VLP functionalized hydrogel

Abstract

A method of preparing a hybrid virus hydrogel, is provided, the method comprising contacting a virus-like particle (VLP) or derivative thereof comprising an alkyne moiety with a swelled hydrogel comprising an azide group. Hydrogel compositions as well as methods of using them are further provided herein. Further provided herein are compositions as well as methods of use for hybrid virus hydrogels comprising additional therapeutic drug agents.

Description

VLP FUNCTIONALIZED HYDROGELCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No.: 63 / 532,868, filed August 15, 2023, the contents of which are incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under CHE2116298 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND OF THE DISCLOSURE

[0003] Plant virus-like particles (VLPs) are protein-based nanomaterials with a highly defined 3D structure, often icosahedral or tubular, that can be produced in the laboratory with yields of tens or hundreds of milligrams.fi] These nanoparticles are highly biocompatible, biodegradable and non-infectious, allowing them to be repurposed for applications such as drug delivery, cancer vaccines, and immunotherapy. [2] For example, intratumoral immunotherapy based on plant VLPs has been used to treat canine cancer patients. [3] Nevertheless, when used as immunotherapy for chronic diseases or cancer vaccines, [4] repeated and even life-long dosing may be required. [5] VLP depots that facilitate slow release over time would avoid this need for repeat administration. [5] This disclosure satisfies this need and provides related advantages as well.SUMMARY OF THE DISCLOSURE

[0004] Plant virus-like particles (VLPs) are biocompatible, non-infectious nanomaterials with promising applications as immunotherapeutics and vaccines. However, slow-release VLP formulations are needed to achieve long-term efficacy without repeated administration. VLP hydrogels allow the encapsulation and sustained delivery of VLPs, but the particles must covalently bind the hydrogel polymers to avoid premature loss. This has been achieved so far by in situ VLP polymerization, which requires high viral concentrations (5-10 mg / ml, 0.5-1 wt%) to form stable hybrid VLP-hydrogel networks and this complicates scalability and clinical translation. As described herein, Applicant developed a novel swell-and-click method that led to successful VLP scaffold formation regardless of the viral load used. As a result, VLP-functionalized hydrogels were fabricated with viral concentrations as low as 0.1-1 mg / ml(0.01-0.1% wt%) without compromising the scaffold stability on the process. The hydrogels incorporate VLPs during swelling, followed by copper-free click chemistry reactions that bind the particles covalently to the polymer. The swell-and-click method also resulted in more than a two-fold enhancement in VLP uptake into the hydrogels and it provides a means of combined burst release and prolonged sustained release, desired traits for cancer immunotherapy treatment. The present work introduces a novel methodology for the design of VLP -based hydrogels, which could facilitate the scalability of the fabrication process and move a significant step forward towards clinical translation of long-term VLP vaccination in cancer disease.

[0005] Applicant discloses herein a Virus-Like Particle (VLP)-long-term delivery system for use in vaccines immunotherapies to target chronic diseases. Applicant designed a hydrogel capable of covalently bonding to the VLP, for example Physalis Mottle Virus (Pym or PhMV), through a novel swell-“ click” methodology. The resulting hybrid virus hydrogel (VHG) can be used for long-term delivery of viral cargo.

[0006] Thus, this disclosure provides a method of preparing a hybrid virus hydrogel, the method comprising, or consisting essentially of, or yet further consisting of contacting a viruslike particle (VLP) or derivative thereof comprising an alkyne moiety with a swelled hydrogel comprising an azide group. In one aspect, the alkyne moiety is part of a strained ring of the VLP. In one aspect, the alkyne is a ring-strained dibenzo-cyclooctyne The VLP is optionally detectably labeled.

[0007] In a further aspect, the VLP is selected from the group of Physalis mottle virus (PhMV), Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), or Cowpea chlorotic mosaic virus (CCMV), that is optionally detectably labeled.

[0008] In one embodiment, the method further comprises, or consists essentially of, or yet further consisting of drying the swelled hydrogel to remove water and form a VLP xerogel. As used herein, the term “drying” intends reducing the water content of the hydrogel which in one aspect, is performed by a method not limited to desiccation, contact or convective drying, lowering the pH of the hydrogel, or freeze drying.

[0009] In another aspect of the above methods, the hydrogel comprises the reaction product of a cross-linking agent and at least one or two or more of an azide-containing monomer or oligomer, a polyalkylene glycol, and a (meth)arylate. In another aspect, the hydrogel comprisesthe reaction product of an acrylate-polyethylene glycol azide, a polyethylene glycol methacrylate, an acrylamide, and polyethylene glycol diacrylate.

[0010] Further provided is a hybrid virus hydrogel prepared by the methods described herein or a VLP xerogel as described herein. In one aspect, the hybrid virus hydrogel is detectably labeled. In a further aspect, the VLP is selected from the group of Physalis mottle virus (PhMV), Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), or Cowpea chlorotic mosaic virus (CCMV), that is optionally detectably labeled.

[0011] Further provided is a hybrid virus hydrogel comprising a polymeric network with a covalently bound VLP, that is optionally detectably labeled and / or processed to a hybrid virus xerogel. In one aspect, the virus particle or VLP is bound to the polymeric network via an azide-yne linkage.

[0012] Also provided herein is hybrid virus hydrogel comprising a polymeric network with a covalently bound VLP, wherein the VLP comprises an alkyne moiety and at least one imino group; and the polymeric network comprises a swelled hydrogel comprising an azide group, and wherein the VLP is conjugated to the polymeric network via the imino group. In some aspects, the VLP further comprises a therapeutic drug agent, optionally a chemotherapeutic drug agent. In some aspects the therapeutic drug agent is conjugated to the VLP via a second imino group.

[0013] Also provided herein is a composition comprising the hydrogel or the xerogel as described herein, and a carrier, optionally a pharmaceutically acceptable carrier. In one aspect, the composition further comprises a stabilizer or a preservative.

[0014] In another aspect, provided herein is a method to deliver a VLP to a cell or tissue in need thereof, comprising, or consisting essentially of, or consisting of contacting the cell or tissue with an effective amount of the hybrid VLP or the composition as described herein. The cell can be a prokaryotic or eukaryotic cell. The contacting can be in vitro or in vivo.

[0015] In another aspect, provided herein is a method to administer a VLP to a subject in need thereof, the method comprising, or consisting essentially thereof, of consisting of administering to the subject an effective amount the hybrid VLP or the composition as described herein. In one aspect, the subject is a mammal, optionally a human patient. In one embodiment, the method induces an immune response in the subject.

[0016] In another aspect, provided herein is a method to deliver a VLP to a cell or tissue in need thereof, comprising, or consisting essentially of, or consisting of contacting the cell or tissue with an effective amount of the hybrid VLP or the composition as described herein, wherein the VLP is released over a time period to the cell or tissue at an amount less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% than the amount released in a hybrid virus hydrogel wherein the VLP is not covalently bound to the hydrogel, e.g., of a corresponding hybrid virus hydrogel which does not comprise an alkyne moiety. In some embodiments, the time period can be up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, up to eight weeks, or greater than eight weeks.

