen, Loyd V., *Journal of Pharmaceutical Press*). The use of conventional excipient media is within the scope of this disclosure unless any conventional excipient medium may be incompatible with the substance or its derivatives, for example, producing any undesirable biological effects or otherwise interacting in a harmful manner with any other component of the pharmaceutical composition. The formulation of pharmaceutically acceptable excipients can be performed using conventional methods in pharmaceutical technology (see Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Eastern Pennsylvania, USA).

[0093] In some embodiments, the composition further comprises a phosphate conjugate. Where it is not desired to be bound by theory, the phosphate conjugate may increase in vivo circulation time and / or increase targeted delivery of the polymer vesicles of this disclosure. The phosphate conjugate for use in this disclosure may be prepared using the methods described in PCT Publication WO2013 / 033438, filed August 30, 2012, or U.S. Publication US2013 / 0196948, filed June 23, 2011, the contents of each of which are incorporated herein by reference in their entirety. As a non-limiting example, the phosphate conjugate may comprise compounds of any of the formulas described in PCT Publication WO2013 / 033438, filed August 30, 2012, which is incorporated herein by reference in its entirety.

[0094] In some embodiments, the composition further comprises a conjugate to enhance the delivery of the polymeric vesicles of this disclosure. Where it is not desired to be bound by theory, the conjugate chosen for use may inhibit phagocytic clearance of the polymeric vesicles in an individual. In some instances, the conjugate may be the human membrane protein CD47 or a “self” peptide derived therefrom (e.g., the “self” particle described by Rodriguez et al. (Science 2013, 339, 971-975, which is incorporated herein by reference in its entirety).

[0095] In some embodiments where the payload is an immunogenic agent or a nucleic acid configured to encode an immunogenic agent, the composition further comprises an immunostimulant to enhance the immune response induced by the immunogenic agent. As a non-limiting example, the composition may comprise a Th1 immunostimulant that enhances a Th1-based response of the immune system (see PCT Publication WO2010 / 123569 and U.S. Publication 2011 / 0223201, each of which is incorporated herein by reference in its entirety).

[0096] In some embodiments, the composition does not include viral components (e.g., viral capsid, viral enzymes, or other viral proteins, such as those required for viral replication), nor is the composition encapsulated within, encapsulated in, linked to, or otherwise bound to a virus or viral particle.

[0097] Kit for preparing polymer vesicles

[0098] One aspect of this disclosure relates to a kit for preparing the polymer vesicles of this disclosure. The kit comprises a first reagent and a second reagent, wherein the first reagent comprises an initiator having a glycan head and an initiator linker portion, and the second reagent comprises an elongator having a functional portion and an elongator linker portion, wherein the functional portion comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the initiator linker portion is configured to be coupled to the elongator linker portion via a disulfide bond.

[0099] In some embodiments, the initiator linking portion is configured to couple with the grower linking portion, thereby forming a bond in the copolymer of this disclosure. In some embodiments, the initiator linking portion and the grower linking portion are independently thiol groups or dithioheterocyclic pentyl groups.

[0100] In some embodiments, the kit further comprises reagents containing a payload to be encapsulated by polymer vesicles prepared using the kit of this disclosure. In some embodiments, the payload may be as described herein.

[0101] Initiator

[0102] The initiator comprises a glycan head and an initiator linker portion. In some embodiments, the glycan head is as described above in the initiator block of the copolymer of this disclosure.

[0103] In some embodiments, the initiator molecule further comprises an initiator spacer group as described herein in the copolymers disclosed. In some embodiments, the initiator spacer group comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0104] In some embodiments, the initiator is selected from the group consisting of:

[0105]

[0106]

[0107] Growth agent

[0108] The grower comprises a functional portion and a grower linking portion, wherein the functional portion comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof. In some embodiments, the functional portion of the grower molecule is as described above in the grower blocks of copolymers disclosed herein.

[0109] In some embodiments, the grower further comprises a grower spacer group as described herein in the copolymers disclosed herein. In some embodiments, the grower spacer group comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0110] In some embodiments, the second reagent comprises two types of growth agents, each different from the other in structure or in providing the desired properties. For example, the second reagent may comprise a first growth agent and a second growth agent, each independently comprising a functional portion comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof. In some embodiments, the first growth agent comprises a guanidine group, and the second growth agent comprises a zwitterionic group. In other embodiments, the first growth agent comprises a guanidine group, and the second growth agent comprises a diethylenetriamine.

[0111] In some other embodiments, the kit further comprises a third reagent, which contains a growth agent that differs from the growth agent of the second reagent in structure or the desired properties it provides. For example, the growth agent of the second reagent is a first growth agent molecule, and the third reagent contains a second growth agent. The first and second growth agents may independently contain functional portions comprising guanidine, zwitterionic groups, diethylenetriamine, or combinations thereof. In some embodiments, the first growth agent contains a guanidine, and the second growth agent contains a zwitterionic group. In other embodiments, the first growth agent contains a guanidine, and the second growth agent contains diethylenetriamine.

[0112] In some embodiments, the growth agent is selected from the group consisting of:

[0113]

[0114] Packaging

[0115] All components of the kit disclosed herein can be individually packaged in physical containers. In some embodiments, the first and second reagents are contained in the same container; in other words, they are in ready-to-use packaging. In some other embodiments, the first and second reagents are contained in separate containers, so the user can decide whether and when to mix the first and second reagents.

[0116] How to use

[0117] One aspect of this disclosure relates to methods using the polymer vesicles of this disclosure. Specifically, the methods are performed to achieve a desired effect, such as targeted delivery of a payload, prevention or treatment of disease, or enhancement of an adaptive immune response in an individual. In some embodiments, the individual may be (but is not limited to) an animal or human for which the payload is designed to demonstrate its efficacy, an animal or human requiring treatment or prevention of disease, or an animal or human requiring enhancement of their adaptive immune response.

[0118] Methods for targeted delivery of payloads to individuals

[0119] In some embodiments, a method for targeted delivery of a payload to an individual is provided, comprising administering an effective amount of the polymer vesicles of this disclosure to the individual. In some embodiments, a method for targeted delivery of a payload to an individual is provided, comprising administering an effective amount of the pharmaceutical formulation of this disclosure to the individual. The polymer vesicles and the payload are as described herein, and the polymer vesicles encapsulate the payload within an internal space defined by a membrane of the polymer vesicle.

[0120] Without being bound by any theory, targeted delivery is achieved through initiator blocks of copolymers that form polymer vesicles. Specifically, the initiator blocks provide the desired binding affinity / specificity to the desired region of the target individual via their glycan heads.

[0121] Methods for preventing or treating diseases in an individual

[0122] In some embodiments, a method for preventing or treating a disease in an individual is provided, comprising administering an effective amount of the polymer vesicles of this disclosure to the individual. The polymer vesicles of this method encapsulate a payload within an internal space defined by a membrane of the polymer vesicle, and the payload is a therapeutic agent or a derivative thereof configured for the prevention and / or treatment of a disease.

[0123] Without being bound by any theoretical constraints, the polymer vesicles of this disclosure provide targeted delivery via a glycan head of its copolymer. Therefore, by using the polymer vesicles of this disclosure to deliver the payload, the efficacy of the payload can be more effectively utilized. For example, in embodiments where the glycan head includes a structure that specifically binds to DC-SIGN on dendritic cells, antigenic or immunogenic payloads can be effectively delivered to dendritic cells to elicit an immune response, thereby preventing the disease of interest. This strategy is beneficial for delivering antigens or nucleic acids encoding antigens in vaccines. Some other examples include glycan heads designed to target cancer cells, enabling the effective delivery of antitumor agents to the cancer microenvironment. This strategy can increase the efficacy of antitumor agents and reduce the side effects of treatment.

[0124] In some embodiments, the disease is characterized by a dysfunction or abnormality in the activity of proteins or peptides. For example, diseases are selected from the group consisting of: rare diseases, infectious diseases, cancers and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0125] In some embodiments, the disease may be cancer or an infectious disease. In some embodiments, the disease may be a virus-related infection, including (but not limited to) human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or SARS-CoV-2 (COVID-19) related infection.

[0126] Methods to enhance adaptive immune response

[0127] In some embodiments, a method for enhancing an adaptive immune response is provided, comprising administering an effective amount of the polymer vesicles of this disclosure to an individual. The polymer vesicles of this method encapsulate a payload within an internal space defined by a membrane of the polymer vesicle, and the payload is a therapeutic agent or a derivative of a therapeutic agent configured to elicit an adaptive immune response in an individual.

[0128] Without wishing to be bound by any theory, the polymer vesicles of this disclosure provide targeted delivery to immune cells via a glycan head of its copolymer. In some embodiments, the glycan head includes a structure that binds to antigen-presenting cells with desired specificity or affinity. For example, the glycan head may include a structure that specifically binds to DC-SIGN on dendritic cells, enabling the polymer vesicles to specifically deliver an immunogenic payload to dendritic cells to promote an adaptive immune response.

[0129] In some embodiments, the enhanced adaptive immune response is directed against a disease, including (but not limited to) cancer or an infectious disease. For example, an infectious disease may be a virus-associated infection, including (but not limited to) human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or SARS-CoV-2 (COVID-19)-associated infection.

[0130] application

[0131] Regarding the methods of this disclosure, in some embodiments, a single dose of the polymeric vesicles or formulation of this disclosure, encapsulated or unencapsulated, is administered to an individual. However, in some embodiments, the polymeric vesicles are administered to an individual at an initial dose, followed by at least one booster dose, such as one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more follow-up doses, wherein the interval between each dose is approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. 7 weeks, 8 weeks, 9 weeks, or 10 weeks, or about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints) 1 to 7 days, 1 to 5 days, 1 to 3 days, 1 to 10 weeks, 1 to 8 weeks, 1 to 6 weeks, 1 to 4 weeks, 1 to 2 weeks, 1 to 12 months, 1 to 8 months, 1 to 6 months, 1 to 4 months, 1 to 2 months, or 6 to 12 months. In some embodiments, the polymer vesicles of this disclosure encapsulating the effective load are administered twice at the same or different doses, with the interval between the two administrations being 1 day, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1 to 5 days, 1 to 2 weeks, 1 to 3 months, 1 to 6 months, 1 month to 1 year, 3 months to 1 year, or 6 months to 1 year.

[0132] Route of administration. The polymer vesicles or compositions described herein can be administered via any route. Suitable routes include (but are not limited to) oral, nasal, mucosal, submucosal, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, percutaneous, and buccal routes. Some practical topical applications include (but are not limited to) application of drops, sprays, aerosols, gels, or ointments to the mucosal epithelium of the eyes, nose, mouth, anus, or vagina. Other possible routes of administration include inhalation of sprays, aerosols, or powders via the respiratory tract.

[0133] Effective amount applied. The effective amount described herein refers to an amount sufficient to provide the desired effect. In embodiments where the purpose of applying the polymer vesicles of this disclosure is to treat a disease, the effective amount refers to a therapeutically effective amount, while in some other embodiments where the purpose is to prevent a disease, the effective amount refers to a preventatively effective amount.

[0134] However, in some other embodiments where the purpose of administering polymeric vesicles and a payload is to enhance an adaptive immune response, the effective amount may be determined as sufficient to induce an antigen-specific immune response in individuals administered the polymeric vesicles and the payload. An antigen-specific immune response can be characterized by measuring the antibody titer against the anti-antigenic peptide (i.e., the payload or the product of the payload) produced in individuals administered the polymeric vesicles and the payload. In some embodiments, the measurement may be performed using an enzyme-linked immunosorbent assay (ELISA).

[0135] In some embodiments, antibody titers are used to assess whether an individual has an infection or to determine whether immunization is required. In some embodiments, antibody titers are used to determine the strength of an autoimmune response, to determine whether booster immunization is needed or has already been administered, to determine the effectiveness of previous vaccines, and / or to identify any current or previous infections.

[0136] The effective amount of the method disclosed herein can be determined based on several factors, including (but not limited to) individual conditions (age, sex, species, weight, health status, etc.), the progression of the disease to be treated, the route of administration, the dosage and interval of administration, and the nature of the payload. Regarding the nature of the payload, for example, in embodiments where the polymer vesicles of this disclosure are used to carry mRNA, such as in an mRNA vaccine, the effective amount can be determined based on the effective amount of mRNA required to elicit a sufficient immune response in the individual. Therefore, in some embodiments where the payload is mRNA, the effective amount of the method disclosed herein is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 micrograms (μg or ug), or any range defined by the foregoing endpoints.For example (including or excluding endpoints): 5 micrograms to 1000 micrograms, 5 micrograms to 900 micrograms, 5 micrograms to 800 micrograms, 5 micrograms to 700 micrograms, 5 micrograms to 600 micrograms, 5 micrograms to 500 micrograms, 5 micrograms to 400 micrograms, 5 micrograms to 300 micrograms, 5 micrograms to 200 micrograms, 5 micrograms to 175 micrograms, 5 micrograms to 150 micrograms, 5 micrograms to 125 micrograms, 5 micrograms to 100 micrograms, 5 micrograms to 90 micrograms, 5 micrograms to 80 micrograms, 5 micrograms to 70 micrograms, 5 micrograms to 60 micrograms, 5 micrograms to 50 micrograms, 5 micrograms to 40 micrograms, 5 micrograms to 30 micrograms, 5 micrograms to 20 micrograms, 5 micrograms to 10 micrograms, 10 micrograms to 1000 micrograms. Micrograms, 10 micrograms to 900 micrograms, 10 micrograms to 800 micrograms, 10 micrograms to 700 micrograms, 10 micrograms to 600 micrograms, 10 micrograms to 500 micrograms, 10 micrograms to 400 micrograms, 10 micrograms to 300 micrograms, 10 micrograms to 200 micrograms, 10 micrograms to 175 micrograms, 10 micrograms to 150 micrograms, 10 micrograms to 125 micrograms, 10 micrograms to 100 micrograms, 10 micrograms to 90 micrograms, 10 micrograms to 80 micrograms, 10 micrograms to 70 micrograms, 10 micrograms to 60 micrograms, 10 micrograms to 50 micrograms, 10 micrograms to 40 micrograms, 10 micrograms to 30 micrograms, 10 micrograms to 20 micrograms, 50 micrograms to 1000 micrograms, 50 micrograms to 90 micrograms 0 micrograms, 50 micrograms to 800 micrograms, 50 micrograms to 700 micrograms, 50 micrograms to 600 micrograms, 50 micrograms to 500 micrograms, 50 micrograms to 400 micrograms, 50 micrograms to 300 micrograms, 50 micrograms to 200 micrograms, 50 micrograms to 175 micrograms, 50 micrograms to 150 micrograms, 50 micrograms to 125 micrograms, 50 micrograms to 100 micrograms, 50 micrograms to 90 micrograms, 50 micrograms to 80 micrograms, 50 micrograms to 70 micrograms or 50 micrograms to 60 micrograms, 100 micrograms to 1000 micrograms, 100 micrograms to 900 micrograms, 100 micrograms to 800 micrograms, 100 micrograms to 700 micrograms, 100 micrograms to 600 micrograms, 100 micrograms to 50 micrograms 0 micrograms, 100 to 400 micrograms, 100 to 300 micrograms, 100 to 200 micrograms, 100 to 175 micrograms, 100 to 150 micrograms, 300 to 1000 micrograms, 300 to 900 micrograms, 300 to 800 micrograms, 300 to 700 micrograms, 300 to 600 micrograms, 300 to 500 micrograms, 300 to 400 micrograms, 500 to 1000 micrograms, 500 to 900 micrograms, 500 to 800 micrograms, 500 to 700 micrograms, 500 to 600 micrograms, 600 to 800 micrograms, or 700 to 900 micrograms.

[0137] However, given the targeted delivery provided by the polymer vesicles of this disclosure, it is anticipated that the effective amount required in the method of this disclosure may be lower than that required in other non-targeted delivery methods. For example, the effective amount required in the method of this disclosure may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or any range defined by the foregoing endpoints, lower than that required in other non-targeted delivery methods. For example (including or excluding endpoints): 1% to 99%, 1% to 95%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 1% to 5%, 5% to 99%, 5% to 95%, 5% to 90%, 5% to 80%, 5% to 70%, 5% to 60%, 5% Up to 50%, 5% to 40%, 5% to 30%, 5% to 20%, 5% to 10%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 30% to 99%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% Up to 60%, 30% to 50%, 30% to 40%, 50% to 99%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 99%, 80% to 95%, 80% to 90%, 90% to 99%, or 95% to 99%.

[0138] Furthermore, in some embodiments in which the polymer vesicles of this disclosure are used to deliver an antigen or a nucleic acid encoding an antigen to induce an antibody against the antigen, the antibody titer induced by the present invention is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 logs, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints) 1 to 10 logs, 1 to 8 logs, 1 to 6 logs, 1 to 4 logs, 2 to 9 logs, 2 to 7 logs, 2 to 5 logs, 3 to 10 logs, 3 to 8 logs, 3 to 5 logs or 4 to 6 logs.

[0139] In some other embodiments, when the polymer vesicles of this disclosure are used to deliver an antigen or a nucleic acid encoding an antigen to induce an immune response against the antigen, the antibody titer induced by the present invention is 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than that induced by non-targeted delivery methods, or any range defined by the aforementioned endpoints, such as (including or excluding the endpoints) 0.1 to 10, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, 0.1 to 5, 0.1 to 4, 0. 1 to 3, 0.1 to 2, 0.1 to 1, 0.1 to 0.5, 0.5 to 10, 0.5 to 9, 0.5 to 8, 0.5 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 7 to 10, 7 to 9, 7 to 8 or 8 to 10 times.

[0140] However, in some embodiments in which the polymer vesicles of this disclosure are used to deliver an antigen or a nucleic acid encoding an antigen to induce an immune response against the antigen, the immune response is induced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 days earlier than an immune response induced by a non-targeted delivery method, or any range defined by the foregoing endpoints, such as (including or excluding the endpoints) 1 to 20, 1 to 18, 1 to 14, 1 to 10, 1 to 6, 2 to 20, 2 to 18, 2 to 14, 2 to 10, 2 to 6, 5 to 20, 5 to 18, 5 to 14, or 5 to 10 days earlier.

[0141] In some embodiments, the polymeric vesicles as described herein in the methods of this disclosure are administered once or more daily at dose levels sufficient to deliver a payload of about 0.0001 mg to about 100 mg, about 0.001 mg to about 0.05 mg, about 0.005 mg to about 0.05 mg, about 0.001 mg to about 0.005 mg, about 0.05 mg to about 0.5 mg, about 0.01 mg to about 50 mg, about 0.1 mg to about 40 mg, about 0.5 mg to about 30 mg, about 0.01 mg to about 10 mg, about 0.1 mg to about 10 mg, or about 1 mg to about 25 mg per kilogram of individual body weight to obtain the desired in vivo effect.

[0142] definition

[0143] Unless otherwise expressly defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, the techniques employed or covered herein are standard methods well-known to one of ordinary skill in the art. Unless otherwise instructed, the practice of this disclosure will employ conventional techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry, and recombinant DNA technology, which are within the skill of the art. Materials, methods, and examples are illustrative and non-limiting only. The following is presented in an illustrative manner and is not intended to limit the scope of this disclosure.

[0144] The figures used to describe and claim certain embodiments of this disclosure representing the amount, characteristics (e.g., molecular weight, reaction conditions, and results, etc.) of components should be understood to be modified by the term "about" in some cases. Those skilled in the art will understand the meaning of the term "about" in its defined value. The numerical values ​​presented in some embodiments of this disclosure may contain some error arising from the standard deviation in their respective test measurements. For example, as used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and is intended to cover variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from a specified value, provided such variations are appropriate.

[0145] As used herein, “substantially” means sufficient to achieve the intended purpose. Therefore, the term “substantially” allows for small, insignificant variations in absolute or perfect state, dimensions, measurements, results, etc., that are expected by those skilled in the art but will not significantly affect overall performance. When used with numerical values ​​or parameters or characteristics expressed as numerical values, “substantially” means within ten percent.

[0146] As used herein, “treat,” “treatment,” and “treating” refer to a method for achieving a beneficial or desired outcome, such as a clinical outcome. For the purposes of this disclosure, a beneficial or desired outcome may include inhibiting or suppressing the onset or progression of an infection or disease; alleviating symptoms of an infection or disease or reducing its development; or a combination thereof.

[0147] As used herein, “preventing” and “prevention” are used interchangeably with “prophylaxis” and can mean complete prevention of infection or prevention of the development of symptoms of said infection, delaying the onset of disease or its symptoms; or reducing the severity of subsequently developed infection or its symptoms.

[0148] As used herein, “polysaccharide” refers to a polysaccharide, oligosaccharide, or monosaccharide. Polysaccharides can be monomers or polymers of sugar residues and can be linear or branched. Polysaccharides may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylenamine glycoside, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, mannose phosphate, 6'-sulfonic acid N-acetylglucosamine, etc.).

[0149] Example

[0150] Example 1: Synthesis of Exemplary Polymer Vesicles

[0151] Chemical Materials and Methods

[0152] For chemical synthesis, unless otherwise specified, all starting materials and commercially available reagents were purchased from Sigma-Aldrich and used as is. All reactions were carried out in dried glassware under a nitrogen atmosphere using anhydrous solvents. 1 H and 13 The 10⁻¹⁴ NMR spectra were recorded on a Brucker AV-600 spectrometer, and referenced to the solvent used (for...). 1 H and 13 C represents CDCl3 at δ 7.24 and 77.23, CD3OD at δ 3.31 and 49.2, D2O at δ 4.80, and DMSO-d6 at δ 2.5 and 39.51, respectively. Chemical shifts (δ) are reported in ppm using the following conventions: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiply), integral, and coupling constant (J), where J is reported in Hz. High-resolution mass spectra are recorded under ESI-TOF mass spectrometry conditions. Silica gel (E, Merck) is used for rapid chromatography. TM The system (containing an Intein-Mediated Purification with Affinity Chitinbinding Tag) was purchased from New England Biotechnology. His-tagged purification resin was purchased from Roche. The HiTrap IMAC column (5 mL) was purchased from GE Healthcare Life Sciences. Gel permeation chromatography (GPC) with an Ultimate 3000 liquid chromatography method associated with a 101 refractive index detector and a Shodex column was performed at 30 °C using THF as eluent at 1 mL / min.-1 Flow rate analysis was performed on the polymer products. Calibration was based on the narrow-linear poly(styrene) Shodex standard (SM-105). The molecular weight (Mw) and dispersion of the polymer products were calculated using DIONX Chromeleon software. Transmission electron microscopy (TEM) images were obtained using a FEITecnai G2 F20 S-Twin.

[0153] The chemical materials and methods described herein apply to all instances described in this disclosure.

[0154] Preparation of the exemplary growth agent disclosed herein

[0155] This article describes five exemplary growth agents, namely growth agents P1, P2, P3, P4, and P5. The preparation methods of growth agents P1 (compound 2), P2 (compound 3), and P4 (compound 8) are described in Scheme 1 below. Growth agent P3 (compound 11) was prepared according to Scheme 2 below. Growth agent P5 (compound 14) was prepared according to Scheme 3 below. The compounds obtained in each step of the scheme were verified by NMR. Additional details and data are described below.

[0156] Option 1

[0157]

[0158] Option 2

[0159]

[0160] Option 3

[0161]

[0162] Compound 1.

[0163]

[0164] Compound 1 was synthesized and characterized using a publicly available procedure. ¹H NMR (600 MHz, CDCl₃): δ 5.92 (br, ¹H), 3.60–3.57 (m, ¹H), 3.29 (dt, J = 11.2 Hz, 2H), 3.21–3.10 (m, 2H), 2.86–2.79 (m, 2H), 2.55–2.31 (m, ¹H), 2.21 (t, 2H, J = 7.4 Hz), 1.90–1.85 (m, ¹H), 1.77–1.41 (m, 8H). The synthesis was performed according to Jiaqi Fu et al., *Journal of the American Chemical Society*, 2015, 137(37), 12153–12160, which is incorporated herein by reference in its entirety.

[0165] Compound 2.

[0166]

[0167] Compound 2 was synthesized and characterized based on a publicly available procedure. 1 ¹H NMR (600MHz, MeOD): δ 3.98 (s, 1H), 3.61–3.39 (m, 1H), 3.30–3.22 (m, 4H), 3.22–2.88 (m, 2H), 2.56–2.30 (m, 1H), 2.21 (t, J = 7.4 Hz, 2H), 1.98–1.72 (m, 1H), 1.79–1.30 (m, 6H). The synthesis was performed according to Fu Jiaqi et al., *Journal of the American Chemical Society*, 2015, 137(37), 12153–12160. This literature is incorporated herein by reference in its entirety.

[0168] Compound 3.

[0169]

[0170] Compound 3 was synthesized and characterized based on a publicly available procedure. 1H NMR (600MHz, CDCl3): δ3.61-3.56(m,1H),3.41-3.34(m,1H),3.31-3.16(m,8H),3.14-3.06(m,2H),2.5 4-2.40(m,1H),2.08(t,J=7.4Hz,2H),1.95-1.86(m,1H),1.69(s,1H),1.55(m,3H),1.47-1.31(m,2H). According to Guo, J. et al., "Rational Design of Poly(disulfide)s as a Universal Platform for Delivery of CRISPR-Cas9 Machineries toward Therapeutic Genome Editing," ACS Central Science, 2021, 7, 990-1000, the synthesis of poly(disulfide)s was carried out, and the aforementioned literature is incorporated herein by reference in its entirety.

[0171] Compound 4.

[0172]

[0173] Compound 4 was synthesized and characterized according to the published protocol. 1 ¹H NMR (600MHz, DMSO-d⁶): δ 7.44–7.12 (m, 5H), 5.02 (s, 2H), 3.62–3.41 (m, 4H), 3.35 (t, 1H, J = 7.2Hz), 2.98 (d, 2H, J = 5.9Hz), 1.79–1.03 (m, 6H). The synthesis was performed according to Fu Jiaqi et al., *Journal of the American Chemical Society*, 2015, 137(37), 12153–12160. This literature is incorporated herein by reference in its entirety.

[0174] Compound 5.

[0175]

[0176] Compound 5 was synthesized and characterized according to the published protocol. 1¹H NMR (600MHz, CDCl₃): δ 11.52 (br, ¹H), 8.62 (s, ¹H), 3.45 (q, J = 4.00Hz, 2H), 2.86 (t, J = 4.00Hz, 2H), 1.49 (s, 9H), 1.48 (s, 9H). The synthesis was based on the work of Kuppusamy, R. et al., *Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl backbone as antibacterial agents*, *Eur. J. Med. Chem.*, 2018, 143, 1702-1722, which is incorporated herein by reference in full.

[0177] Compound 6.

[0178]

[0179] The solution of 4 (0.126 mmol) in DMF (1 mL) was pre-activated under nitrogen atmosphere with EDC (0.506 mmol), HOBt (0.506 mmol), and trimethylamine (0.57 mmol) for 30 minutes. Then, DMF (1 mL) containing 5 (0.506 mmol) was added to the above solution, and the resulting solution was stirred at room temperature for 12 hours. The mixture was concentrated to dryness under vacuum and then diluted with ethyl acetate. The organic layer was washed three times with H₂O and dried over MgSO₄, filtered, and concentrated. The crude product was purified by silica gel column chromatography (MeOH / DCM 1:20) to give 6 (134 mg, 74%). 1 H NMR (600MHz, CDCl3): δ11.45-11.33(m,3H),8.53-8.45(m,3H),8.35(t,J=4.9Hz,2H),7.33-7.25(m,5H),5.04(s,2H) ,3.59-3.19(m,16H),3.15-3.12(q,J=6.0Hz,2H),3.06-3.04(t,J=6.9Hz,1H),1.77-1.71(m,1H),1.55-1.32(m,59H). C 57 H 97 N 14 O 17 [M+H] + HRMS (ESI) calculated value for m / z: 1249.7156; experimental value: 1249.7166.

[0180] Compound 7.

[0181]

[0182] Palladium / charcoal (Pd / C, 10% Pd content, 13 mg) was added to a solution of 6 (0.12 mmol) in MeOH (2 mL). The mixture was stirred at room temperature under a hydrogen atmosphere for 6 hours. The solution was filtered through a diatomaceous earth pad. The residue was concentrated to dryness under vacuum and dissolved in DCM (2 mL). Lipoic acid (0.152 mmol), EDCI (0.304 mmol), HOBt (0.304 mmol), and TEA (0.304 mmol) were added to the mixture, followed by stirring at room temperature for 2 hours. The mixture was concentrated to dryness under vacuum and then diluted with ethyl acetate. The organic layer was washed three times with H2O, dried over MgSO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (MeOH / DCM 1:50) to give 7 (94 mg, 82%). 1 H NMR (600MHz, CDCl3): δ11.48-11.30(m,3H),8.57-8.46(m,3H),8.40(t,J=5.2Hz,2H),8.15-8.12(m,1H),6.22-6.18(m,1H),3.60-3.34 (m,16H),3.28-3.04(m,7H),2.48-2.38(m,1H),2.17-2.14(t,J=7.4Hz,2H),1.92-1.71(m,11H),1.71-1.59(m,4H),1.57-1.34(m,64H). C 57 H 103 N 14 O 16 S2[M+H] + HRMS (ESI) calculated value for m / z: 1303.7118; experimental value: 1303.7129.

[0183] Compound 8.

[0184]

[0185] Compound 7 (0.08 mmol) was added to a solution of 4 M HCl (0.5 mL) in 1,4-dioxane (0.5 mL), and the mixture was stirred at room temperature for 12 hours. The solution was then removed and dried under vacuum to give compound 8 (24 mg, 89%). 1HNMR (600MHz, D2O): δ3.60-3.40(m,3H),3.4-3.16(m,16H),3.02(s,2H),2.76(s,2H),2.20-2.03(m,2H),1.98-1.76(m,2H),1.62-1.16(m,11H). 13 C NMR (150MHz, D2O): δ176.61,175.62,173.68,172.92,156.88(x3),66.01,65.73,56.57,55.36,55. 24,40.40(x2),40.27,38.69,37.99(x3),37.82,35.44,33.57,28.66,28.18,27.73,24.99,22.71. C 27 H 55 N 14 O4S2[M+H] + HRMS (ESI) calculated value for m / z: 703.3967; experimental value: 703.3995.