[0017] In another aspect, provided herein is a method to deliver a VLP to a subject in need in need thereof, comprising, or consisting essentially of, or consisting of administering to the subject in need an effective amount of the hybrid VLP or the composition as described herein, wherein the VLP is released over a time period to the subject at an amount less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% than the amount released in a hybrid virus hydrogel wherein the VLP is not covalently bound to the hydrogel, e.g., of a corresponding hybrid virus hydrogel which does not comprise an alkyne moiety. In some embodiments, the time period can be up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, up to eight weeks, or greater than eight weeks. In some aspects, the VLP further comprises a therapeutic drug agent, optionally wherein the therapeutic drug agent is conjugated to the VLP via an imino bond, and optionally wherein the therapeutic drug agent is a chemotherapeutic drug agent. In one aspect, in the methods described herein the hybrid virus hydrogel or composition is implanted in the subject in need thereof, wherein the implantation is adjacent to a tumor in the subject in need thereof. In one aspect, the subject in need thereof has a tumor, optionally wherein the tumor is a solid tumor selected from bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer.BRIEF DESCRIPTION OF THE FIGURES

[0018] Figures 1A-1H. Synthesis and characterization of VLPs. FIG. 1A) Strategy for the synthesis of VLPs. Analysis of VLPs by FIG. IB) SDS-PAGE and FIG. 1C) native gelelectrophoresis under white light, stained with Coomasie Brilliant Blue and superimposed with red fluorescence. FIG. ID) Photograph of VLP solutions: PhMV-cy5.5 (left) and PhMV-yne (right). Distribution of VLP particle size as determined by FIG. IE) size exclusion chromatography, FIG. IF) dynamic light scattering, and FIG. 1G) transmission electron microscopy (TEM). FIG. 1H) TEM images of negatively stained VLPs.

[0019] Figures 2A-2D. Swell-and-click synthesis of the VLP-laden hydrogels (VHGs). FIG. 2A components of the hydrogel. Before adding VLPs, the monomers, crosslinker and initiator were photopolymerized, washed and dried, to produce FIG. 2B) the control hydrogel (HG). The latter was swollen in a PhMV-yne solution (swell-and-click) to produce FIG. 2C) the NLP -clicked hydrogel (VC-HG). In parallel, the hydrogel was swollen in a PhMV-cy5.5 solution to produce FIG. 2D) the VLP-native hydrogel (VN-HG). For simplicity, the polymer backbone and crosslinker are depicted as chains and round junctions, respectively.

[0020] Figures 3A-3B. VLP loading test for the control hydrogel (HG): FIG. 3A) SDmax and FIG. 3B) VLP loading. In FIG. 3B, initial concentrations of 0.1, 0.5 and 0.75 mg / mL correspond to 0.01, 0.05 and 0.075 wt%, respectively. In each set of columns in FIG. 3A and 3B, left is PhMV-cy5.5 (SD or loading) and right is PhMV-yne (SD or loading).

[0021] Figures 4A-4D. Differential thermograms (DTGs) for the control hydrogel (HG), the VLP-laden hydrogels (VHGs) and their respective components. FIG. 4A) Acrylamide (AAm,), AC-PEG2K-N3and HG in the range 250-450 °C. FIG. 4B) PhMV, PhMV-DBCO and PhMV-cy5.5 VLPs in the range 150-450 °C. FIG. 4C) The VLP-native hydrogel (VN-HG), HG and PhMV-cy5.5 in the range 0-600 °C. FIG. 4D) The N P-clicked hydrogel (VC-HG), HG , PhMV-yne and PhMV-C(lick) in the range 0-600 °C. Chemical structures of the monomers, HG and simplified VLP structures are shown next to the figures. In FIGS. 4A-4BArrows leading from the hydrogel names indicate the hydrogel curves. Arrows next to a temperature indicate the temperature. In FIG. 4C, the arrow leading from Lysines)- NH2 PhMV-cy5.5 indicates the PhMV-cy5.5-HG DTG curve and the arrow from AAm indicates the HG-DTG curve. The curve without an arrow in FIG. 4C is VN-HG DTG. In FIG. 4D, the arrow leading from Lysines)-NH2 PhMV-C(lick) indicates the PhMV-C(lick) DTG curve, the arrow leading from AAm indicates the HG DTG curve, and the arrow leading from Lysines)-NH2 PhMV-yne indicates the PhMV-yne DTG curve. The curve without an arrow in FIG. 4D is VC-HG DTG.

[0022] Figures 5A-5B. Compressive strength of the control hydrogel (HG) and VLP- laden hydrogel (VHG) samples. FIG. 5A) Experimental setup. FIG. 5B) Compressive stressstrain curves and Young’s modulus (inset). Upper line is HG, middle line is VLP -native hydrogel, and lower line is VLP-clicked hydrogel. In the inset, 16.31 kPa is HG, 15.97 kPa is VN-HG, and 12.57 kPa is VC-HG.

[0023] Figures 6A-6D. Swelling and porosity studies. FIG. 6A) Swelling degree (SD) of the control hydrogel (HG) and VLP-laden hydrogels (VN-HG, VC-HG) samples in water. Pore sizes for FIG. 6B) HG, FIG. 6C) VN-HG (incorporating native PhMV particles) and FIG. 6D) VC-HG (incorporating VLP-clicked particles). In FIG. 6A, at final time point, top line is HG, middle line is VN-HG, and lower line is VC-HG.

[0024] Figures 7A-7B. Cumulative release of VLPs from VLP-laden hydrogels in vitro. FIG. 7A) weekly release FIG. 7B) monthly release. In both FIGS. 7A and 7B the upper curve is VN-HG (VLP-native hydrogel) and the lower curve is VC-HG (VLP-clicked hydrogel).

[0025] Figures 8A-8B. Validity test of SPAAC reaction for PhMV-cy5.5 (left) and PhMV-yne (right) FIG. 8A) SDS-PAGE and FIG. 8B) native electrophoresis.

[0026] Figures 9A-9B. UV / VIS spectroscopic data for VLP loading experiments. FIG. 9A) calibration curves and FIG. 9B) UV / VIS spectra in the region of 500-750 nm. In FIG. 9A the upper calibration curve is PhMV-cy5.5 (dashed line is the line of best fit) and the lower calibration curve is PhMV-yne. In FIG. 9B the upper curve is PhMV-cy5.5 and the lower curve is PhMV-yne.

[0027] Figures 10A-10B. Thermograms (TGs, upper panel) and differential thermograms (DTGs, lower panel) in the 0-1200 °C range. FIG. 10A) Acrylamide (AAm), AC-PEG2K-N3 and HG. FIG. 10B) PhMV, PhMV-DBCO and PhMV-cy5.5 VLPs . The chemical structure of the monomers and HG as well as the simplified struture of the different VLPs are depicted in each of FIGS. 10A and 10B.

[0028] Figure 11. Thermograms (TGs, upper) and differential thermograms (DTGs, lower) in the 0-1200 °C range. To the left: PhMV-cy5.5 VLPs, HG (hydrogel) and VN-HG (VLP-native hydrogel). To the right: PhMV-yne, PhMV-C(lick), HG (hydrogel) and VC-HG(VLP-clicked hydrogel). The chemical structure of the different VLPs are depicted in the far right panel.

[0029] Figures 12A-12B. Rheology test under frequency sweep conditions. FIG. 12A) storage and loss modulus. FIG. 12B) phase angle

[0030] Figures 13A-13B. FTIR spectra of the FIG. 13A) control hydrogel (HG) and VLP- laden hydrogel (VN-HG and VC-HG) samples and FIG. 13B) commercially available AC- PEGIK-N3 and AC-PEG2K-N3 monomer.

[0031] Figure 14. Control hydrogel (HG) and VLP-laden hydrogels (VN-HG, VC-HG) before in vitro release.

[0032] Figure 15. Burst VLP release from virus hydrogels (VHGs) during early hours. In FIG.15 the upper line is VN-HG (VLP -native hydrogel) and the lower line is VC-HG (VLP- clicked hydrogel).

[0033] Figures 16A-16D. UV / vis spectra of VLPs before and after release from the hydrogel. FIG. 16A) PhMV-cy5.5 and FIG. 16B) PhMV-yne in the 195-295 nm range. FIG. 16C) PhMV-cy5.5 and FIG. 16D) PhMV-yne in the 530-740 nm range.

[0034] Figure 17. Validity test of dye labeling for PhMV-WT (left) and PhMV Cys 3x (right) with SDS-PAGE and native electrophoresis. Left to right is increasing amounts of Cy5 dye.

[0035] Figures 18A-18C. Loading of VLPs with increasing amounts of aldoxorubicin. FIG. 18A) PhMV-WT (left) and PhMV Cys 3x (right) with SDS page and native electrophoresis. Left to right is increasing amounts of aldoxrubicin. FIG. 18B) shows a schematic of doxorubicin-loaded VLP in a VLP-loaded hydrogel. The aldoxorubicin is conjugated to the VLP via an imino bond. FIG. 18C) shows the FPLC results of PhMV conjugated to aldoxorubicin at multiple wavelengths. 488 nm is the lowest curve, 647 nm is the middle curve, and 280 nm is the upper curve.

[0036] Figures 19A-19D. Acid-cleavable and clickable VLP. FIG. 19 A) Process for generating a PhMV with a cleavable handle and a clickable handle. FIG. 19B) Agarose and SDS-page electrophoresis of virus-based hydrogels with cleavable handle and a clickable handle. FIG. 19C) shows a schematic of the acid-cleavable and clickable VLP within a VLP-conjugated hydrogel. In FIG. 19C 1 is the cleavable handle and 2 is the clickable handle. FIG 19D) shows the FPLC results of PhMV-DBCO (which includes both a cleavable handle and a clickable handle). PhMV-DBC0-P3 is the lower curve and prep-20-A AKTA is the upper curve. Prep-20-A AKTA refers to PhMV without the DBCO handle.DETAILED DESCRIPTION OF THE DISCLOSUREDefinitions

[0037] Embodiments according to the present disclosure will be described more fully hereinafter. Aspects of the disclosure may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0038] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. While not explicitly defined below, such terms should be interpreted according to their common meaning.

[0039] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0040] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0041] Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.

[0042] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of + / - 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0043] Throughout this disclosure, various publications, patents and published patent specifications may be referenced by an identifying citation or by an Arabic numeral or first author name. The full citation for the publications identified by an Arabic numeral or first author name are found immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this disclosure pertains.

[0044] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989)); Current Protocols In Molecular Biology (F. M. Ausubel, etal. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988)) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

[0045] As used in the description of the disclosure and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0046] The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.

[0047] As used herein, the term “comprising” is intended to mean that the compositions or methods include the recited steps or elements, but do not exclude others. “Consisting essentially of’ shall mean rendering the claims open only for the inclusion of steps or elements, which do not materially affect the basic and novel characteristics of the claimed compositions and methods. “Consisting of’ shall mean excluding any element or step not specified in the claim. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0048] The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.

[0049] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0050] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals.

[0051] As used herein, "biologies” or "biological drug" and their plurals are used interchangeably and refer to drugs consisting of or comprising biological molecules or material, i.e., proteins, polypeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, oligosaccharides and fragments thereof, as well as cells, tissues, biological fluids or extracts thereof. In some embodiments, biological drugs may include proteins such as monoclonal antibodies, cytokines, soluble receptors, growth factors, hormones, enzymes, adhesion molecules and fusion proteins and peptides that are specific to certain targets known to modulate disease mechanisms. In yet some further embodiments, biological drugs may include or target any component participating in molecular and / or cellular processes such as, cell cycle, cell survival, apoptosis, immunity and the like. In more specific embodiments, biological drugs may be any checkpoint protein / s or any modulators or inhibitors thereof, or any combinations thereof. In yet some further embodiments, biological drugs (or their precursors or components) may be isolated from living sources human, animal, plant, fungal, or microbial.

[0052] Still further in some embodiments, "biologies" refers to a class of therapeutics that are produced by means of biological processes involving recombinant DNA technology which are usually one of three types: (a) substances that are similar to the natural occurring proteins:(b) monoclonal antibodies; and (c) receptor constructs or fusion proteins, usually based on a naturally occurring receptor linked to the immunoglobulin frame.

[0053] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method, cell or composition described herein. Non-limiting examples of mammals include humans, non -human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. A mammal can be a pregnant female. In some embodiments a subject is a human. In some embodiments, a subject has or is suspected of having a cancer or neoplastic disorder.

[0054] “Eukaryotic cells” comprise, or alternatively consist essentially of, or yet further consist of all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human,

[0055] “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called on episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0056] A “composition” typically intends a combination of the active agent, e.g., the nanoparticle of this disclosure and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and includepharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; di saccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0057] The compositions used in accordance with the disclosure, including cells, treatments, therapies, agents, drugs and pharmaceutical formulations can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, z.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0058] The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or whenmanipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0059] As used herein, the term “isolated cell” generally refers to a cell that is substantially separated from other cells of a tissue. The term includes prokaryotic and eukaryotic cells.

[0060] As used herein, the term “vector” refers to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. In some embodiments, plasmid vectors may be prepared from commercially available vectors. In other embodiments, viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Further details as to modem methods of vectors for use in gene transfer may be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.).

[0061] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound.

[0062] As used herein, “homology” or “identical”, percent “identity” or “similarity”, when used in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. (1987)) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non- redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. The terms “homology” or “identical,” percent “identity” or “similarity” also refer to, or can be applied to, the complement of a test sequence. The terms also include sequences that have deletions and / or additions, as well as those that have substitutions. As described herein, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences disclosed herein.

[0063] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’ when referring to a reference protein, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unlessspecifically recited herein, it is contemplated that any of the above also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and / or exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0064] The phrase “equivalent polypeptide” or “equivalent peptide fragment” refers to protein, polynucleotide, or peptide fragment encoded by a polynucleotide that hybridizes to a polynucleotide encoding the exemplified polypeptide or its complement of the polynucleotide encoding the exemplified polypeptide, under high stringency and / or which exhibit similar biological activity in vivo, e.g., approximately 100%, or alternatively, over 90% or alternatively over 85% or alternatively over 70%, as compared to the standard or control biological activity. Additional embodiments within the scope of this disclosure are identified by having more than 60%, or alternatively, more than 65%, or alternatively, more than 70%, or alternatively, more than 75%, or alternatively, more than 80%, or alternatively, more than 85%, or alternatively, more than 90%, or alternatively, more than 95%, or alternatively more than 97%, or alternatively, more than 98% or 99% sequence homology. Percentage homology can be determined by sequence comparison using programs such as BLAST run under appropriate conditions. In one aspect, the program is run under default parameters.

[0065] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. (1987)) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR.Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi- bin / BLAST.

[0066] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single selfhybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0067] Examples of stringent hybridization conditions include: incubation temperatures of about 25 °C to about 37 °C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40 °C to about 50 °C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. A high stringency hybridization refers to a condition in which hybridization of an oligonucleotide to a target sequence comprises no mismatches (or perfect complementarity). Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, 0. lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[0068] The term “isolated” as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials. In one aspect, the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.

[0069] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0070] The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by nonnucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and singlestranded molecules. Unless otherwise specified or required, any aspect of this technology that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0071] A “marker gene” or a “selectable marker gene” refers to a gene whose expression in a plant cell gives the cell a selective advantage. The selective advantage possessed by thecells transformed with the marker gene may be due to their ability to grow in presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the ability to grow of non-transformed cells. The selective advantage possessed by the transformed cells may also be due to their enhanced capacity, relative to non-transformed cells, to utilize an added compound as a nutrient, growth factor or energy source. A selective advantage possessed by a transformed cell may also be due to the loss of a previously possessed gene in what is called “negative selection”. In this, a compound is added that is toxic only to cells that did not lose a specific gene (a negative selectable marker gene) present in the parent cell (typically a transgene).