[0186] Compound 9.

[0187]

[0188] A solution of lipoic acid (2.5 mmol) in DMF (3 mL) was pre-activated with EDCI (3 mmol), HOBt (3 mmol), and trimethylamine (3 mmol) under nitrogen atmosphere for 30 minutes. Then, DMF (3 mL) containing lysine (1 mmol) was added to the above solution, and the resulting solution was stirred at room temperature for 2 hours. The mixture was concentrated to dryness under vacuum and then diluted with ethyl acetate. The organic layer was washed three times with H₂O, dried over MgSO₄, filtered, and concentrated. The crude product was purified by silica gel column chromatography (MeOH / DCM 1:20) to give 9 (230 mg, 90%). 1 H NMR (600MHz, MeOD): δ4.37-4.32 (dd, J=4.6, 9.2Hz, 1H), 3.63-3.57 (qui, J=6.8Hz, 2H), 3.22-3.17 (m, 4H), 3.14-3.09 (m, 2H ),2.51-2.45(m,2H),2.29(t,J=7.0Hz,2H),2.21(t,J=7.0Hz,2H),1.95-1.86(m,3H),1.79-1.62(m,9H),1.60-1.40(m,8H). C 57 H 97 N 14 O 17 [M+H]+ HRMS (ESI) calculated value for m / z: 1249.7156; experimental value: 1249.7166.

[0189] Compound 10.

[0190]

[0191] The solution of 9 (0.141 mmol) in DMF (1 mL) was pre-activated under nitrogen atmosphere with EDC (0.211 mmol), HOBt (0.211 mmol), and trimethylamine (0.282 mmol) for 30 minutes. Then, DMF (1 mL) containing 5 (0.183 mmol) was added to the above solution, and the resulting solution was stirred at room temperature for 2 hours. The mixture was concentrated to dryness under vacuum and then diluted with ethyl acetate. The organic layer was washed three times with H₂O, dried over MgSO₄, filtered, and concentrated. The crude product was purified by silica gel column chromatography (MeOH / DCM 1:20) to give 10 (92 mg, 84%). 1 H NMR (600MHz, CDCl3): δ11.39 (s, 1H), 8.58 (t, J = 6.0Hz, 1H), 7.98 (t, J = 4.6Hz, 1H), 6.41 (d, J = 7. 4Hz,1H),5.79-5.74,(m,1H),4.37(q,J=7.5Hz,1H),3.57-3.48(m,4H),3.44-3.35(m,2H),3.26- 3.12(m,4H),3.11-3.05(m,2H),2.46-2.39(m,2H),2.18(t,J=7.4Hz,2H),2.13(t,J=7.4Hz,2H), 1.91-1.84(m,2H),1.82-1.75(m,1H),1.71-1.57(m,9H),1.54-1.35(m,20H),1.33-1.25(m,2H). 13 C NMR (150MHz, CDCl3): δ172.91,172.55,171.72,162.87,157.42,153.03,83.72,79.97,56.46,56.44,52.78,41.19,40.27,40.26,40.20 ,38.96,38.48(x2),36.48,36.37,36.30,34.64,34.61,32.60,29.18,28.95,28.88,28.29(x2),28.05(x2),25.46,25.36,25.32,22.29. C 35 H 63 N6O7S4[M+H]+ HRMS (ESI) calculated value for m / z: 807.3641; experimental value: 807.3655.

[0192] Compound 11.

[0193]

[0194] Compound 10 (0.1 mmol) was added to a solution of 4 M HCl (0.5 mL) in 1,4-dioxane (0.5 mL), and the mixture was stirred at room temperature for 12 hours. The solution was then removed and dried under vacuum to give compound 8 (59 mg, quantified). The compound was pure and could be used directly without further purification.

[0195] Compound 12.

[0196]

[0197] A solution of lipoic acid (3 mmol) and CDI (3.9 mmol) was dissolved in 25 mL of anhydrous DCM. This solution was added dropwise to 5 mL of anhydrous DCM containing 8 mmol of N-methyl-1,3-propanediamine at 0 °C. The reaction mixture was stirred at 0 °C for 40 min and then at room temperature for 30 min. It was then washed three times with H2O, dried over MgSO4, filtered, and concentrated to give 12 (589 mg, 66%). 1 H NMR (600MHz, CDCl3): δ3.57-3.50(m,1H),3.31(q,J=5.8Hz,2H),3.17-3.12(m,1H),3.11-3.05(m,1H),2.65(t,J=6.0 Hz,2H),2.46-2.40(m,1H),2.39(s,3H),2.15-2.11(m,2H),1.91-1.84(m,1H),1.72-1.54(m,6H),1.49-1.37(m,2H). C 12 H 26 N2OS2[M+H] + HRMS (ESI) calculated value for m / z: 277.1408; experimental value: 277.1399.

[0198] Compound 13.

[0199]

[0200] Compound 13 was synthesized and characterized according to the published protocol. 1¹H NMR (600MHz, CDCl₃): δ 4.50–4.38 (m, 4H), 4.19–4.15 (m, 2H), 1.74–1.70 (m, 2H), 1.38–1.20 (m, 12H), 0.89 (t, J = 7.2 Hz, 3H). According to Liu et al., zwitterionic phospholipidation of cationic polymers facilitates systemic mRNA delivery to the spleen and lymph nodes. (J. Am. Chem. Soc., 2021, 143, 21321-21330). This work was synthesized using this literature, which is incorporated herein by reference in its entirety.

[0201] Compound 14.

[0202]

[0203] A solution of 12 (0.25 mmol) in 1 mL of anhydrous DMF was added to 13 (0.25 mmol), and the reaction mixture was stirred at 70 °C for 24 hours. The mixture was concentrated to dryness under vacuum to give 14 (116 mg, 90%). 1 H NMR (600MHz, MeOD): δ7.90(s,1H),4.14-4.01(m,1H),3.91-3.83(m,1H),3.73-3.67(m,1H) ,3.62-3.55(m,1H),3.28(t,J=6.6Hz,2H),3.21-3.15(m,1H),3.13-3.06(m,1H),2.98(t,J =7.2Hz,2H),2.69(s,3H),2.50-2.41(m,1H),2.24(t,J=7.2Hz,2H),1.93-1.84(m,1H),1.7 6-1.59(m,6H),1.51-1.43(m,2H),1.43-1.37(m,1H),1.36-1.25(m,5H),0.92-0.88(m,3H). 13C NMR (150MHz, MeOD): δ177.08,79.68,57.79,48.91,48.77,48.04,41.54,39.55,37.06,36 .91,35.91,33.85,33.20,32.09,32.05,30.44,30.12,27.73,27.16,26.81,23.92,14.65. C 22 H 44 N2O5PS2[MH] - HRMS (ESI) calculated value for m / z: 511.2429; experimental value: 511.2423.

[0204] Preparation of the exemplary initiator disclosed herein

[0205] This paper describes nine exemplary initiators, namely initiators I2, I3, I4, I5, I6, I7, I8, I9, and I10. Initiators I2 (compound 34), I3 (compound 39), and I4 (compound 37) were prepared according to schemes 4, 5, and 6. Initiators I5 (compound 16), I6 (compound 43), I7 (compound 44), I8 (compound 40), I9 (compound 49), and I10 (compound 54) were prepared according to schemes 7, 8, 9, and 10. The compounds obtained in each step of the schemes were verified by NMR. Additional details and data are described below.

[0206] Additionally, a control initiator I1 was prepared in this example. I1 without the glycan head is an initiator without specific targeting preference and was used to prepare copolymers for forming the exemplary growers of this disclosure to characterize the growers. Initiator I1 has the following structure:

[0207]

[0208] Option 4

[0209]

[0210] Option 5

[0211]

[0212] Option 5 (continued)

[0213]

[0214] Option 6

[0215]

[0216] Option 7

[0217]

[0218] Option 8

[0219]

[0220] Option 9

[0221]

[0222] Option 10

[0223]

[0224] Compound 15 8 .

[0225]

[0226] Compound 15 was synthesized and characterized according to the published protocol. ¹H NMR (600 MHz, MeOD): δ 6.93 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 5.3 (s, 1H), 4.00–3.99 (m, 1H), 3.89–3.88 (m, 1H), 3.81–3.79 (m, 1H), 3.79–3.70 (m, 2H), 3.69–3.67 (m, 1H).

[0227] Compound 16.

[0228]

[0229] EDC (0.24 mmol), HOBt (0.24 mmol), trimethylamine (0.4 mmol), and 3-mercaptopropionic acid (0.2 mmol) were added to a solution of 15 (0.24 mmol) in DMF (2 mL), and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was concentrated to dryness under vacuum, and the crude product was purified by silica gel column chromatography (MeOH / DCM 1:2) to give 16 (72%). 1 H NMR(600MHz,MeOD)δ6.94(d,J=8.6Hz,2H),6.72(d,J=8.6Hz,2H),5.30(s,1H),4.00(dd,J=3.4,1.8H z, 1H), 3.89 (dd, J = 9.7, 3.4Hz, 1H), 3.81-3.67 (m, 4H), 2.72 (t, J = 6.8Hz, 2H), 2.60 (t, J = 6.8Hz, 2H). 13C NMR (150MHz, MeOD) δ176.55,151.13,143.23,119.25(x2),117.93(x2),101.49,101.32,75.10,72.43,72.18,68.42,62.66,40.34,20.70. C 15 H 22 NO7S[M+H] + The calculated HRMS (ESI) value is 360.1117, and the experimental value is 360.1101.

[0230] Compounds 17 to 20. According to Peng, W.; Paulson, JC, CD22 ligands on a natural N-Glycan scaffold efficiently deliver toxins to B-Lymphoma cells. JAMA 2017, 139, 12450-12458. The synthesized compounds 17 to 20 are cited in full hereof and are incorporated herein by reference.

[0231] Compounds 21 to 25. Based on the sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions (Chem. Commun.), 2014, 50, 5786-5789, compounds 21 to 25 were synthesized.

[0232] Compound 26. EDC (0.52 mmol), HOBt (0.52 mmol), trimethylamine (0.86 mmol), and 3-mercaptopropionic acid (0.47 mmol) were added to DCM (5 mL) containing compound 25 (0.43 mmol), and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was concentrated to dryness under vacuum, and the crude product was purified by silica gel column chromatography (MeOH / DCM 1:10) to give 26 (184 mg, 80%). 1H NMR (600MHz, CDCl3) δ5.96(d,J=8.6Hz,1H),5.28(t,J=9.6Hz,1H),5.05(t,J=9.4Hz,1H),4.64(d,J=8.4H z,1H),4.24(dd,J=12.3,4.7Hz,1H),4.11(dd,J=12.3,2.5Hz,1H),3.86-3.80(m,2H),3.68-3.66(m,1H), 3.47-3.43(m,1H),3.34-3.29(m,1H),3.22-3.16(m,1H),2.81(t,J=8.2Hz,2H),2.51-2.45(m,2H),2.06( s,3H),2.01(s,3H),2.00(s,3H),1.93(s,3H),1.57-1.51(m,4H),1.48(t,J=7.0Hz,2H)1.37-1.29(m,4H). C 23 H 38 N2O 10 S[M+H] + The calculated HRMS (ESI) value was 535.2325, and the experimental value was 535.2314. This data is based on the work of Maklakova et al. (SY, 2020), who investigated the cellular uptake of N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, and d-mannose-containing fluorescent glycoconjugates by liver intravital microscopy. The literature cited in *Carbohydr. Res.*, 2020, 489, 107928, is incorporated herein by reference in its entirety.

[0233] Compound 27. NaOMe was added to MeOH containing compound 26, and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was neutralized by IR-120, then filtered, and concentrated to dryness under vacuum to give 27 (92 mg, quantified). 1HNMR(600MHz,MeOD)δ4.38(d,J=9.2Hz,1H),3.90-3.86(m,2H),3.68(dd,J=12 .9,5.9Hz,1H),3.65-3.62(m,1H),3.48-3.43(m,2H),3.28-3.24(m,1H),3.20 -3.14(m,2H),2.94(t,J=7.3Hz,1H),2.73(t,J=7.3Hz,1H),2.59(t,J=7.3Hz, 1H), 2.47 (t, J = 7.3Hz, 1H), 1.97 (s, 3H), 1.56-1.48 (m, 4H), 1.39-1.35 (m, 4H). 13 C NMR (150MHz, MeOD): δ173.70,169.75,102.74,77.92,76.07,70.50,62.76,57.35,41.02,40.32,36.45,35.19,30.49,27.66,26.73,23.03. C 17 H 32 N2O 17 S[M+H] + The calculated HRMS (ESI) value is 409.2008, and the experimental value is 409.2017.

[0234] 9 Am Neu5Ac-α2,3-SCT 30, 9 AmPreparation of Neu5Ac-α2,6-SCT 31 and Neu5Ac-α2,3-SCT 38. According to Lin, C.-W. et al., Homogeneous antibody and CAR-T cells with improved effector functions targeting SSEA-4 glycan on pancreatic cancer (Proceedings of the National Academy of Sciences, 2021, 118, e2114774118, which is incorporated herein by reference in its entirety), 30 mg of sialylated glycopeptide (SGP) was digested in Tris-HCl buffer at 37°C with Endo-S WT (300 μg) for 48 hours. The product was purified by Sephadex G-25 gel filtration chromatography and analyzed by ESI-MS to obtain compound 28, sialylated glycopeptide. Tris-HCl buffer containing neuraminidase (5 U / ml, 12 μL) was added to the mixture at 37 °C for 12 hours, and the mixture was purified by desaminoglycan gel filtration chromatography (G-25) to obtain desialylated N-glycan 29. Subsequently, it was purified in 0.5 mL of 100 mM 9... Am The reaction was carried out in a HEPES buffer (50 mM, pH 8.5) containing Neu5Ac (14.5 mg, 47 μmol), 110 mM CTP (27.2 mg, 52 μmol), and 20 mM MgCl2. The pH of the reaction mixture was adjusted to 8.5 by adding 2N NaOH. Subsequently, 0.5 mg / mL NmCSS was added to the above solution. The resulting mixture was incubated at 37 °C for 8 hours, and CMP-9 was monitored by TLC analysis. Am The formation of Neu5Ac.

[0235] For 9 AmNeu5Ac-α2,6-SCT, according to Peng, W. et al., recent H3N2 viruses have evolved specificity for extended, branched human-type receptors, thus possessing the potential for increased affinity (Recent H3N2 Viruses Have Evolved Specificity for Extended, Branched Human-type Receptors, Conferring Potential for Increased Avidity). Cell Host Microbe, 2017, 21, 23-34 (which is incorporated herein by reference in its entirety), added hST6Gal-I (0.5 mg / mL) to a 9-cell N-glycan-containing... Am The mixture was incubated in the Neu5Ac reaction mixture at 37°C. For 9 Am Neu5Ac-α2,3-SCT, adding PmST3 (0.3 mg / mL) to 9-cell N-glycans Am The Neu5Ac reaction mixture was added to the Neu5Ac reaction mixture and incubated at 37°C. For Neu5Ac-α2,3-SCT, PmST3 (0.3 mg / mL) was added to the Neu5Ac reaction mixture containing N-glycans and incubated at 37°C. The reaction was monitored by mass spectrometry and TLC. After acceptor depletion, the reaction mixture was centrifuged, and the supernatant was passed through a centrifugal filter (Amicon Ultra, Millipore) with a molecular weight cutoff of 10 kDa to remove proteins. The filtrate was purified by P-2 gel filtration chromatography to obtain 9 Am Neu5Ac-α2,3-SCT 30, 9 Am Neu5Ac-α2,6-SCT 31 and Neu5Ac-α2,3-SCT 38.