[0072] As used herein, a “nucleoprotein complex” is a complex comprising proteins conjugated with nucleic acids (either DNA or RNA).

[0073] As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid, peptide, protein, biological complexes or other active compound is one that is isolated in whole or in part from proteins or other contaminants. Generally, substantially purified peptides, proteins, biological complexes, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, biological complex or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other coingredient in a complete pharmaceutical formulation. More typically, the peptide, protein, biological complex or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.

[0074] As used herein, the term “overexpress” with respect to a cell, a tissue, or an organ expresses a protein to an amount that is greater than the amount that is produced in a control cell, a control issue, or an organ. A protein that is overexpressed may be endogenous to the host cell or exogenous to the host cell.

[0075] As used herein, the term “enhancer”, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed. An enhancer may enhancetranscription from a single promoter or simultaneously from more than one promoter. As long as this functionality of improving transcription is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of wild-type activity, that is, activity of a full- length sequence), any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition.

[0076] The term “promoter” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.

[0077] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0078] The term “introduce” as applied to methods of producing modified cells refers to the process whereby a foreign (i.e. extrinsic or extracellular) agent is introduced into a host cell thereby producing a cell comprising the foreign agent. Methods of introducing nucleic acids include but are not limited to transduction, retroviral gene transfer, transfection, electroporation, transformation, viral infection, and other recombinant DNA techniques known in the art. In some embodiments, transduction is done via a vector (e.g., a viral vector). In some embodiments, transfection is done via a chemical carrier, DNA / liposome complex, or micelle (e.g., Lipofectamine (Invitrogen)). In some embodiments, viral infection is done via infecting the cells with a viral particle comprising the polynucleotide of interest.

[0079] The term “culturing” refers to growing cells in a culture medium under conditions that favor expansion and proliferation of the cell. The term “culture medium” or “medium” is recognized in the art and refers generally to any substance or preparation used for the cultivation of living cells. The term “medium”, as used in reference to a cell culture, includes the components of the environment surrounding the cells. Media may be solid, liquid, gaseous or a mixture of phases and materials. Media include liquid growth media as well as liquid mediathat do not sustain cell growth. Media also include gelatinous media such as agar, agarose, gelatin and collagen matrices. Exemplary gaseous media include the gaseous phase to which cells growing on a petri dish or other solid or semisolid support are exposed. The term “medium” also refers to material that is intended for use in a cell culture, even if it has not yet been contacted with cells. In other words, a nutrient rich liquid prepared for culture is a medium. Similarly, a powder mixture that when mixed with water or other liquid becomes suitable for cell culture may be termed a “powdered medium.”

[0080] As used herein, the term “hydrogel” refers to a polymeric material that exhibits the ability to swell and retain a significant fraction of water within its structure, without dissolving in the water. The polymeric structure may contain greater than 20 wt% water. For example, the polymer structure may contain greater than 30 wt% water, greater than 40 wt% water, greater than 50 wt% water, or greater than 60 wt% water, or greater than 70 wt% water.

[0081] As used herein, the term “xerogel” refers to a dried hydrogel, and which can be rehydrated to reform the hydrogel. The xerogel structure may contain less than 20 wt% water. For example, the polymer structure may contain less than 10 wt% water, less than 5 wt% water, or less than 1 wt% water. In some embodiments, xerogels contain less than 2 wt% water, less than 1 wt% water, or less than 0.5 wt% water.

[0082] As used herein, a “hybrid hydrogel” is a synthetic polymeric network (i.e. hydrogel) with a non-synthetic component embedded or conjugated in the network.

[0083] As used herein, a “hybrid virus hydrogel” is a system or network that is comprised of both a hydrogel and a VLP. In a hybrid virus hydrogel, the VLP is encapsulated in the hydrogel. Hybrid virus hydrogels can be formed by in situ VLP polymerization, or by using the novel methods as described herein. As used herein, “hybrid virus hydrogel” and “virus hydrogel” are used interchangeably.

[0084] As used herein, a “swelled hydrogel” is a hydrogel which has absorbed a solvent. A swelled hydrogel is swollen compared to a non-swelled hydrogel.

[0085] As used herein, an “azide-yne linkage” refers to the bond between an alkyne moiety of a VLP with an azide group of an hydrogel in a hybrid virus hydrogel . As used herein, azideyne linkage may also be referred to as an “azide-alkyne linkage.”

[0086] As used herein “imine bonds” refer to the imine group connecting a VLP to the hydrogel, and / or connecting a VLP to another therapeutic agent, for example achemotherapeutic agent. Imine bonds belong to a class of covalent bonds termed “dynamic covalent bonds.” Dynamic covalent bonds, although covalent in nature, are also reversible under specific conditions or chemical cue. These “chemical cues” are small molecules or atoms that displace the equilibrium of the imine tautomer towards the aldehyde tautomer (imine hydrolysis, mediated my acidic pH) or displace the imine of the virus towards an imine of a smaller amine compound (transimination, mediated by primary amines). Imine-bond reversible hydrogels are also known as “Schiff-base hydrogels” (Duceac, I. and Coseri, S.( 2022) Gels, 8:779).

[0087] As used herein, the term “an equivalent thereof’ when referring to sequence identity comprises at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identify to the respective reference sequence of which it is compared to, while still retaining a functional activity. In some instances, a functional activity refers to the modulation of an immunostimulatory effect on an immune cell.

[0088] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0089] “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response such as passive immunity; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations ofthe specified disorder or disease in the patient. A therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0090] “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include systemic or localized administration, e.g., oral administration, intratumorally, or localized at the site of the cancer or tumor, nasal administration, injection, and topical application. Additional non-limiting routes of administration include transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, intratumoral, epidural and intrathecal. In one aspect, administration is systemic or intraperitoneal.

[0091] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (z.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. When the disease is cancer, the following clinical end points arenon-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor, or delay, slowing, or prevent of relapse. In one aspect, treatment excludes prophylaxis. In one aspect, treatment provides a longer progression free survival or a longer overall survival.

[0092] In one embodiment, the term “disease” or “disorder” as used herein refers to a cancer or a tumor (which are used interchangeably herein), a status of being diagnosed with such disease, a status of being suspect of having such disease, or a status of at high risk of having such disease.

[0093] Cancer” or “malignancy” are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasize) as well as any of a number of characteristic structural and / or molecular features. In some embodiments, the term “cancer” is used interchangeably with the term “tumor”. Non-liming examples of cancers include carcinomas, sarcomas, and hematological cancers. In one aspect, the cancer is ovarian cancer such as ovarian serous carcinoma.

[0094] A “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. In some embodiments, a solid tumor comprises bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer.

[0095] Virus and Virus-like Particles (VLPs)

[0096] As utilized herein, a VLP is a non-native VLP that comprise, or consists essentially of, or yet further consists of, one or more viral particles, e.g., a capsid, derived from a plant virus. In some instances, the plant virus is from the genus Bromovirus, Comovirus, Tymoviriis, or Sobemovirus. In some cases, the VLP is derived from Cowpea chlorotic mottle virus (CCMV), Cowpea mosaic virus (CPMV), Physalis mottle virus (PhMV), or Sesbania mosaic virus (SeMV).

[0097] In some instances, the VLP comprise, or consists essentially of, or yet further consists of, a capsid protein derived from a plant virus. In some instances, the capsid protein is a wild-type protein derived from the plant virus. In other instances, the capsid protein is a variant of the wild-type protein derived from the plant virus. In additional instances, the capsid protein is a modified protein, either full-length or truncated version.

[0098] As used herein, the term “Virus-like particle” or “VLP” refers to a non-replicating, viral shell, derived from one or more viruses (e.g., one or more plant viruses described herein). VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification. Methods for producing VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445- 1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012).