[0236] 9 BPC Neu5Ac-α2,6-SCT-SH 34, 9 TCC Preparation of Neu5Ac-α2,3-SCT-SH37 and Neu5Ac-α2,3-SCT-SH39. 9 AmNeu5Ac-α2,6-SCT and DIEA (5.0 equivalents) were dissolved in H2O, followed by the addition of THF containing biphenyl-N-hydroxysuccinimide ester (BPC-NHS) (3 equivalents). The reaction mixture was stirred at 0°C until the starting material was exhausted. Subsequently, the reaction mixture was purified by passing it through a Sep-Pak C18 column (2 g, Waters Corp.) and eluted with H2O-MeOH to give compound 32 in 91% yield. Similarly, 9 was prepared by stirring with 4H-thieneno[3,2-c]chromene-2-carbamoyl-NHS (TCC-NHS) in THF and H2O. Am Neu5Ac-α2,3-SCT yielded compound 35. After purification, the product was obtained in 91% yield.

[0237] To 9 BPC Neu5Ac-α2,6-SCT 32 or 9 TCC A mixture of Neu5Ac-α2,3-SCT 35 and water was incubated with CDMBI and TEA at 4°C for 1 hour to produce the corresponding oxazoline N-glycans 33 and 36, respectively. These were then purified by dextran gel filtration chromatography (G-25) and characterized by ESI-MS. GlcNAc-SH 27 (0.25 mg) and Endo-M (N175Q) (1.6 U / mL) were added to a solution containing 9... BPC The product was incubated in a 50 mM phosphate buffer (pH 7) solution of Neu5Ac-α2,6-SCT-oxazoline at 30 °C for 30 min. The transglycosylated product was separated by P-2 gel filtration chromatography to obtain compound 34, which was characterized by ESI-MS. TCC The preparation of Neu5Ac-α2,3-SCT-SH 37 and Neu5Ac-α2,3-SCT-SH 39 is the same as described above.

[0238] Compound 34.9 BPC Neu5Ac-α2,6-SCT-SH. 1H NMR (600MHz, DMSO-d6): δ = 8.33 (s, 3H, NH), 8.00 (d, J = 8.6Hz, 4H), 7.72 (dd, J = 8.0, 14.2Hz, 9H), 7.49 (t, J = 8.3Hz, 4H), 7.40 ( t,J=8.3Hz,2H),4.98-4.95(m,2H),4.76(s,1H),4.54(s,1H),4.44-4.39(m,2H),4.25-4.22(m,3H),3.99(s,1H),3.87(s,1H) ,3.83-3.73(m,5H),3.41-3.25(m,58H),3.21-3.16(m,3H),3.11-2.99(m,4H),2.87(t,J=6.9Hz,2H),2.61(d,J=8.4Hz,2H),2 .44(t,J=7.4Hz,2H),1.91-1.77(m,18H),1.44(t,J=6.9Hz,2H),1.39-1.28(m,4H),1.28-1.19(m,4H),1.12(t,J=6.9Hz,1H). C 119 H 171 N9O 63 S 2- [M-2H] 2- The calculated HRMS (ESI) value is 1383.0093, and the experimental value is 1383.0077.

[0239] Compound 37.9 TCC Neu5Ac-α2,3-SCT-SH. 1H NMR (600MHz, DMSO-d6): δ=8.22(s,3H,NH),8.10-8.09(m,1H,NH),7.96-7.93(m,1H,NH),7.81-7.79(m,1H,NH),7.74-7.72(m,1H,NH),7.35(d,J=7.8Hz, 2H),7.29(s,2H),7.20(t,J=7.8Hz,2H),6.97(t,J=7.8Hz,2H),6.92(d,J=7 .9Hz,2H),5.29(s,4H),5.01-4.95(m,2H),4.77(s,1H),4.54(s,1H),4.44-4 .36(m,2H),4.27-4.19(m,3H),3.99(s,1H),3.88(s,1H),3.85-3.71(m,3H) ,3.50-3.22(m,52H),3.19(d,J=9.5Hz,2H),3.10-3.00(m,4H),2.87(t,J=6. 9Hz,2H),2.64-2.58(m,2H),2.44(t,J=7.4Hz,2H),1.93-1.78(m,18H),1.4 5-1.39(m,2H),1.39-1.29(m,4H),1.28-1.20(m,4H),1.16(t,J=6.9Hz,1H). C 117 H 169 N9O 65 S3 2- [M-2H] 2- The calculated HRMS (ESI) value is 1416.9606, and the experimental value is 1416.9623.

[0240] Compound 39. Neu5Ac-α2,3-SCT-SH 1H NMR (600MHz, DMSO-d6): δ=8.33(s,3H,NH),8.10(s,1H,NH),7.96-7.94(m,1H,NH),7.81-7.80(m,1H,NH),7.74-7.73(m,1H ,NH),4.98-4.95(m,2H),4.76-4.75(m,1H),4.54(s,1H),4.46-4.38(m,2H),4.25-4.22(m,3H),3.99(s,1H),3.87(s,1H),3 .80-3.71(m,4H),3.41-3.25(m,59H),3.23-3.18(m,3H),3.08-3.01(m,3H),2.87(t,J=6.9Hz,2H),2.63-2.60(m,2H),2.4 4(t,J=7.4Hz,2H),1.87-1.79(m,18H),1.44(t,J=6.9Hz,2H),1.39-1.28(m,4H),1.28-1.19(m,4H),1.07(t,J=6.9Hz,1H). C 93 H 153 N7O 63 S 2- [M-2H] 2- The calculated HRMS (ESI) value is 1203.9357, and the experimental value is 1203.9377.

[0241] Compound 40.

[0242]

[0243] EDC (0.12 mmol), HOBt (0.12 mmol), DMAP (0.12 mmol), trimethylamine (0.2 mmol), and CT (PEG) were added to 1 mL of DMF containing compound 15 (0.12 mmol). 12 (0.1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 12 hours. The mixture was concentrated to dryness under vacuum, and the crude product was purified by silica gel column chromatography to give 40 (59%). 1H NMR (600 MHz, MeOD): δ 6.93 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 5.3 (s, 1H), 4.00–3.99 (m, 1H), 3.89–3.88 (m, 1H), 3.81–3.70 (m, 5H), 3.70–3.59 (m, 48H), 2.68 (t, J = 6.8 Hz, 2H), 2.50–2.47 (m, 2H). 13C NMR (150MHz, MeOD): δ170.17,151.03,143.50,119.26(x2),117.82(x2),101.36,75.10,74.0 8,72.45,72.19,71.45,71.41,71.37,71.30,71.24,71.11,70.93(x18),68.43,62.68,24.67. C 39 H 70 NO 19 S[M+H] + The calculated HRMS (ESI) value is 888.4263, and the experimental value is 888.4241.

[0244] Compounds 41 and 42. Based on the Reactivity-Based One-Pot Synthesis of Oligomannoses: Defining Antigens Recognized by 2G12, a Broadly Neutralizing Anti-HIV-1 Antibody. Angewandte Chemie International Edition, 2004, 43, 1000-1003. The above-mentioned literature is incorporated herein by reference in its entirety.

[0245] Compound 43.

[0246]

[0247] EDC (0.24 mmol), HOBt (0.24 mmol), trimethylamine (0.4 mmol), and 3-mercaptopropionic acid (0.2 mmol) were added to a solution of 42 (0.24 mmol) in DMF (2 mL), and the resulting solution was stirred at room temperature under nitrogen for 2 hours. The mixture was concentrated to dryness under vacuum, and the crude product was purified by silica gel column chromatography (MeOH / DCM 1:3) to give 16 (81%). 1 H NMR(600MHz,MeOD)δ4.91(s,1H),3.84(dd,J=3.4,1.8Hz,1H),3.82-3.76(m,2H),3.75-3.67(m,2H), 3.62(m,1H),3.56-3.41(m,2H),2.94(t,J=7.0Hz,2H),2.70(t,J=7.2Hz,2H),2.48(t,J=7.2Hz,2H). C14 H 28 NO7S[M+H] + The calculated HRMS (ESI) value is 354.1586, and the experimental value is 354.1602.

[0248] Compound 44.

[0249]

[0250] EDC (0.12 mmol), HOBt (0.12 mmol), trimethylamine (0.2 mmol), and CT (PEG) were added to 1 mL of DMF containing compound 42 (0.12 mmol). 12 (0.1 mmol), and the resulting solution was stirred under nitrogen and at room temperature for 12 hours. The mixture was concentrated to dryness under vacuum and the crude product was purified by silica gel column chromatography to give 44 (62%). 1 H NMR (600MHz, D2O): δ4.77(d,J=8.6Hz,2H),3.85-3.82(m,1H),3.81-3.77(m,1H),3.69-3.67(m,1H),3.66-3.64(m,2H),3.63-3.57(m ,41H),3.55-3.45(m,4H),2.88(t,J=6.8Hz,2H),2.57(t,J=7.2Hz,2H),2.37(t,J=7.2Hz,2H),1.66-1.51(m,4H),1.40-1.31(m,2H). 13 C NMR (150MHz, D2O): δ171.01,99.62,72.72,70.57,69.99,69.24(x22),69. 04,67.90,67.39,66.73,60.92,39.36,37.57,27.96,26.70,22.91,22.43. C 38 H 76 NO 19 S[M+H] + The calculated HRMS (ESI) value is 882.4732, and the experimental value is 882.4750.

[0251] Compound 45.

[0252]

[0253] MeOH containing 5 mmol of compound 15b was added to NaOMe (0.2 equivalents), and the resulting solution was stirred at nitrogen and room temperature for 2 hours. The mixture was neutralized by IR-120, filtered, and concentrated to dryness under vacuum. It was then dissolved in anhydrous DCM (40 mL) and treated with imidazole (7.5 mmol) at 0 °C, followed by the addition of TBDPSCl (5.5 mmol). The mixture was stirred at room temperature and under nitrogen atmosphere for 2.5 hours. The reaction was quenched by the addition of MeOH. After stirring at room temperature for 10 minutes, the solvent was removed under reduced pressure to obtain a dry residue, which was purified by column chromatography with MeOH / DCM (1 / 10) to give compound 45 (75%). 1 H NMR (600MHz, CDCl3) δ7.61(m,4H),7.41-7.29(m,11H),7.23-7.18(m,2H),6.92(d,J=9.3Hz,2H),5.41(s,1H),5.16(s,2H),4 .09(s,1H),4.02-4.00(dd,J=9.3,3.4Hz,1H),3.91(t,J=9.3Hz,1H),3.85(d,J=5.1Hz,2H),3.72-3.69(m,1H),1.01,(s,9H). 13 C NMR(150MHz, CDCl3):162.61,135.64(x4),135.54(x4),132.73,129.95(x4),128.64(x4),128.38,128.3 3,127.84(x2),127.81(x2),98.09,71.41,71.22,70.22,70.13,64.89,36.53,31.48,26.83(x3),19.19. C 36 H 42 NO8Si[M+H] + The calculated HRMS (ESI) value is 644.2680, and the experimental value is 644.2699.

[0254] Compound 46.

[0255]

[0256] Trimethyl orthobenzoate (9 mmol) was added to a solution of compound 45 (3 mmol) and a catalytic amount of CSA (0.3 mmol) in CH3CN (60 mL) at room temperature and atmospheric nitrogen. After stirring for 30 min, Et3N was added to quench the reaction, and the resulting mixture was dried under reduced pressure. The residue was purified by column chromatography with EA / hexane (1 / 2) to give compound 46 (81%).1 H NMR (600MHz, CDCl3) δ7.65-7.59(m,2H),7.57-7.52(m,4H),7.41-7.27(m,14 H),7.26-7.20(m,2H),6.93(d,J=9.2Hz,2H),5.77(s,1H),5.17(s,2H),4.70 (d,J=6.1Hz,1H),4.58(dd,J=9.3,3.4Hz,1H),3.79-3.76(m,2H),3.74-3.70 (m,1H),3.69-3.66(m,1H),3.22(s,3H),2.53(d,J=3.9Hz,1H),0.93,(s,9H). 13 C NMR(150MHz, CDCl3):171.23,153.49,152.24,137.09,136.09,135.68(x4), 135.48(x4),132.98,132.72,129.86,129.84(x2),129.18,128.65(x2),128. 39,128.37,128.34,127.78(x2),127.71(x2),126.23,121.11,117.18,95.69 ,79.52,69.57,69.45,67.03,63.75,60.44,51.16,26.76(x3),19.15,14.22. C 44 H 48 NO9Si[M+H] + The calculated HRMS (ESI) value is 762.3098, and the experimental value is 762.3072.

[0257] Compound 47.

[0258]

[0259] Compound 46 (2 mmol) was dissolved in DCM (20 mL) and mixed sequentially with DIPEA (6 mmol), benzoic anhydride (4 mmol), and DMAP (0.2 mmol). After stirring for 30 min, the solvent was evaporated under reduced pressure to obtain a dry residue, which was then poured into EA (20 mL) and 2N HCl (20 mL) and stirred vigorously for 30 min. The solvent was removed by evaporation, followed by extraction with EA. The collected organic layer was washed with ice-cold saturated NaHCO3 (aqueous solution), water, and brine, and dried over MgSO4. The filtrate was evaporated under reduced pressure and redissolved in THF (20 mL). AcOH (4 mmol) and 1M TBAF (2.4 mmol in THF) were added at 0 °C. The resulting mixture was gradually heated to room temperature and stirred for 2 h, then diluted with EA. The organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The dried residue was purified by column chromatography with EA / hexane (1 / 2) to give compound 47 (65%). 1 H NMR (600MHz, CDCl3) δ8.10-8.05(m,4H),7.61-7.56(m,2H),7.48-7.41(m,4H),7.39-7.27(m,7H),7.03(d,J=9.2Hz,2 H),5.69(s,1H),5.62-5.55(m,2H),5.16,(s,2H),4.62(dd,J=9.3,3.4Hz,1H),4.06-4.01(m,1H),3.76-3.67(m,2H). 13 C NMR(150MHz, CDCl3):167.42,166.05,153.55,152.01,136.03,133.84,133.76,130.00(x4),129.09,128. 96,128.69(x4),128.65(x4),128.62(x4),128.34,117.11(x2),96.18,72.61,71.30,70.01,68.53,61.16. C 34 H 32 NO 10 [M+H] + The calculated HRMS (ESI) value is 614.2026, and the experimental value is 614.2038.

[0260] Compound 48.