[0099] In some embodiments, the VLP is derived from a plant Picornavirus, for example a Comovirus, a Tymovirus or other plant virus with an icosahedral shaped plasmid.

[0100] In some embodiments, the VLP is derived from Cowpea chlorotic mottle virus (CCMV). CCMV is a spherical plant virus that belongs to the Bromovirus genus. Several strains have been identified and include, but not limited to, Carl (Ali, et al., 2007. J. Virological Methods 141 :84-86), Car2 (Ali, et al., 2007. J. Virological Methods 141 :84-86, 2007), type T (Kuhn, 1964. Phytopathology 54: 1441-1442), soybean (S) (Kuhn, 1968. Phytopathology 58: 1441-1442), mild (M) (Kuhn, 1979. Phytopathology 69:621-624), Arkansas (A) (Fulton, et al., 1975. Phytopathology 65: 741-742), bean yellow stipple (BYS) (Fulton, et al., 1975. Phytopathology 65: 741-742), R (Sinclair, ed. 1982. Compendium of Soybean Diseases. 2nded. The American Phytopathological Society, St. Paul. 104 pp.), and PSM (Paguio, et al., 1988. Plant Diseases 72(9): 768-770).

[0101] In some instances, the VLP from CCMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wildtype CCMV capsid, optionally expressed by Carl, Car2, type T, soybean (S), mild (M), Arkansas (A), bean yellow stipple (BYS), R, or PSM strain. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the CCMV capsid comprise, or consists essentially of, or yet further consists of, s the sequence as set forth in the UniProtKB ID P03601 :

[0102] MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTG YSVSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVK SCVTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAA LTEGDVIVHLEVEHVRPTFDDSFTPVY (SEQ ID NO: 1), or an equivalent thereof.

[0103] In some cases, the VLP from CCMV is prepared by the method as described in Ali et al., “Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation,” Journal of Virological Methods 141 : 84-86 (2007).

[0104] In some embodiments, the VLP is derived from Cowpea mosaic virus (CPMV). CPMV is a non-enveloped plant virus that belongs to the Comovirus genus. CPMV strains include, but are not limited to, SB (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1) and Vu (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1).

[0105] In some instances, the VLP from CPMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, CPMV produces a large capsid protein and a small capsid protein precursor (which generates a mature small capsid protein). In some cases, CPMV capsid is formed from a plurality of large capsid proteins and mature small capsid proteins. In some cases, the large capsid protein is a wild-type large capsid protein, optionally expressed by SB or Vu strain. In other instances, the large capsid protein is a modified large capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the large capsid protein comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 460-833):

[0106] MEQNLFALSLDDTSSVRGSLLDTKFAQTRVLLSKAMAGGDVLLDEYLYD VVNGQDFRATVAFLRTHVITGKIKVTATTNISDNSGCCLMLAINSGVRGKYSTDVYT ICSQDSMTWNPGCKKNFSFTFNPNPCGDSWSAEMISRSRVRMTVICVSGWTLSPTTDVIAKLDWSIVNEKCEPTIYHLADCQNWLPLNRWMGKLTFPQGVTSEVRRMPLSIGG GAGATQAFLANMPNSWISMWRYFRGELHFEVTKMSSPYIKATVTFLIAFGNLSDAFG FYESFPHRIVQFAEVEEKCTLVFSQQEFVTAWSTQVNPRTTLEADGCPYLYAIIHDST TGTISGDFNLGVKLVGIKDFCGIGSNPGIDGSRLLGAIAQ (SEQ ID NO: 2), or an equivalent thereof.

[0107] In some cases, the mature small capsid protein is a wild-type mature small capsid protein, optionally expressed by SB or Vu strain. In other instances, the mature small capsid protein is a modified mature small capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the mature small capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 834-1022):

[0108] GPVCAEASDVYSPCMIASTPPAPFSDVTAVTFDLINGKITPVGDDNWNTHI YNPPIMNVLRTAAWKSGTIHVQLNVRGAGVKRADWDGQVFVYLRQSMNPESYDA RTFVISQPGSAMLNFSFDIIGPNSGFEFAESPWANQTTWYLECVATNPRQIQQFEVNM RFDPNFRVAGNILMPPFPLSTETPPL (SEQ ID NO: 3), or an equivalent thereof.

[0109] In some embodiments, the VLP is derived from Physalis mottle virus (PhMV). PhMV is a single stranded RNA virus that belongs to the genus Tymovirus. In some instances, the VLP from PhMV comprises, or consists essentially of, or yet further consists of, a plurality of coat proteins. In some instances, the coat protein is a wild-type PhMV coat protein. In other instances, the coat protein is a modified coat protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the PhMV coat comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P36351 :

[0110] MDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQV SLQTADPITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILR VYGGQSFVLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIP TASIQISGRIRLSKPMLIAN (SEQ ID NO: 4), or an equivalent thereof.[OHl] In some embodiments, the VLP is derived from Sesbania mosaic virus (SeMV). SeMV is a positive stranded RNA virus that belongs to the genus Sobemovirus. In some instances, the VLP from SeMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type SeMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, orconsisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the SeMV capsid comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q9EB06:

[0112] MAKRLSKQQLAKAIANTLETPPQPKAGRRRNRRRQRS AVQQLQPTQAGIS MAPSAQGAMVRIRNPAVSSSRGGITVLTHSELSAEIGVTDSIVVSSELVMPYTVGTWL RGVAANWSKYSWLSVRYTYIPSCPSSTAGSIHMGFQYDMADTVPVSVNQLSNLRGY VSGQVWSGSAGLCFINGTRCSDTSTAISTTLDVSKLGKKWYPYKTSADYATAVGVD VNIATPLVPARLVIALLDGSSSTAVAAGRIYCTYTIQMIEPTASALNN (SEQ ID NO: 5), or an equivalent thereof.

[0113] In some embodiments, the plant virus nanoparticle or VLP is derived from Tobacco Mosaic Virus (TMV). It is a member of the Virgaviridae family. This virus is among the first if not the first characterized virus and has been utilized as a model virus for decades for life science applications.

[0114] TMV has a rod-like appearance. Its capsid is made from 2130 molecules of coat protein and one molecule of genomic single strand RNA, 6400 bases long. The coat protein self-assembles into the rod-like helical structure (16.3 proteins per helix turn) around the RNA, which forms a hairpin loop structure. The protein monomer consists of 158 amino acids which are assembled into four main alpha-helices, which are joined by a prominent loop proximal to the axis of the virion. Virions are -300 nm in length and -18 nm in diameter. The RNA is located at a radius of -4 nm and is protected from the action of cellular enzymes by the coat protein. X-ray fiber diffraction structure of the intact virus was studied based on an electron density map at 3.6 A resolution. Inside the capsid helix, near the core, is the coiled RNA molecule, which is made up of 6,395 ±10 nucleotides.

[0115] In some embodiments, a TMV coat protein comprises, or alternatively consists essentially of, or yet further consists of the sequence as set forth in the UniProtKB ID : P69687 :MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVWKPSPQ VTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLDATRRV DDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT (SEQ ID NO: 6), or an equivalent thereof.

[0116] In some cases, the virus or VLP from CCMV is prepared by the method as described in Ali et al., “Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation,” Journal of Virological Methods 141 : 84-86 (2007).

[0117] In some embodiments, the VLP comprises an engineered or modified coat protein. Coat proteins in VLP can be engineered to have an increased number of cysteines, which increases the loading capacity. Coat proteins with an increased number of cysteines can be made, for example, according to the methods described in Barkovich, et al. (2023) Bioconjugate Chemistry. 34. 10.1021 / acs.bioconjchem.3c00269.