[0261]

[0262] A stirred solution of 0.2 mmol of 47 and 0.2 g of 4A molecular sieve in anhydrous DCM (2 mL) was cooled to -40 °C, and then 0.02 mmol of BF3(OEt)2 was added dropwise to the solution. A solution of 15a in anhydrous DCM was added dropwise to the above mixture, and the mixture was stirred at -40 °C for 1 hour. The reactants were then gradually heated to room temperature and stirred for another hour. The solution was quenched by adding triethylamine, filtered, and extracted with DCM after adding a saturated aqueous solution of NaHCO3. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by silica gel column chromatography to obtain the trisaccharide product. The product was then dissolved in MeOH, NaOMe (0.2 equivalents) was added, and the resulting solution was stirred at room temperature for 2 hours. The mixture was neutralized by IR-120, filtered, and concentrated to dryness under vacuum. The deacetylated mixture was purified by Bio-Gel P-2 gel electrophoresis with H2O as the eluent to obtain pure trisaccharide. The compound was lyophilized, then dissolved in MeOH (2 mL), and 10% Pd-C (30 mg) was added. The mixture was stirred vigorously overnight under a H2 atmosphere. The solution was filtered through diatomaceous earth and concentrated to dryness to give compound 48 (42%). 1 HNMR(600MHz,D2O)δ7.03(d,J=9.2Hz,2H),6.88(d,J=9.2Hz,2H),5.48(s,1H),5.19(s,1H ), 4.76 (s, 1H), 4.32 (s, 1H), 4.14 (dd, J = 9.3, 3.0Hz, 1H), 4.11 (s, 1H), 3.95-3.63 (m, 15H). 13 C NMR(150MHz,D2O):151.38,143.39,121.23,120.70,105.19,101.60,101.39,80.93,76.13, 75.38,74.04,73.27,73.12,72.79,72.66,72.22,69.50,69.41,68.70,67.90,63.71,63.65. C 24 H 37 NO 16 Na[M+Na] + The calculated HRMS (ESI) value is 618.2010, and the experimental value is 618.2029.

[0263] Compound 49.

[0264]

[0265] EDC (0.12 mmol), HOBt (0.12 mmol), DMAP (0.12 mmol), trimethylamine (0.2 mmol), and CT (PEG) were added to 1 mL of DMF containing compound 48 (0.12 mmol). 12 (0.1 mmol), and the resulting solution was stirred at room temperature under nitrogen for 12 hours. The mixture was concentrated to dryness under vacuum and the crude product was purified by Bio-Gel P-2 gel with H2O as the eluent to give 49 (54%). 1 H NMR(600MHz,D2O):7.20(d,J=9.2Hz,2H),7.13(d,J=9.2Hz,2H),5.53(s,1H),5.09(s,1H),4.65(s,1H),4.25(s,1H),4.06 (dd,J=9.3,3.0Hz,1H),4.01(m,1H),3.83-3.67(m,11H),3.61-3.56(m,52H),2.64(t,J=6.4Hz,2H),2.48(t,J=6.4Hz,2H). 13 C NMR(150MHz,D2O):178.06,153.84,149.84,123.02,118.11,120.42,98.8,97.78,78.05,73.39,72.61,72.17,71.47,7 0.52,70.35,70.01,69.85,69.55,69.38,69.31,69.17,66.97,66.74,66.60,65.82,65.10,60.96,60.86,35.82,23.03. C 51 H 89 NO 29 SNa[M+Na] + The calculated HRMS (ESI) value is 1234.5139, and the experimental value is 1234.5114.

[0266] Compound 51.

[0267]

[0268] Trichloroacetonitrile and DBU were added to a stirred solution of 50 (1 mmol) in anhydrous DCM (10 mL), and the solution was stirred at room temperature for 2 hours. The solvent was removed, and the residue was purified by silica gel rapid column chromatography to give the imino ester product. A stirred solution of (4-hydroxyphenyl)carbamate (1.2 mmol) and 4A molecular sieve (1 g) in anhydrous DCM (10 mL) was cooled to -40 °C, and then BF3(OEt)2 (0.1 mmol) was added dropwise to the solution. A solution of the imino ester donor (1 mmol) in anhydrous DCM was added dropwise to the above mixture, and the mixture was stirred at -40 °C for 1 hour. Subsequently, the reaction mixture was gradually heated to room temperature and stirred for another 1 hour. The solution was quenched by adding triethylamine, then filtered, and extracted with DCM after adding saturated NaHCO3 aqueous solution. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by silica gel rapid column chromatography to give compound 51 (72%). 1 H NMR (600MHz, CDCl3) δ7.40-7.28(m,18H),7.19(d,J=7.9Hz,2H),7.10(d,J=8.1Hz,2H),7.00(d,J=8.1Hz,2H ),5.57-5.55(m,2H),5.12(s,2H),4.91(d,J=10.5Hz,1H),4.80(d,J=10.5Hz,1H),4.69(d,J=10.5Hz,1H),4. 65(d,J=10.5Hz,1H),4.53(d,J=10.5Hz,1H),4.46(d,J=10.5Hz,1H),4.22(dd,J=9.4,3.6Hz,1H),4.07-4.0 4(t,J=9.7Hz,1H),3.93(d,J=9.5Hz,1H),3.83(dd,J=10.9,3.9Hz,1H),3.68(d,J=10.8Hz,1H),2.21(s,3H). 13 C NMR(150MHz, CDCl3):170.44,156.25,154.62,138.27,138.04,137.82,136.51,132.71,129.79,128.50,128.43,128.31,128.27,128.1 0,128.08,127.83,127.80,127.64,127.60,116.63,96.13,77.95,76.66,75.22,74.00,73.34,71.99,71.92,68.55,68.5,66.63,21.09. C 43 H 43 NO 10[M+H] + The calculated HRMS (ESI) value is 718.3016, and the experimental value is 718.3041.

[0269] Compound 52.

[0270]

[0271] Add 0.1 mmol of NaOMe to a stirred solution of 51 (0.6 mmol) in MeOH and stir the resulting solution at room temperature for 1 hour. Neutralize the mixture by IR-120, filter, and concentrate to dryness under vacuum. Then dissolve the deacetylated product in anhydrous DCM (5 mL) and add 0.5 g of 4A molecular sieve. Cool the solution to -40 °C, and then add BF3(OEt)2 (0.05 mmol) dropwise. Add a solution of imine ester donor (0.5 mmol) in anhydrous DCM dropwise to the above mixture and stir at -40 °C for 1 hour. Subsequently, gradually heat the reactants to room temperature and stir for another 1 hour. Quench the solution with triethylamine, filter, add saturated NaHCO3 aqueous solution, and extract with DCM. Dry the organic layer with MgSO4 and evaporate to dryness. Then dissolve the product in MeOH, add NaOMe (0.1 mmol), and stir the resulting solution at room temperature for 2 hours. The mixture was neutralized by IR-120, filtered, and concentrated to dryness under vacuum. The residue was purified by silica gel rapid column chromatography to give compound 52 (69%). 1 H NMR (600MHz, CDCl3) δ7.36-7.25(m,25H),7.22-7.13(m,10H),6.99-6.93(m,4H),5.66(s,1H ),5.17(s,1H),5.06(s,2H),4.86(d,J=10.5Hz,1H),4.79(d,J=10.5Hz,1H),4.73,(s,2H),4 .65(d,J=10.5Hz,1H),4.58-4.57(m,2H),4.55-4.52(m,2H),4.47-4.43(m,3H),4.19-4.14( m,2H),3.99-3.96(m,2H),3.88(dd,J=9.1,3.1Hz,1H),3.84-3.76(m,3H),3.67-3.63(m,3H). 13C NMR(150MHz, CDCl3):156.22,154.70,138.47,138.35,138.18,138.14,138.05,137.90,136.51,132.30, 129.74,129.63,128.46,128.44,128.31,128.28,128.26,128.19,128.06,127.93,127.85,127.83,127.7 4,127.68,127.60,127.54,127.46,127.38,127.32,116.63,101.12,96.95,79.97,79.42,77.21,77.00,7 6.78,75.12,75.02,74.66,74.45,74.33,73.23,73.16,72.44,72.41,72.16,71.69,68.99,68.45,66.57. C 68 H 70 NO 13 [M+H] + The calculated HRMS (ESI) value is 1108.4847, and the experimental value is 1108.4819.

[0272] Compound 53.

[0273]

[0274] 0.3 mmol of 52 was added to a stirred solution in 2.5 mL of anhydrous DCM containing 0.25 g of 4A molecular sieve. The solution was cooled to -40 °C, and then 0.03 mmol of BF3(OEt)2 was added dropwise. A solution of 0.3 mmol of imine ester donor in anhydrous DCM was added dropwise to the mixture and stirred at -40 °C for 1 hour. Subsequently, the reaction mixture was gradually heated to room temperature and stirred for another 1 hour. The solution was quenched by adding triethylamine, then filtered and extracted with DCM after adding saturated NaHCO3 aqueous solution. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by silica gel rapid column chromatography to give the trisaccharide product. The product was then dissolved in MeOH, NaOMe (0.1 equivalent) was added, and the resulting solution was stirred at room temperature for 2 hours. The mixture was neutralized by IR-120, filtered, and concentrated to dryness under vacuum. The compound was then dissolved in MeOH (2 mL), and 10% Pd-C (30 mg) was added. The mixture was stirred vigorously overnight under a H2 atmosphere. The solution was filtered through diatomaceous earth and concentrated to dryness to give compound 53 (60%). 1H NMR(600MHz,D2O)δ7.03(d,J=9.0Hz,2H),6.83(d,J=9.0Hz,2H),5.09(s,1H) ,4.81(s,1H),3.94-3.55(m,16H),3.48(t,J=9.6Hz,1H),3.29-3.27(m,1H). 13 C NMR(150MHz,D2O):151.36,143.37,121.20,120.67,101.59,101.23,96.33,78.46,75.3 4,74.69,73.51,73.11,72.97,72.52,71.53,69.28,69.14,68.90,65.51,63.26,62.91. C 24 H 38 NO 16 [M+H] + The calculated HRMS (ESI) value is 596.2191, and the experimental value is 596.2044.

[0275] Compound 54.

[0276]

[0277] EDC (0.02 mmol), HOBt (0.02 mmol), DMAP (0.02 mmol), trimethylamine (0.04 mmol), and CT (PEG) were added to DMF (0.2 mL) containing compound 53 (0.02 mmol). 12 (0.02 mmol), and the resulting solution was stirred at room temperature under nitrogen for 12 hours. The mixture was concentrated to dryness under vacuum and the crude product was purified by Bio-Gel P-2 gel with H2O as the eluent to give 54 (58%). 1 H NMR(600MHz,D2O):7.03(d,J=9.2Hz,2H),6.82(d,J=9.2Hz,2H),5.09(s,1H),4.81 (s,1H),3.84-3.46(m,65H),3.30-3.26(m,1H),2.65(t,J=6.5Hz,2H),2.52(s,2H). 13C NMR(150MHz,D2O):173.22,151.36,143.37,121.37,120.59,101.20,101.67,97.04,79.17,76.06,75. 40,75.22,74.23,73.69,73.23,72.60,72.45,72.22,71.68,71.21,69.85,69.61,63.97,38.52,26.08. C 51 H 90 NO 29 S[M+H] + The calculated HRMS (ESI) value is 1212.5319, and the experimental value is 1212.5146.

[0278] Preparation of polymer vesicles.

[0279] In this example, polymer vesicles of this disclosure are prepared by encapsulating mRNA to form mRNA-polymer nanoparticles (PNPs) for delivery. The terms "PNP" and "polymer vesicle" are used interchangeably to describe the polymer vesicles of this disclosure. Exemplary PNPs include I2-P1 / P5 mRNA-PNP, I3-P1 / P5 mRNA-PNP, I4-P1 / P5 mRNA-PNP, I5-P1 / P5 mRNA-PNP, I6-P1 / P5 mRNA-PNP, I7-P1 / P5 mRNA-PNP, I8-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA PNP, and I10-P1 / P5 mRNA PNP, illustrated in [illustration missing]. Figure 1B middle.

[0280] WT spike DNA construction.pMRNA XP The mRNA synthesis vector was obtained from SystemBiosciences. The spike DNA sequence of WT (Wuhan / WH01 / 2019 virus strain) with K986P and K987P mutations (2P) was codon-optimized for Homo sapiens. 1 pMRNA XP The vector was digested with EcoRI and BamHI at 37°C for 1 hour. (The result was obtained via KODOne.) TMThe spike protein DNA sequence was amplified using the PCR master mix (TOYOBO Bio-Technology). PCR fragments of the linearized pMRNAXP vector and spike protein DNA were removed using a Wizard SV gel and PCR cleanup system (Promega). The PCR fragments of the spike protein DNA were cloned into the linearized pMRNAXP vector using the In-Fusion HD cloning kit (Clontech Laboratories, Inc.). The cloning mix was then converted to One Shot. TM TOP10 Chemically Formed Escherichia coli (E. coli) (Invitrogen) TM The cultures were incubated overnight at 37°C. Successful constructs were screened using Quick Taq HSDyeMix (Toyobo Biotechnology). Insertion-specific and backbone-specific primers were designed for colony PCR. Single clones were selected for PCR using pipette tips. PCR products were analyzed using agarose gel electrophoresis. Potential candidates were selected and analyzed by DNA sequencing.

[0281] Polymer synthesis. The polymerization process was carried out according to a publicly available procedure, such as that described in Fu et al., Journal of the American Chemical Society, 2015, 137(37), 12153-12160, which is incorporated herein by reference in its entirety. Modifications may be made where applicable. Briefly, stock solutions of monomer (i.e., the extender, 2 M in DMF), initiator (50 mM in DMF, freshly prepared), terminator (iodoacetamide, 0.5 M in H2O, freshly prepared) and triethanolamine (TEOA) buffer (1 M, pH = 7.0) were prepared. The initiator was added to 80 μL of mixed buffer (DMF / TEOA = 1 / 1) and 10 μL of monomer stock solution (using a 1:1 v / v ratio for heteropolymers P1 / P3, P2 / P3, P1 / P4, P2 / P4, P1 / P5, and P2 / P5). See also Figure 1A After stirring at room temperature for 30 minutes, the polymerization reaction was quenched by adding 1.9 mL of a terminator stock solution. The resulting polymer was dialyzed against H₂O on the same day. The solution was lyophilized and the polymer was kept at -20°C. For in vitro and in vivo experiments, the initiator was mixed with 5% IP, followed by the same protocol described above.

[0282] For the preparation of mRNA-PNP, a self-assembly process is used to encapsulate the mRNA in the appropriate polymer; that is, the polymer in the ethanol phase (10 mg / mL) is mixed with an aqueous solution of mRNA at pH 4.0 (1 mg / mL) at a 3:1 N / P ratio. See [link to product]. Figure 1BmRNA-PNP was dialyzed overnight at 4°C against PBS buffer (pH 7.4) using Micro Float-A-Lyzer (10kDa MWCO, Spectrum Labs) and stored at -40°C until further use.

[0283] Characterization of polymer vesicles. To examine the morphology of the mRNA-polymer complex, we first mixed the P1 polymer with mRNA and found that it formed near-spherical nanoparticles. The P1 / P5 copolymer complex with mRNA exhibited liposome-like characteristics. The polymer may form a positively charged layer that can encapsulate mRNA and promote subsequent cellular uptake through zwitterionic bonding.

[0284] Molecular weight and polymerization index were characterized by gel permeation chromatography (GPC). The polymers showed a single peak but a fairly broad molecular weight distribution in the GPC chromatograms and eluted with relatively short elution times, indicating their polymerization state. The peak molecular weight of mRNA-PNP (P1 / P5) was 10.2 kDa (PDI = 1.33).