[0118] As used herein, the term “an equivalent thereof’ in reference to a polynucleotide or a protein (e.g., a capsid or coat protein) include a polynucleotide or a protein that comprise, or consists essentially of, or yet further consists of, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identify to the respective polynucleotide or protein of which it is compared to, while still retaining a functional activity. In the instances with reference to a capsid or coat protein, a functional activity refers to the formation of a virus or VLP.

[0119] As used herein, the term “modification” include, for example, substitutions, additions, insertions and deletions to the amino acid sequences, which can be referred to as “variants.” Exemplary sequence substitutions, additions, and insertions include a full length or a portion of a sequence with one or more amino acids substituted (or mutated), added, or inserted, for example of a capsid derived from the plant virus. In some instances, a capsid described herein includes, e.g., a modified capsid comprising, or consisting essentially of, or yet further consisting of, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to its respective wild-type version.

[0120] The term “sequence identity” refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. The term “reference sequence” refers to a molecule to which a test sequence is compared. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to a reference sequence means that, when aligned, that percentage of bases (or amino acids) at each position in the test sequence areidentical to the base (or amino acid) at the same position in the reference sequence. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60, expect = 10, Matrix ~ BLOSUM62; Descriptions =::50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / blast / Blast.cgi.

[0121] Modified capsid polypeptides include, for example, non-conservative and conservative substitutions of the capsid amino acid sequences.

[0122] As used herein, the term “conservative substitution” denotes the replacement of an amino acid residue by another, chemically or biologically similar residue. Biologically similar means that the substitution does not destroy a biological activity or function, e.g., assembly of a viral capsid. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Particular examples of conservative substitutions include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the substitution of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Such proteins that include amino acid substitutions can be encoded by a nucleic acid. Consequently, nucleic acid sequences encoding proteins that include amino acid substitutions are also provided.

[0123] Modified proteins also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond.

[0124] Modified forms further include “chemical derivatives,” in which one or more amino acids has a side chain chemically altered or derivatized. Such derivatized polypeptides include, for example, amino acids in which free amino groups form amine hydrochlorides, p-toluene sulfonyl groups, carobenzoxy groups; the free carboxy groups form salts, methyl and ethyl esters; free hydroxl groups that form O-acyl or O-alkyl derivatives as well as naturally occurring amino acid derivatives, for example, 4-hydroxyproline, for proline, 5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine etc. Also included are amino acid derivatives that can alter covalent bonding, for example, the disulfide linkage that forms between two cysteine residues that produces a cyclized polypeptide.

[0125] In some instances, a virus or VLP described herein further comprise, or consists essentially of, or yet further consists of, a label or a tag, e.g., such as a detectable label. A detectable label can be attached to, e.g., to the surface of a virus or VLP.

[0126] Non-limiting exemplary detectable labels also include a radioactive material, such as a radioisotope, a metal or a metal oxide. Radioisotopes include radionuclides emitting alpha, beta or gamma radiation. In particular embodiments, a radioisotope can be one or more of3H,10B,18F,nC,14C,13N,18O,150,32P, P33,35S,35Cl,45Ti,46Sc,47Sc,51Cr,52Fe,59Fe, -57Co,60Cu,61Cu,62Cu,64Cu,67Cu,67Ga,68Ga,72As76Br,77Br,81mKr,82Rb,85Sr,89Sr,86Y,90Y,95Nb,94mTc, "mTc,97RU,103RU,105Rh,109Cd,n iIn,113Sn,113mIn,114In, I125, 1131,140La,141Ce,149Pm,153Gd,157Gd,153Sm,161Tb,166Dy,166Ho,169Er,169Y,175Yb,177Lu,186Re,188Re,2O1T1,203Pb,211At,212Bi or225Ac.

[0127] Additional non-limiting exemplary detectable labels include a metal or a metal oxide. In particular embodiments, a metal or metal oxide is one or more of gold, silver, copper, boron, manganese, gadolinium, iron, chromium, barium, europium, erbium, praseodynium, indium, or technetium. In additional embodiments, a metal oxide includes one or more of Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Ffe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), or Er(III).

[0128] Further non-limiting exemplary detectable labels include contrast agents (e.g., gadolinium; manganese; barium sulfate; an iodinated or noniodinated agent; an ionic agent or nonionic agent); magnetic and paramagnetic agents (e.g., iron-oxide chelate); nanoparticles; an enzyme (horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase); a prosthetic group (e.g., streptavidin / biotin and avidin / biotin); a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); a luminescent material (e.g., luminol); or a bioluminescent material (e.g., luciferase, luciferin, aequorin).

[0129] Additional non-limiting examples of tags and / or detectable labels include enzymes (horseradish peroxidase, urease, catalase, alkaline phosphatase, beta-galactosidase, chloramphenicol transferase); enzyme substrates; ligands (e.g., biotin); receptors (avidin); GST-, T7-, His-, myc-, HA- and FLAG®-tags; electron-dense reagents; energy transfer molecules; paramagnetic labels; fluorophores (fluorescein, fluorscamine, rhodamine, phycoerthrin, phycocyanin, allophycocyanin); chromophores; chemi-luminescent (imidazole, luciferase, acridinium, oxalate); and bio-luminescent agents.

[0130] Modes for Carrying Out the Disclosure

[0131] Applicant developed a novel method for the covalent functionalization of hydrogels with VLPs without relying on viral concentration to achieve successful gelation. VLP concentrations as low as 0.1-1 mg / mL (0.01-0.1 wt%) were used to fabricate VLP -based hydrogels with excellent mechanical stability. The hydrogels were chemically crosslinked to ensure mechanical stability and integrity in the absence of VLPs, which were incorporated using a post-synthesis swell-and-click approach. Solutions of alkyne-functionalized VLPs were added to dehydrated xerogels of azide-functionalized polymer networks, and the VLPs were efficiently taken up into the polymer network during the xerogel-to-hydrogel transition due to the swellability of the xerogels. The alkyne-functionalized VLPs then underwent copper-free click chemistry with the polymer to form robust covalent bonds even at very low VLP concentrations. The hydrogels retained the covalently attached VLPs, which lead to a combined partial premature particle release followed by sustained release in vitro over a period of several months. The novel materials are used for the development of extended controlled-release formulations, such as single-dose VLP implants to tackle chronic diseases where repeated administration is typically required.

[0132] Provided herein are hybrid virus hydrogels comprising a polymeric network and a VLP, and methods of use thereof.

[0133] Methods to Prepare Hybrid Virus Hydrogel

[0134] As used herein, a “hybrid virus hydrogel” is a system or network that is comprised of both a hydrogel and a VLP. In a hybrid virus hydrogel, the VLP is encapsulated in the hydrogel. Hybrid virus hydrogels can be formed by in situ VLP polymerization, or by usingthe novel methods as described herein. As used herein, “hybrid virus hydrogel” and “virus hydrogel” are used interchangeably.

[0135] Provided herein is a method to prepare a hybrid virus hydrogel, the method comprising, or alternatively consisting essentially of, or yet further consisting of contacting a virus-like particle (VLP) or derivative thereof comprising an alkyne moiety with a swelled hydrogel comprising an azide group (-N3), thereby preparing the hybrid virus hydrogel (see FIGS. 1 and 2). In the hybrid virus hydrogel, the VLP can be covalently bound to a polymer backbone of the swelled hydrogel. In one aspect, the alkyne moiety is part of a strained ring of the VLP. In one aspect, the alkyne moiety is a ring-strained dibenzo-cyclooctyne (DBCO) (FIG. 1). DBCO has a structure according to:

[0136] In some aspects, the VLP or derivative thereof further comprises an imino group. In some aspects, the VLP further comprises a therapeutic agent, for example a chemotherapeutic agent conjugated to the VLP via the imino group (FIG. 18B). In yet some other aspects, the VLP is conjugated to the swelled hydrogel via the imino group (FIG. 19). In yet another aspect, the therapeutic agent is linked via a first imino group to the VLP and the VLP is linked via a second imino group to the swelled hydrogel. The imino bond can be targeted by chemical cues to degrade the hybrid virus hydrogel. Cues can include changes in the pH (e.g. increasing the acidity) or addition of small molecules comprising primary amines.

[0137] In yet some other aspects, the VLP or derivative thereof further comprises a different acid-cleavable bond. In some aspects, the VLP further comprises a therapeutic agent conjugated to the VLP via the acid-cleavable group. In some aspects, the VLP is conjugated to the swelled hydrogel via the acid-cleavable group. The acid-cleavable bond can be targeted by acid to degrade the hybrid virus hydrogel and release the VLP and / or therapeutic agent.

[0138] In some aspects, the hydrogel or swelled hydrogel comprises the reaction product of a cross-linking agent and at least one azide-containing monomer or oligomer, a polyalkylene glycol, and a (meth)acrylate. In other aspects, the hydrogel or swelled hydrogel comprises thereaction product of an acrylate-polyethylene glycol azide, a polyethylene glycol methacrylate, an acrylamide, and polyethylene glycol diacrylate.

[0139] The VLP is selected from a plant virus, an animal virus, or a bacteriophage. In one aspect, the VLP is selected from a Picornavirus, for example a Comovirus, a Tymovirus or other plant virus with an icosahedral shaped plasmid. In some aspects, the VLP is selected from the group of Physalis mottle virus (PhMV), Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), or Cowpea chlorotic mosaic virus (CCMV). In one aspect, the VLP is or is derived from a PhMV.

[0140] In some embodiments, the diameter of the VLP is selected from: about 10 nm to about 100 nm, or from about 20 nm to about 80 nm, or from about 30 nm to about 60 nm, or from about 30 nm to about 40 nm, or from about 30 nm to about 35 nm, or about 33 nm. In one aspect, the diameter of the VLP is about 33nm (FIG. IF).

[0141] In one aspect, the VLP is detectably labelled. In one aspect the VLP is labelled with a marker molecule.

[0142] In one aspect, the method further comprises, or consists essentially of, or yet further consists of drying the swelled hydrogel to remove water and form a VLP xerogel. In some aspects the xerogel is a hydrogel that has been fully dried. In some aspects the xerogel may contain less than 20 wt% water. For example, the polymer structure may contain less than 10 wt% water, less than 5 wt% water, or less than 1 wt% water. In some embodiments, xerogels contain less than 2 wt% water, less than 1 wt% water, or less than 0.5 wt% water.

[0143] Hybrid Virus Hydrogel

[0144] Provided herein is a hybrid virus hydrogel, comprising a virus-like particle (VLP) or derivative thereof comprising an alkyne moiety with a swelled hydrogel comprising an azide group (-N3). Also provided herein is a composition comprising the hybrid virus hydrogel. In one aspect, the hybrid virus hydrogel is formed using the methods as described herein. In one aspect, the alkyne moiety is part of a strained ring of the VLP. In one aspect, the alkyne moiety is a ring-strained dibenzo-cyclooctyne (DBCO) (FIG. 1).

[0145] Also provided herein is a hybrid virus hydrogel comprising a polymeric network with a covalently bound VLP. As used herein, the term “polymeric network” refers to the hydrogels or swelled hydrogels, which are comprised of polymers. In one aspect, the VLP is bound to the polymeric network via an azide-yne linkage. As used herein, an “azide-ynelinkage” or “azide-alkyne linkage” refers to the bond between the alkyne moiety of the VLP with the azide group of the hydrogel or swelled hydrogel. In one aspect, the alkyne moiety of the VLP is DBCO (FIGS. 1A and 19A).

[0146] In some aspects, the VLP or derivative thereof further comprises a therapeutic drug agent. The therapeutic drug agent can be conjugated to the surface of the VLP, or be formulated to fit the pores of the VLP. In some aspects, the pores on the surface of the VLP are approximately 1 nm -10 nm in diameter. In some aspects, the pores on the surface of the VLP are approximately 5 nm - 10 nm in diameter. In some aspects, the pores on the surface of the VLP are approximately 6nm in diameter. In some aspects the therapeutic drug agent is a chemotherapeutic agent.

[0147] In some aspects, the VLP or derivative thereof further comprises at least one imino group. The imino group can be used to covalently attach the VLP to the swelled hydrogel and / or a therapeutic drug agent. Imino bonds are dynamic covalent bonds, which are cleaved under cues, such as an acidic pH. In one aspect, a therapeutic drug agent can be linked via the imino group to the VLP (FIG. 18). In yet another aspect, the VLP can be linked via the imino group to the swelled hydrogel (FIG. 19). In yet another aspect, the therapeutic agent is linked via a first imino group to the VLP and the VLP is linked via a second imino group to the swelled hydrogel. The imino group can be targeted by cues to degrade the hybrid virus hydrogel and release the VLP and / or therapeutic drug agent. Non-limiting examples of cues can include acidic pH and / or small molecules containing primary amine groups.

[0148] In some aspects, the VLP or derivative thereof further comprises a different acid- cleavable bond, and a therapeutic drug agent bound to the acid-cleavable bond. In some aspects, the VLP is conjugated to the swelled hydrogel via the acid-cleavable group. The acid-cleavable bond can be targeted by acid to degrade the hybrid virus hydrogel and release the VLP and / or therapeutic drug agent.

[0149] In some aspects, the therapeutic drug agent is bound to the VLP via a different cleavable bond, such as a labile ester bond, a reducible thiol bond, or an enzyme cleavable bond. In some aspects, the VLP is bound to the hydrogel or swelled hydrogel via a different cleavable bond, such as a labile ester bond, a reducible thiol bond, or an enzyme cleavable bond.

[0150] The VLP is selected from a plant virus, an animal virus, or a bacteriophage. In one aspect, the VLP is selected from a Picornavirus, for example a Comovirus, a Tymovirus or other plant virus with an icosahedral shaped plasmid. In some aspects the VLP is selected from the group of Physalis mottle virus (PhMV), Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), or a Cowpea chlorotic mosaic virus (CCMV). In one aspect, the VLP is derived from a PhMV.

[0151] In some embodiments, the hybrid virus hydrogel further comprises a therapeutic drug agent. In one aspect, the therapeutic drug agent is a chemotherapeutic agent, for example doxorubicin, mitoxantrone, 5-fluorouracil-l -acetic acid (5-FA), cis-platin, oxaliplatin, or irinotecan.

[0152] In one aspect, the diameter of the VLP is selected from: about 10 nm to about 100 nm, or from about 20 nm to about 80 nm, or from about 30 nm to about 60 nm, or from about 30 nm to about 40 nm, or from about 30 nm to about 35 nm, or about 33 nm.

[0153] In some embodiments, the VLP concentration is approximately 0.01-1 wt% of the hydrogel or swelled hydrogel, approximately 0.01-0.5 wt% of the hydrogel or swelled hydrogel, approximately 0.01-0.25 wt% of the hydrogel or swelled hydrogel, or approximately 0.01-0.1 wt% of the hydrogel or swelled hydrogel.

[0154] In some embodiments, the hydrogel or swelled hydrogel has at least a, 30%, at least a 40%, or at least a 50% increased VLP loading value compared to a hydrogel or swelled hydrogel in which the VLP is not bound to the polymeric network via the adize-yne linkage (FIG. 3). As used herein, VLP loading value refers to the ability of the hydrogel or swelled hydrogel to incorporate the VLP. As used herein, VLP loading value may also be referred to as VLP loading.

[0155] In one aspect, the VLP is detectably labelled. In one aspect the VLP is labelled with a marker molecule.