[0285] Example 2: Encapsulation and transfection efficiency of polymer vesicles

[0286] To identify the optimal polymer for efficient intracellular delivery of GFP-mRNA as a model, homopolymers and heteropolymers were synthesized by copolymerization with different growth agents, and their encapsulation ability and transfection efficiency in HEK293T cells were evaluated.

[0287] Quantification of encapsulated mRNA.

[0288] Through Quant-iT TM RiboGreen TM RNA reagents and kits (Thermo Scientific) TM Encapsulation efficiency was determined using Quant-iT buffer. The prepared mRNA polymeric vesicles (mRNA-PNPs) were treated overnight with 10 mM GSH. Subsequently, the solution was diluted 250-fold with 1×TE buffer and then further diluted 2-fold with TE buffer or TE buffer containing 2% Triton X-100. mRNA was prepared at concentrations of 100, 50, 25, 12.5, and 0 ng / ml in TE or TE buffer containing 1% Triton X-100 to establish a standard curve. After incubation at 37°C for 10 minutes, the encapsulation efficiency was determined. TM RiboGreen TM RNA reagent was added to the wells. (The process was repeated.) Plus (BMG Labtech) measures fluorescence intensity.

[0289] Results: All copolymers containing guanidine groups were able to encapsulate GFP-mRNA. Specifically, the I1-P2 / P3, I1-P2 / P4, and I1-P2 / P5 copolymers with trivalent guanidine moieties exhibited higher mRNA encapsulation capabilities and were comparable to those with the conventional transfection agent polyethyleneimine (PEI). See also Figure 2A .

[0290] WT spike mRNA was prepared to form mRNA-PNP. To obtain WT spike mRNA, according to the manufacturer's protocol, it was prepared at 37°C using TOOLS Ultra high-fidelity DNA polymerase (BIOTOOLS Co., Ltd.) with 1 μL DNA template in mMESSAGE mMACHINE. TM Linear DNA containing the T7 promoter, 50 untranslated regions, 30 untranslated regions, S-2P, and poly(A) tail signal sequences was amplified in the kit (Thermo Scientific) for 1 hour. mRNA was purified using an RNA clearance kit (BioLabs) according to the manufacturer's protocol and stored at -80°C until further use. For mRNA-PNP formulation, mRNA was encapsulated in the appropriate polymer using a self-assembly process; i.e., the polymer (10 mg / mL) in the ethanol phase was mixed with an aqueous solution of mRNA at pH 4.0 (1 mg / mL) at different N / P ratios. mRNA-PNP was dialyzed overnight at 4°C against PBS buffer (pH 7.4) using Micro Float-A-Lyzer (10 kDa MWCO, Sterling), and stored at -40°C until further use.

[0291] Results. The results showed that the P1 / P5 copolymer exhibited excellent ability to capture spike mRNA at N / P ratios of 1, 5, 10, or 20. Figure 3A The average particle size of the resulting mRNA-P1 / P5 copolymer complex was approximately 127 nm, as revealed by CryoEM, TEM, and dynamic light scattering (DLS) analysis. Figure 3B The measured zeta potential was approximately -4.2 mV. An N / P ratio of 3 was chosen for the following experiments to ensure complete encapsulation. mRNA release and translation were attributable to GSH-mediated polymer degradation, as it occurred in a time-dependent manner in the presence of 10 mM GSH (data not shown).

[0292] HEK293T cell transfection.

[0293] Transfection of polymeric vesicles encapsulating GFP-mRNA. Next, we evaluated the transfection efficiency of GFP-mRNA in HEK293T cells using different copolymers. HEK293T cells were cultured in 2.5 mL of DMEM at a rate of 5 × 10⁶ cells / mL. 5 Cells / well were seeded into 6-well plates. 1 μg GFP mRNA or 3 μg WT spike mRNA was prepared with the corresponding polymer according to the above procedure and then added to the cells. GFP and spike expression were monitored by fluorescence microscopy and Western blotting 18 hours post-transfection. For WB, cells containing spike protein were lysed with 200 μL LRIPA rehydration buffer (including protease inhibitors) and incubated for 10 min. Cells were then vortexed, centrifuged, and analyzed by Western blotting using a polyclonal anti-SARS-CoV-2S protein antibody (1:5000, 1% BSA) and an HRP-bound anti-rabbit antibody (1:10000). Spike protein was detected using a chemiluminescent HRP substrate and visualized using a reverse illuminator (FUJIFILM LAS3000).

[0294] Results showed that copolymer P1 exhibited comparable transfection efficiency to the conventional transfection agent polyethyleneimine (PEI) in HEK293T cells, while hybrids P1 / P4, P2 / P4, P1 / P5, and P2 / P5 demonstrated superior transfection efficiency and were able to release mRNA for translation into GFP. Specifically, polymers P1 / P5 and P2 / P5 (containing zwitterionic groups) showed superior transfection efficiency. Figure 2B The results were highly efficient, likely due to membrane fusion as described above. Compared to the conventional transfection agent PEI, all tested polymeric vesicles showed less impact on cell viability, and none exhibited significant cytotoxicity (data not shown).

[0295] Transfection of polymeric vesicles encapsulating WT spike mRNA. Subsequently, we transfected HEK293T cells with the spike mRNA-P1 / P5 complex (3 μg) and performed proteoblotting. Forty-eight hours post-transfection, spike protein translation in cells was analyzed by proteoblotting using a spike-specific antibody. A significant band corresponding to the SARS-CoV-2 spike protein at approximately 250 kDa was observed (second lane) compared to the spike mRNA used as a negative control (lane 1). Figure 3C This study confirms that the P1 / P5 copolymer is an effective nanocarrier for in vitro mRNA transfection.

[0296] Location of mRNA-PNP in cells.

[0297] To observe the location of mRNA-PNP in cells, a FITC-labeled polymer was synthesized from P1 / P4 / P5, where FITC is conjugated to the polymer via an amine group on P4. For example... Figure 4 As shown in the study, the colocalization of lysosomes with mRNA-PNP (I1-P1 / P4 / P5) is lower than that of mRNA-PNP (I1-P1 / P4), indicating that alkylation of zwitterionic residues can significantly improve membrane fusion and lysosomal escape.

[0298] Example 3: Targeted (Selective) Delivery

[0299] To selectively deliver mRNA vaccines to antigen-presenting cells (APCs), particularly dendritic cells, we designed initiators with different glycan heads recognized by lectin receptors (e.g., Siglec-1, Siglec-2, Siglec-5 / E, and DC-SIGN), which are primarily expressed on dendritic cells (DCs) and macrophages. To evaluate mRNA-PNP uptake via Siglecs, we compared the binding and internalization of mRNA-PNP to T cells, B cells, and bone marrow-derived dendritic cells (BMDCs).

[0300] method

[0301] Spleen cell preparation and BMDC culture. To prepare spleen cells, mouse spleens were homogenized with the ground end of a glass slide and treated with RBC lysis buffer (Sigma) to consume red blood cells (RBCs), followed by passing through a cell filter (BD Biosciences). Bone marrow-derived dendritic cells (BMDCs) were prepared as described. 3 In short, bone marrow was isolated from the femur and tibia of mice and treated with RBC lysis buffer (Sigma-Aldrich) to consume RBCs. Cells were then cultured at a density of 2 × 10⁵ cells / mL in RPMI-1640 containing 10% heat-inactivated FBS (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Thermo Fisher Scientific), and 20 ng / mL recombinant mouse GM-CSF (eBioscience). On day 3, cells were replenished with an equal volume of the complete culture medium described above, and on day 6, the medium was replenished with half the volume. On day 8, suspension cells were collected.

[0302] Treatment of spleen cells and BMDCs with PNP. Spleen cells or BMDCs were incubated with 1:2000 mRNA-PNP (diluted to 10 mg / mL) in RPMI-1640 at 37°C for 24 hours. Cells were blocked for 20 minutes with an Fc receptor binding inhibitor (clone: ​​93, Biolegend). Spleen cells were stained with antibodies against CD3 (clone: ​​17A2, BV421 conjugated, Biolegend) and against CD19 (clone: ​​1D3, PECy7 conjugated, Biolegend). BMDCs were stained with an antibody against CD11c (clone N418 APC conjugated, Biolegend). The labeled cells were analyzed using FACSC and flow cytometry (Biolegend).

[0303] C2C12 Cell Culture. The mouse muscle myoblast cell line C2C12 was purchased from Taiwan's Bioresource Collection and Research Center, China. C2C12 cells were cultured in high-glucose DMEM (ATCC) supplemented with 10% FBS and 1× antibiotic-antifungal agent. Cells were incubated at 37°C under controlled humidification with 5% CO2. The medium was changed every 2 to 3 days.

[0304] Treatment of C2C12 cells with polymeric vesicles. Cultured C2C12 myoblasts were isolated from culture dishes using 0.25% trypsin-EDTA (Gibco) and neutralized with growth medium containing 10% FBS. mRNA-PNP (I1-P1 / P4-FITC-P5) or mRNA-PNP (I9-P1 / P4-FITC / P5) was added to 200 μL of C2C12 cells (2 × 10⁶ cells) in growth medium. 5 In 1000 cells, the final dilutions were achieved at 1:1000, 1:2000, 1:4000, or 1:8000 of the original stock solution (10 mg / mL). Measurements were taken at three time points: 5 minutes, 1 hour, and 24 hours.

[0305] Flow cytometry. After incubation with mRNA-PNP, BMDC cells were washed with ice-cold FACS buffer (1×DPBS containing 1% FBS with 0.1% sodium azide) and incubated on ice for 20 min in FACS buffer with purified anti-mouse CD16 / 32 antibody (Baijin), followed by washing with FACS buffer. BMDCs were stained with APC anti-mouse CD11c antibody (BioLengend) at 4°C for 30 min and washed with FACS buffer. Finally, BMDCs were stained with propidium iodide (Sigma-Aldrich). C2C12 cells were centrifuged and washed with FACS buffer. Cells were stained with propidium iodide. Flow cytometry was performed on a FACSCanto flow cytometer (BD Biosciences).

[0306] The binding of glycan-PNP and DC-SIGN was determined by ELISA. To assess the binding of DC-SIGN to mannoside-modified PNPs, ELISA plates were plated overnight in PBS at 4°C with mRNA-PNP (I5-P1 / P5), mRNA-PNP (I6-P1 / P5), mRNA-PNP (I7-P1 / P5), mRNA-PNP (I8-P1 / P5), mRNA-PNP (I9-P1 / P5), or mRNA-PNP (I10-P1 / P5) (10 mg / mL), respectively. The plates were then incubated with diluted DC-SIGN ECD (15 to 0.075 nM in HEPES buffer containing 20 mM HEPES, 150 mM NaCl, 10 mM CaCl2, and 0.1% BSA) at room temperature for 1 hour at pH 7.4, 6.0, and 5.0. The binding of DC-SIGN ECD was detected using an HRP-conjugated anti-DC-SIGN(B2) IgG antibody (Santa Cruz Biotechnology). After incubation at room temperature for 1 hour, the plate was treated with tetramethlybenzidine (TMB) for 10 minutes. Optical density was measured at 450 nm after adding 0.5 M sulfuric acid to the plate using a microplate reader. Apparent Kd was calculated using a nonlinear regression curve fitted to the total binding using a GraphPad Prism.

[0307] The binding of glycan-PNPs of DC-SIGN-Fc, MMR-Fc, MINCLE-Fc, lectin-2-Fc, and Langerhansin-Fc was measured by ELISA. To assess the binding of receptor proteins to mannoside-modified PNPs, ELISA plates were plated overnight in PBS at 4°C with mRNA-PNP (I1-P1 / P5), mRNA-PNP (I8-P1 / P5), mRNA-PNP (I9-P1 / P5), or mRNA-PNP (I10-P1 / P5) (10 mg / mL), respectively. The plates were then incubated with diluted DC-SIGN-Fc, MMR-Fc, MINCLE-Fc, lectin-2-Fc, and Langerhansin-Fc (0.625 μg / mL in buffer) at pH 7.4 and room temperature for 1 hour. The bound proteins were detected using HRP-conjugated anti-Fc IgG antibodies. After incubation at room temperature for 1 hour, the plate was treated with tetramethylbenzidine (TMB) for 10 minutes. After adding 0.5M sulfuric acid to the plate using a microplate reader, the average optical density was measured at 450 nm.

[0308] result

[0309] FITC-conjugated mRNA-PNPs were incubated with each cell line for 1 hour using flow cytometry. Compared to I1 mRNA-PNPs without glycan modification, I2 mRNA-PNPs with 9BPCNeu5Ac conjugated N-glycans, intended to target Siglec-2, showed higher cellular uptake across all APCs. Figure 5 Specifically, in this experiment, I2 polymeric vesicles showed increased uptake of BMDCs, B cells, and T cells by 33.1%, 32.6%, and 27.8%, respectively, compared to I1 polymeric vesicles. Similar results were obtained by targeting Siglec-5 / E and Siglec-1 with I3 and I4 mRNA-PNPs, respectively, where all glycan-decorated polymeric vesicles showed better uptake of all APCs than those without glycans. Similarly, I9-P1 / P4-FITC / P5 mRNA-PNP showed approximately 1-fold higher uptake compared to I1-P1 / P4-FITC-P5 mRNA-PNP. Figure 9 C2C12 muscle cell uptake.

[0310] like Figure 6As shown, compared to I1 polymeric vesicles without glycan heads (I1-P1 / P4-FITC / P5), I5 polymeric vesicles with arylmannose heads (I5-P1 / P4-FITC / P5) exhibited approximately 34% higher cellular uptake of BMDCs (an increase of 33.8%). On the other hand, when treated with polymeric vesicles, B cells and T cells with insignificant DC-SIGN expression showed only a slight increase in fluorescence signal (an increase of 16.2% and 12.8%, respectively). This data demonstrates that efficient internalization and selective uptake of mRNA-PNPs (I5-P1 / P4-FITC / P5) by dendritic cells can be achieved through DC-SIGN receptor targeting.

[0311] Evaluation of DC-SIGN binding to mRNA-polymer vesicles generated from I5-P1 / P5, I6-P1 / P5, I7-P1 / P5, I8-P1 / P5, I9-P1 / P5, and I10-P1 / P5 at different pH values ​​showed that those polymer vesicles could bind at low K+ levels. D Binding to DC-SIGN (Table 1). mRNA-polymer vesicles from I5-P1 / P5, I8-P1 / P5, I9-P1 / P5, and I10-P1 / P5 bound to the extracellular domain (ECD) of DC-SIGN with nearly identical affinity at pH 7.4 and 5.0. In contrast, binding to mRNA-polymer vesicles from I6-P1 / P5 and I7-P1 / P5 was not detected at lower pH values, suggesting reduced coordination with calcium ions at low pH.

[0312] Table 1

[0313]

[0314] Without being bound by theory, these binding results indicate that aryl-trimannoside interacts with DC-SIGN in the acidic endosome compartment. This type of binding stability enhances DC-SIGN-mediated signaling and its synergistic effect with endosome-like receptors (e.g., TLR7) residing in the endosome. The strong binding of aryl-mannoside-containing PNPs to DC-SIGN can be attributed to the dense display of ligands, and the aryl group can participate in CH-π and hydrophobic interactions. Furthermore, for receptor-targeted delivery, the ligand is typically linked to the vector, and the distance between the vector and ligand is modulated by the presence of a spacer. mRNA-PNPs carrying longer ligands (I8-P1 / P5 with Man-Ar-PEG12 and I7-P1 / P5 with Man-PEG12) exhibit slightly higher affinity for DC-SIGN. Overall, mRNA-PNPs with aryl-trimannoside (I9-P1 / P5) exhibit the highest affinity and lowest KD for DC-SIGN. The increased affinity can be attributed to the clustering effect and spatial arrangement of ligands in the polymer, and the aryl moiety can promote their hydrophobic interactions.

[0315] Further binding affinity assays were performed on DC-SIGN, macrophage mannose receptor (MMR), MINCLE, lectin-2, and Langerhansin to investigate whether the uptake of mannosylated mRNA-PNP depends on DC-SIGN. Figure 7 As shown, compared to I1, all I8, I9, and I10 polymeric vesicles exhibit selective binding. Among them, the branched aryl-trimannoside I9 shows a greater preference for DC-SIGN compared to the linear aryl-trimannoside I10. The fact that I9 exhibits more selective binding to DC-SIGN supports the conclusion that efficient uptake of mRNA-I9-P1 / P5 by DCs is more likely mediated by DC-SIGN.

[0316] Example 4: Immunization using polymer vesicles of this disclosure

[0317] Subsequently, we evaluated the effects of WT spike mRNA-PNPs with or without aryl-mannoside heads on vaccination and immune response.

[0318] Methods and immunization design.

[0319] Animals. Balb / c mice (8 weeks old) were purchased from the Laboratory Animal Center, Taiwan, China. All mice were maintained in a specific pathogen-free environment. Eight-week-old Balb / c mice were immunized twice with an intramuscular injection (IM) two weeks apart. Each immunization contained 100 μl of PBS. Serum collected from immunized mice was analyzed by ELISA 10 days after the last immunization. The experimental protocol was approved by the Animal Care and Utilization Committee, Taiwan, China (Approval No. 22-08-1901).

[0320] Animal immunization: BALB / c mice (n=5) aged 6 to 8 weeks were intramuscularly immunized with phosphate-buffered saline (PBS) containing 15 μg mRNA-PNP. Animals were immunized at week 0 and given a booster vaccination at week 2. Figure 8A Serum samples were collected from each mouse one week after the second immunization. Positive controls included groups treated with spike mRNA-LNPs (including ALC-0315, DSPC, ALC-0159, and cholesterol, commonly used in current mRNA vaccine formulations).

[0321] Measurement of serum IgG titer. The IgG titer of mouse serum was determined using ELISA. Each well of a 96-well ELISA plate (Greiner Bio-One) was coated overnight at 4°C with 100 ng of SARS-CoV-2 spike protein (ACROBiosystems) in 100 mM sodium bicarbonate at pH 8.8. Each well was then blocked at 37°C for 1 hour with 200 μl of 1×PBS containing 5% skim milk and washed three times with 200 μl of PBST (1×PBS, 0.05% Tween 20, pH 7.4). Serially diluted mouse serum samples (2-fold) were added to each well and incubated at 37°C for 2 hours, followed by six washes with 200 μl of PBST. At 37°C, each well was incubated with 100 μl of HRP-conjugated anti-mouse secondary antibody (1:10000, in PBS) for 1 hour and washed six times with 200 μl of PBST. 100 μl of horseradish peroxidase substrate (1-Step) was then added. TM Ultra TMB-ELISA substrate solution (Thermo Scientific) TMAdd 100 μl of 1M H2SO4 to each well. After incubation for 30 minutes, measure the absorbance (OD 450 nm) using a SpectraMax M5.

[0322] Pseudovirus Neutralization Assay. The pseudoviruses were constructed using a procedure similar to that previously described by the RNAi Core Facility in Taiwan. Briefly, pseudolentiviruses carrying the SARS-CoV-2 spike protein were generated by transiently transfecting HEK-293T cells with pCMV-ΔR8.91 and pLAS2w.Fluc.Ppuro. HEK-293T cells were seeded one day prior to transfection, and the specified plasmids were delivered into the cells using the TransITR-LT1 transfection reagent (Mirus). The culture medium was refreshed 16 hours post-transfection, and cells were collected at 48 and 72 hours. Cell debris was removed by centrifugation at 4,000 × g for 10 min, and the supernatant was passed through a 0.45 μm syringe filter (Pall Corporation). The pseudolentiviruses were aliquoted and subsequently stored at -80°C. To estimate lentiviral titers using the AlarmaBlue assay (Thermo Fisher Scientific), transduction units (TUs) of SARS-CoV-2 pseudolentivirus were estimated by reacting the limited dilutions of lentivirus with a cell viability assay. Briefly, HEK-293T cells stably expressing the human ACE2 gene were seeded into 96-well plates one day prior to lentiviral transduction. To titrate the pseudolentivirus, varying amounts of lentivirus were added to medium containing polybrene (final concentration 8 μg / ml). Spin infection was performed at 1,100 × g for 30 min in 96-well plates at 37°C. After incubation at 37°C for 16 h, the medium containing the virus and polybrene was removed and replaced with fresh, complete DMEM containing 2.5 μg / ml puromycin. After 48 h of puromycin treatment, the medium was removed, and cell viability was assessed using 10% AlarmaBlue reagent according to the manufacturer's instructions. The viability of uninfected cells (without puromycin treatment) was set at 100%. Viral titers (transduction units) were determined by plotting the relationship between surviving cells and diluted viral doses. For the neutralization assay, heat-inactivated serum or antibody was serially diluted and incubated in DMEM at 37°C for 1 hour with 1,000 TU of SARS-CoV-2 pseudotyped lentivirus. The mixture was then seeded in 96-well plates with 10,000 stable HEK-293T cells expressing the human ACE2 gene. Sixteen hours post-infection, the medium was replaced with fresh, complete DMEM supplemented with 10% FBS and 100 U / mL penicillin / streptomycin and cultured for another 48 hours. Luciferase gene expression levels were determined using the Bright-Glo luciferase assay system (Promega). Relative light units (RLU) were detected using the Tecan i-control (Infinite500). The formula (RLU) was used. 对照 -RLU 血清The RLU / RLU control group calculated the percentage inhibition as the ratio of RLU reduction in the presence of diluted serum to the RLU value in the serum-free control group.

[0323] result

[0324] The results showed that both I1-P1 / P5 polymer vesicle treatment and mRNA-LNP treatment could produce anti-spike antibody levels of 10,000 in serum on day 28. In contrast, I9-P1 / P5 polymer vesicle treatment produced approximately 30,000 anti-spike antibody levels, which was at least 3 times higher. Figure 8B The ability of serum to neutralize pseudovirus-mediated entry into ACE2-expressing cells was then tested. Antiserum levels from the I1-P1 / P5 polymeric vesicles were similar to those in the mRNA-LNP group; both were below 2000. In contrast, significantly higher levels (over 6000) of neutralizing antibodies were observed in the I9-P1 / P5 polymeric vesicle group. Figure 8C Furthermore, spike-specific antibody titers and pseudovirus neutralizing activity were well correlated.

[0325] Exemplary Examples

[0326] Example 1. A copolymer for forming polymer vesicles, wherein the copolymer comprises: an initiator block comprising a glycan head; an elongator block comprising a functional portion comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof; and a linker covalently connecting the initiator block and the elongator block, wherein the linker comprises a disulfide bond.

[0327] Example 2. A copolymer as in Example 1, wherein the polysaccharide head comprises a terminal mannoside.

[0328] Example 3. A copolymer as in Example 1 or Example 2, wherein the polysaccharide head comprises O-arylmannoside, wherein the O-arylmannoside comprises an optionally substituted benzene ring.

[0329] Example 4. A copolymer of any one of Examples 1 to 3, wherein the polysaccharide head comprises monomannoside, dimannoside, or trimannoside.

[0330] Example 5. A copolymer as in Example 4, wherein the trimannoside is a linear or branched trimannoside.

[0331] Example 6. A copolymer as in Example 5, wherein the branched trimannoside is α-1,3-α-1,6-trimannoside.

[0332] Example 7. A copolymer as described in any one of Examples 1 to 6, wherein the initiator block further comprises an initiator spacer group.

[0333] Example 8. A copolymer as in Example 7, wherein the initiator spacer group comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0334] Example 9. A copolymer as in Example 8, wherein the saturated carbon moiety comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons (optionally, 2 to 6 carbons).

[0335] Example 10. A copolymer as in Example 8 or Example 9, wherein the PEG portion comprises 2 to 72 (OCH2CH2) subunits.

[0336] Example 11. A copolymer as in Example 10, wherein the PEG portion has a linear, branched, or star-shaped configuration.

[0337] Example 12. A copolymer of any one of Examples 1 to 11, wherein the polysaccharide head is configured to bind dendritic cells.

[0338] Example 13. A copolymer as in Example 12, wherein the polysaccharide head is configured to selectively bind DC-SIGN.

[0339] Example 14. A copolymer as in Example 13, wherein the polysaccharide head is configured with K in the range of 5 to 8000 nM at pH 7.4. D Combined with DC-SIGN.

[0340] Example 15. The copolymer as in Example 14, wherein the K is obtained at pH 7.4. D In the range of 5 to 500 nM.

[0341] Example 16. A copolymer as described in any one of Examples 13 to 15, wherein the polysaccharide head is configured to have a K+ content in the range of 1 to 2000 nM at pH 5. D Combined with DC-SIGN.

[0342] Example 17. The copolymer as in Example 16, wherein the K at pH 5 D In the range of 1 to 600 nM.

[0343] Example 18. A copolymer as in Example 1, wherein the polysaccharide head comprises 9 BPC Neu5Ac-conjugated N-glycans, Neu5Ac-conjugated N-glycans, 9 TCC Neu5Ac conjugated N-glycans or combinations thereof.

[0344] Example 19. A copolymer as in Example 18, wherein the initiator block is configured to combine Siglec-2, Siglec-5 / E, Siglec-1 or a combination thereof.

[0345] Example 20. A copolymer of any one of Examples 1 to 19, wherein the initiator block is selected from the group consisting of:

[0346]

[0347] Solid circles represent mannosides, hollow circles represent galactose, solid squares represent GlcNAc, and rhombuses represent Neu5Ac.

[0348] Example 21. A copolymer of any one of Examples 1 to 20, wherein the grower block comprises more than one guanidine group.

[0349] Example 22. A copolymer as in Example 21, wherein the grower block comprises three guanidine groups.

[0350] Example 23. A copolymer of any one of Examples 1 to 22, wherein the growth agent block comprises a growth agent spacer group, the growth agent spacer group comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0351] Example 24. A copolymer as in Example 23, wherein the saturated carbon moiety comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons (optionally, 2 to 6 carbons).

[0352] Example 25. A copolymer as in Example 23 or Example 24, wherein the PEG portion comprises 2 to 72 (OCH2CH2) subunits.

[0353] Example 26. A copolymer as in Example 25, wherein the PEG portion has a linear, branched, or star-shaped configuration.

[0354] Example 27. A copolymer of any one of Examples 1 to 26, comprising a plurality of growth block and a plurality of links, wherein each of the plurality of growth block is connected to at least one other growth block or the initiator block via one of the plurality of links.

[0355] Example 28. A copolymer of any one of Examples 1 to 27, wherein the growth block is a first growth block, the linking is a first linking, and the copolymer further comprises a second growth block connected to the first growth block via a second linking; wherein the first growth block and the second growth block independently comprise a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the second linking comprises a disulfide bond.

[0356] Example 29. A copolymer as in Example 28, wherein the first growth agent block comprises the guanidine group, and the second growth agent block comprises the zwitterionic group.

[0357] Example 30. A copolymer as in Example 28, wherein the first growth agent block comprises the guanidine group and the second growth agent block comprises the diethylenetriamine.

[0358] Example 31. A copolymer of any one of Examples 28 to 30, wherein the copolymer further comprises a third growth block, wherein the third growth block is connected to the first growth block or the second growth block via a third bond comprising a disulfide bond.

[0359] Example 32. A copolymer of any one of Examples 1 to 31, wherein the growth agent block is selected from the group consisting of:

[0360]

[0361] Example 33. A copolymer as in Example 32, comprising at least two growth block, wherein the at least two growth block is (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, (4) PB2 and PB5, or (5) P1, P4 and P5.

[0362] Example 34. A copolymer as in Example 33, wherein the initiator block and the at least two propagator blocks are selected from the group consisting of: IB5-PB1 / PB5, IB5-PB1 / PB4, IB5-PB2 / PB5, IB5-PB2 / PB4, IB5-PB1 / PB4 / PB5, IB6-PB1 / PB5, IB6-PB1 / PB4, IB6-PB2 / PB5, IB6-PB2 / PB4, IB6-PB1 / PB4 / PB5, IB7-PB1 / PB5, IB7-PB1 / PB4, IB7-PB2 / PB5, IB7-PB 2 / PB4, IB7-PB1 / PB4 / PB5, IB8-PB1 / PB5, IB8-PB1 / PB4, IB8-PB2 / PB5, IB8-PB2 / PB4, IB8-PB1 / PB4 / PB5, IB9-PB1 / PB5, IB9-PB1 / PB4, IB9-PB2 / PB5, IB9-PB2 / PB4, IB9-PB1 / PB4 / PB5, IB10-PB1 / PB5, IB10-PB1 / PB4, IB10-PB2 / PB5, IB10-PB2 / PB4 and IB10-PB1 / PB4 / PB5.

[0363] Example 35. A polymer vesicle comprising a membrane defining an internal space, wherein the membrane comprises a copolymer as described in any one of Examples 1 to 34.

[0364] Example 36. A polymer vesicle as described in Example 35, wherein the copolymer comprises at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0365] Example 37. Polymer vesicles as in Example 35 or Example 36, wherein the membrane encapsulates the payload therein.

[0366] Example 38: Polymer vesicles as in Example 37, wherein the effective load is nucleic acid, compound, polypeptide, protein, polysaccharide or combination thereof.

[0367] Example 39. Polymer vesicles as in Example 38, wherein the nucleic acid is RNA or DNA.

[0368] Example 40. A polymer vesicle as described in Example 39, wherein the payload encodes a polypeptide.

[0369] Example 41. A polymer vesicle as described in any of Examples 37 to 40, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0370] Example 42. A polymer vesicle as described in any one of Examples 35 to 41, wherein the copolymer is a first copolymer, and the membrane further comprises a second copolymer, wherein the first copolymer and the second copolymer are independent of any one of Examples 1 to 34.

[0371] Example 43. A polymer vesicle as described in any of Examples 35 to 42, having a diameter of 0.001 to 5 micrometers or 0.01 to 5 micrometers.

[0372] Example 44. A polymer vesicle as described in any of Examples 35 to 43, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load.

[0373] Example 45. A polymer vesicle as described in Example 44, wherein the second effective load is a nucleic acid, compound, polypeptide, protein, polysaccharide, or a combination thereof.

[0374] Example 46. A polymer vesicle as in Example 45, wherein the first effective load and the second effective load are different.

[0375] Example 47. A formulation comprising polymeric vesicles as described in any one of Examples 35 to 46.

[0376] Example 48. A formulation as in Example 47, comprising 0.01 to 95% (w / w) of the polymer vesicles.

[0377] Example 49. A formulation as in Example 47 or Example 48, wherein the polymer vesicle is a first polymer vesicle, and the composition further comprises a second polymer vesicle.

[0378] Example 50. A formulation as in Example 49, wherein the first polymer vesicle and the second polymer vesicle differ in size, the copolymer in which they form a film, the effective load encapsulated within the polymer vesicle, or a combination thereof.

[0379] Example 51. A formulation of any one of Examples 47 to 50, further comprising a pharmaceutically acceptable excipient, adjuvant, or combination thereof.

[0380] Example 52. A formulation as in Example 51, wherein the excipient comprises a solvent, a dispersion medium, a diluent, a dispersion, a suspending agent, a surfactant, an isotonic agent, a thickener or emulsifier, a preservative, a polymer, a peptide, a protein, a cell, hyaluronidase, or a mixture thereof.

[0381] Example 53. A formulation as described in Example 51 or Example 52, wherein the adjuvant comprises C34, glucose-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants that can be used in some vaccines in the compositions of this disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03, MF59, and CpG 1018 or combinations thereof.

[0382] Example 54. A kit for preparing polymer vesicles, comprising: a first reagent comprising an initiator, wherein the initiator comprises a glycan head and an initiator linker portion; and a second reagent comprising an elongator, wherein the elongator comprises a functional portion and an elongator linker portion, wherein the functional portion comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the initiator linker portion is configured to be coupled to the elongator linker portion via a bond comprising a disulfide bond.

[0383] Example 55. A kit as in Example 54, wherein the glycan head comprises a terminal mannoside.

[0384] Example 56. A kit as described in Example 54 or Example 55, wherein the polysaccharide head comprises O-arylmannoside.

[0385] Example 57. A kit as described in any of Examples 54 to 56, wherein the polysaccharide head comprises monomannoside, dimannoside, or trimannoside.

[0386] Example 58. A kit as described in Example 57, wherein the trimannoside is a linear or branched trimannoside.

[0387] Example 59. The kit as in Example 58, wherein the branched trimannoside is α-1,3-α-1,6-trimannoside.

[0388] Example 60. A kit as described in any one of Examples 54 to 59, wherein the initiator further comprises an initiator spacer group comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0389] Example 61. A kit as described in Example 60, wherein the saturated carbon portion comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons (optionally, 2 to 6 carbons).

[0390] Example 62. A kit as described in Example 61, wherein the PEG portion comprises 2 to 72 (OCH2CH2) subunits.

[0391] Example 63. A kit as in Example 62, wherein the PEG portion has a linear, branched, or star-shaped configuration.

[0392] Example 64. A kit as described in any one of Examples 54 to 63, wherein the glycan head is configured to bind dendritic cells.

[0393] Example 65. A kit as in Example 64, wherein the glycan head is configured to selectively bind DC-SIGN.

[0394] Example 66. A kit as described in Example 65, wherein the glycan head is configured to have a K+ concentration in the range of 5 to 8000 nM at pH 7.4. D Combined with DC-SIGN.

[0395] Example 67. The kit as described in Example 66, wherein the K is prepared at pH 7.4. D In the range of 5 to 500 nM.

[0396] Example 68. A kit as described in any one of Examples 65 to 67, wherein the glycan head is configured to have a K+ concentration in the range of 1 to 800 nM at pH 5. D Combined with DC-SIGN.

[0397] Example 69. The kit as described in Example 68, wherein the K at pH 5 D In the range of 1 to 600 nM.

[0398] Example 70. A kit as described in any one of Examples 54 to 69, wherein the glycan head comprises 9 BPC Neu5Ac-conjugated N-glycans (I2), Neu5Ac-conjugated N-glycans (I3), 9 TCC Neu5Ac conjugated N-glycans (I4) or combinations thereof.

[0399] Example 71. A kit as described in Example 70, wherein the glycan head is configured to bind Siglec-2, Siglec-5 / E, Siglec-1, or a combination thereof.

[0400] Example 72. A kit as described in any of Examples 54 to 71, wherein the initiator linking portion is a thiol group or a dithiohexacyclopentyl group.

[0401] Example 73. A kit as described in any one of Examples 54 to 72, wherein the initiator is selected from the group consisting of:

[0402]

[0403]

[0404] Example 74. A kit as described in any of Examples 54 to 73, wherein the growth agent comprises more than one guanidine group.

[0405] Example 75. A kit as in Example 74, wherein the growth agent comprises three guanidine groups.

[0406] Example 76. A kit as described in any of Examples 54 to 75, wherein the growth agent further comprises a growth agent spacer group comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0407] Example 77. A kit as in Example 76, wherein the saturated carbon portion comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons (optionally, 2 to 6 carbons).

[0408] Example 78. A kit as described in Example 76 or Example 77, wherein the PEG portion comprises 2 to 72 (OCH2CH2) subunits.

[0409] Example 79. The kit as in Example 78, wherein the PEG portion is linear PEG.

[0410] Example 80. A kit as described in any one of Examples 54 to 79, wherein the growth agent of the second reagent is a first growth agent, and wherein the second reagent further comprises a second growth agent, or the kit further comprises a third reagent comprising the second growth agent; and wherein the first growth agent and the second growth agent independently comprise a functional portion comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof.

[0411] Example 81. A kit as described in Example 80, wherein the first growth agent comprises the guanidine group, and the second growth agent comprises the zwitterionic group.

[0412] Example 82. The kit of Example 80, wherein the first growth agent comprises the guanidine group and the second growth agent comprises the diethylenetriamine.

[0413] Example 83. A kit as described in any of Examples 54 to 82, wherein the growth agent linking portion is a thiol group or a dithiohexacyclopentyl group.

[0414] Example 84. A kit as described in any one of Examples 54 to 83, wherein the growth agent is selected from the group consisting of:

[0415]

[0416] Example 85. A kit as described in any one of Examples 54 to 84, wherein the first reagent and the second reagent are contained in the same container.

[0417] Example 86. A kit as described in any of Examples 54 to 84, wherein the first reagent and the second reagent are contained in separate containers.

[0418] Example 87. A kit as described in any of Examples 54 to 86, further comprising a payload, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a polysaccharide, or a combination thereof.

[0419] Example 88. A kit as described in Example 87, wherein the nucleic acid is RNA or DNA.

[0420] Example 89. A kit as described in Example 88, wherein the payload encodes a polypeptide.

[0421] Example 90. A kit as described in any of Examples 87 to 89, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0422] Example 91. A method for targeted delivery of a payload to an individual, comprising administering to the individual an effective amount of a pharmaceutical formulation comprising polymeric vesicles, wherein the polymeric vesicles comprise a membrane encapsulating the payload, and wherein the membrane comprises a copolymer as described in any one of Examples 1 to 34.

[0423] Example 92. The method of Example 91, wherein the copolymer comprises at least 50%, 70%, 80%, 90%, 95%, or 99% of the film.

[0424] Example 93. The method of Example 92, wherein the copolymer is a first copolymer, and the film further comprises a second copolymer, wherein the first copolymer and the second copolymer are independent of any one of Examples 1 to 34.

[0425] Example 94. The method of any one of Examples 91 to 93, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a polysaccharide, or a combination thereof.

[0426] Example 95. The method of Example 94, wherein the nucleic acid is RNA or DNA.

[0427] Example 96. The method of Example 95, wherein the payload encodes a polypeptide.

[0428] Example 97. The method of any one of Examples 91 to 96, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0429] Example 98. The method of any one of Examples 91 to 97, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load.

[0430] Example 99. The method of Example 98, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0431] Example 100. The method of Example 99, wherein the first payload and the second payload are different.

[0432] Example 101. A method for preventing or treating a disease in an individual, comprising administering to the individual an effective amount of a pharmaceutical preparation comprising polymer vesicles, wherein the polymer vesicles comprise a membrane, wherein the membrane comprises a polymer component as described in any one of Examples 1 to 34; and an effective load encapsulated within the membrane; and wherein the effective load is a therapeutic agent or a derivative therapeutic agent.

[0433] Example 102. The method of Example 101, wherein the polymer component comprises at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0434] Example 103. The method of Example 101 or Example 102, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a polysaccharide, or a combination thereof.

[0435] Example 104. The method of Example 103, wherein the nucleic acid is RNA or DNA.

[0436] Example 105. The method of Example 104, wherein the payload encodes a polypeptide.

[0437] Example 106. The method of any one of Examples 101 to 105, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load.

[0438] Example 107. The method of Example 106, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0439] Example 108. The method of Example 107, wherein the first payload and the second payload are different.

[0440] Example 109. The method of any one of Examples 101 to 108, wherein the polymer vesicles are administered at an initial dose, followed by one, two, three, four, five or more booster doses.

[0441] Example 110. The method of Example 109, wherein the booster dose is administered approximately one month, two months, three months, four months, five months, or six months or longer after the initial dose.

[0442] Example 111. The method of any one of Examples 101 to 110, wherein the effective amount is in the range of about 5 μg to 1000 μg.

[0443] Example 112. A method for enhancing an adaptive immune response, comprising administering to an individual an effective amount of a pharmaceutical formulation comprising polymeric vesicles; wherein the polymeric vesicles comprise a membrane encapsulating an effective load, and wherein the membrane comprises a copolymer as described in any one of Examples 1 to 34; wherein the effective load is an immunogenic or derived immunogenic biomolecule.

[0444] Example 113. The method of Example 112, wherein the polymer component comprises at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0445] Example 114. The method of Example 83 or Example 84, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a polysaccharide, or a combination thereof.

[0446] Example 115. The method of Example 85, wherein the nucleic acid is RNA or DNA.

[0447] Example 116. The method of any one of Examples 112 to 115, wherein the biomolecule is a polypeptide or a protein.

[0448] Example 117. The method of any one of Examples 112 to 116, wherein the effective load is a first effective load, and the membrane further encapsulates a second effective load.

[0449] Example 118. The method of Example 117, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a polysaccharide, or a combination thereof.

[0450] Example 119. The method of Example 118, wherein the first payload and the second payload are different.

[0451] Example 120. The method of any one of Examples 112 to 119, wherein the polymer vesicles are administered at an initial dose, followed by one, two, three, four, five or more booster doses.

[0452] Example 121. The method of Example 120, wherein the booster dose is administered approximately one month, two months, three months, four months, five months, or six months or longer after the initial dose.

[0453] Example 122. The method of any one of Examples 112 to 121, wherein the effective amount is in the range of about 5 μg to 1000 μg.

Claims

1. A copolymer for use in forming a polymeric vesicle, wherein the copolymer comprises: an initiator block comprising a glycan head; a propagator block comprising a functional moiety comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof; and a linkage covalently connecting the initiator block and the propagator block, wherein the linkage comprises a disulfide bond.

2. The copolymer of claim 1, wherein the glycan head comprises a terminal mannose.

3. The copolymer of claim 1, wherein the glycan head comprises an O-aryl mannose comprising an optionally substituted phenyl ring.

4. The copolymer of claim 1, wherein the glycan head comprises a mono-mannose, a di-mannose, or a tri-mannose.

5. The copolymer of claim 4, wherein the tri-mannose is an alpha-1,3-alpha-1,6- tri-mannose.

6. The copolymer of claim 1, wherein the initiator block further comprises an initiator spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

7. The copolymer of claim 1, wherein the glycan head is configured to bind to a dendritic cell.

8. The copolymer of claim 7, wherein the glycan head is configured to selectively bind to DC-SIGN.

9. The copolymer of claim 8, wherein the glycan head is configured to have a Kd in the range of 5 to 8000 nM at pH 7.4 D binds to DC-SIGN.

10. The copolymer of claim 1, wherein the glycan head comprises 9 BPC Neu5Ac-conjugated N-glycans, Neu5Ac-conjugated N-glycans, 9 TCC Neu5Ac-conjugated N-glycans, or a combination thereof.

11. The copolymer of claim 1, wherein the initiator block is selected from the group consisting of: wherein a solid circle represents a mannose, an open circle represents a galactose, a solid square represents a GlcNAc, and a diamond represents a Neu5Ac.

12. The copolymer of claim 1, wherein the propagator block comprises more than one guanidine group.

13. The copolymer of claim 12, wherein the propagator block comprises three guanidine groups.

14. The copolymer of claim 1, wherein the propagator block comprises a propagator spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

15. The copolymer of claim 1, wherein the propagator block is a first propagator block, the linkage is a first linkage, and the copolymer further comprises a second propagator block connected to the first propagator block via a second linkage; wherein the first propagator block and the second propagator block independently comprise a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof; and wherein the second linkage comprises a disulfide bond.

16. The copolymer of claim 15, wherein the first propagator block comprises the guanidine group and the second propagator block comprises the zwitterionic group; or wherein the first propagator block comprises the guanidine group and the second propagator block comprises the diethylenetriamine.

17. The copolymer of claim 1, wherein the propagator block is selected from the group consisting of:

18. The copolymer of claim 17, comprising at least two extender blocks, wherein the at least two extender blocks are (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, (4) PB2 and PB5, or (5) P1, P4, and P5.

19. The copolymer of claim 18, wherein the initiator block and the at least two extender blocks are selected from the group consisting of: IB5-PB1 / PB5, IB5-PB1 / PB4, IB5-PB2 / PB5, IB5-PB2 / PB4, IB5-PB1 / PB4 / PB5, IB6-PB1 / PB5, IB6-PB1 / PB4, IB6-PB2 / PB5, IB6-PB2 / PB4, IB6-PB1 / PB4 / PB5, IB7-PB1 / PB5, IB7-PB1 / PB4, IB7-PB2 / PB5, IB7-PB2 / PB4, IB7-PB1 / PB4 / PB5, IB8-PB1 / PB5, IB8-PB1 / PB4, IB8-PB2 / PB5, IB8-PB2 / PB4, IB8-PB1 / PB4 / PB5, IB9-PB1 / PB5, IB9-PB1 / PB4, IB9-PB2 / PB5, IB9-PB2 / PB4, IB9-PB1 / PB4 / PB5, IB10-PB1 / PB5, IB10-PB1 / PB4, IB10-PB2 / PB5, IB10-PB2 / PB4, and IB10-PB1 / PB4 / PB5.

20. A polymersome comprising a membrane defining an internal space, wherein the membrane comprises the copolymer of any one of claims 1-19.

21. The polymersome of claim 20, wherein the membrane encapsulates a payload within.

22. The polymersome of claim 21, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

23. The polymersome of claim 22, wherein the nucleic acid is RNA or DNA.

24. The polymersome of claim 21, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

25. The polymersome of claim 20, wherein the copolymer is a first copolymer, and the membrane further comprises a second copolymer, wherein the first copolymer and the second copolymer are independent of claim 1.

26. The polymersome of claim 21, wherein the payload is a first payload, and the membrane further encapsulates a second payload.

27. A formulation comprising the polymersome of claim 20.

28. The formulation of claim 27, comprising 0.01 to 95% (w / w) of the polymersome.

29. The formulation of claim 27, wherein the polymersome is a first polymersome, and the composition further comprises a second polymersome, wherein the first polymersome and the second polymersome differ in size, the copolymer from which it forms a membrane, the payload encapsulated within the polymersome, or a combination thereof.

30. The formulation of claim 27, further comprising a pharmaceutically acceptable excipient, adjuvant, or a combination thereof.

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