[0156] Therapeutic Methods of Use of the Hybrid Virus Hydrogel

[0157] In some aspects, the VLP in the hybrid virus hydrogel has therapeutic potential, used to treat a subject in need. In some aspects, the VLPs are immunomodulators and can be used as adjuvants. In other aspects the VLPs can function as a vaccine. In one aspect, the hybrid virus hydrogel is the sole active agent (e.g., drug or therapeutic) in the vaccine. In other aspects, the hybrid virus hydrogel is not the sole active agent in the vaccine and is combined with other active agents, drugs or therapeutics.

[0158] In the methods of use described herein, according to some aspects, the hybrid virus hydrogel is used as a drug delivery vehicle. Thus, in one aspect, the hybrid virus hydrogel further comprises a therapeutic drug agent such as a chemotherapeutic agent. The methods described herein can be used to administer a therapeutic drug agent to a subject in need. According to some aspects, the chemotherapeutic agent is selected from doxorubicin, mitoxantrone, 5-fluorouracil-l -acetic acid (5-FA), cis-platin, oxaliplatin, or irinotecan. In some aspects, the therapeutic drug agent is a different drug.

[0159] In some aspects, both the VLP and a therapeutic drug agent conjugated to the VLP have therapeutic potential.

[0160] In some aspects, the therapeutic drug agent is bound to the VLP via an imino bond (FIG. 18). In some aspects, the VLP is bound to the hydrogel or swelled hydrogel via an imino bond (FIG. 19). In some aspects, the therapeutic drug agent is bound to the VLP via a first imino bond and the VLP is bound to the hydrogel or swelled hydrogel via a second imino bond. The imino bond(s) can be targeted by chemical cue to degrade the hybrid virus hydrogel, thereby releasing the therapeutic drug agent bound to the VLP. The rate of degradation can be altered by the chemical cue, thereby altering the rate of release of the therapeutic drug agent from the hybrid virus hydrogel. Chemical cue can include acidic pH, protons, amnio-based small molecules including lysine or pyridoxal phosphate, or other cues.

[0161] In some aspects, the therapeutic drug agent is bound to the VLP via a different cleavable bond, such as a labile ester bond, a reducible thiol bond, or an enzyme cleavable bond. In some aspects, the VLP is bound to the hydrogel or swelled hydrogel via a different cleavable bond, such as a labile ester bond, a reducible thiol bond, or an enzyme cleavable bond.

[0162] Provided herein is a method to deliver a VLP or hybrid virus hydrogel to a cell or tissue, comprising contacting the cell or tissue with an effective amount of the hybrid virus hydrogel, or the composition of this disclosure, thereby delivering the VLP or hybrid virus hydrogel to the cell or tissue. The contacting is in vitro, ex vivo, or in vivo, and / or the cell is animal or plant cell or tissue.

[0163] Also provided herein are methods to administer a VLP or hybrid virus hydrogel to a subject in need thereof, comprising administering to the subject an effective amount of the hybrid virus hydrogel, or the composition of this disclosure, thereby delivering the VLP orhybrid virus hydrogel to the subject. In one aspect, the method induces an immune response in the subject in need. The subject can be a mammal, optionally a human. According to some embodiments, the subject in need thereof has cancer. In some embodiments, the subject in need thereof is in remission.

[0164] In one aspect, provided herein is a method inhibiting, delaying, slowing down, or preventing relapse of cancer in a subject in need thereof. The method comprises, consists essentially of, or consists of administering to the subject a hybrid virus hydrogel, thereby inhibiting, delaying, slowing down, or preventing the relapse of the cancer in the subject.

[0165] In another aspect, provided is a method for treating cancer in a subject in need thereof. The method comprises, consists essentially of, or consist of administering to the subject a hybrid virus hydrogel, thereby treating the cancer in the subject.

[0166] The method can further comprise, consist essentially of, or yet consist of resection of the cancer prior to, or after administration of the hybrid virus hydrogel, or the composition of this disclosure. Alternatively, a different cancer therapy can be administered before, after, or at the same time as the disclosed hybrid virus hydrogel.

[0167] According to another aspect, provided herein is a method to deliver a VLP to a cell or tissue in need thereof, comprising contacting the cell or tissue with an effective amount of the hybrid virus hydrogel or composition as described herein, wherein the VLP and / or therapeutic drug agent is released over a time period to the cell or tissue at an amount less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% than the amount released in a hybrid virus hydrogel wherein the VLP is not covalently bound to the hydrogel, e.g., of a corresponding VLP in a hydrogel or swelled hydrogel which does not comprise an alkyne moiety. According to one embodiment wherein the time period is up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, or up to eight weeks. See FIG. 7 and FIG. 15. In some embodiments, the time period is shorter than one hour. In some embodiments the time period is longer than two months. In some aspects, the contacting is ex vivo, in vitro or in vivo and / or the cell is animal or plant cell or tissue.

[0168] According to one aspect, provided herein is a method to deliver a VLP to a subject in need thereof, comprising administering to a subject in need thereof an effective amount of the hybrid virus hydrogel or composition as described herein, wherein the VLP and / or therapeutic drug agent is released over a time period to the subject at an amount less than 40%,less than 50%, less than 60%, less than 70%, or less than 80% than the amount released in a corresponding hybrid virus hydrogel wherein the VLP is not covalently bound to the hydrogel, e.g., VLP in a hydrogel or swelled hydrogel which does not comprise an alkyne moiety. According to one embodiment wherein the time period is up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, or up to eight weeks. See FIG. 7 and FIG. 15. In some embodiments, the time period is shorter than one hour. The subject can be a mammal, optionally a human. According to some embodiments, the subject in need thereof has cancer. In some embodiments, the subject in need thereof is in remission.

[0169] In one aspect, the hybrid virus hydrogel is administered to the subject as an implant, wherein the hybrid virus hydrogel is inserted next to a target tumor. The implant can be removably implanted next to the tumor. In this way, the hybrid virus hydrogel releases the VLP and / or therapeutic drug agent to the tumor over time.

[0170] In the methods of use described herein, the cancer may be a solid tumor such as bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer. In some aspects, the tumor is a tumor that is difficult to operate on, for example pancreatic cancer or prostate cancer.

[0171] In the methods of use described herein, the VLP and / or therapeutic drug agent release rate can be slowed or hastened. For example, 1) by decreasing the pore sizes of the hydrogels in order to slow down the release of the VLPs (Nat Rev Mater 1, 16071 (2016)), 2) tailoring the polymer backbone of the hydrogel so that it forms weak interactions with the VLPs that slow down the release of the latter (Adv. Sci. 2018, 5, 1700991) (for example, designing negatively charged hydrogels that interact electrostatically with the positively charged PhMV VLP surface.) and / or 3) tailor the hydrogel so that it interacts covalently with the VLP (Materials Today Chemistry 38 (2024) 102100). Further, as previously mentioned, the VLP and / or therapeutic drug release rate can be influenced by acidity and small molecule cues.

[0172] In the methods of use, in one aspect the VLP is selected from a plant virus, an animal virus, or a bacteriophage. In one aspect, the VLP is selected from a Picornavirus, for example a Comovirus, a Tymovirus or other plant virus with an icosahedral shaped plasmid. In some aspects, the VLP is selected from the group of Physalis mottle virus (PhMV), Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), or Cowpea chlorotic mosaic virus (CCMV). Inone aspect, the VLP is derived from a PhMV. In another aspect, the diameter of the VLP is selected from: about 10 nm to about 100 nm, or from about 20 nm to about 80 nm, or from about 30 nm to about 60 nm, or from about 30 nm to about 40 nm, or from about 30 nm to about 35 nm, or about 33 nm.

[0173] Compositions

[0174] Compositions, including pharmaceutical compositions comprising, consisting essentially of, or consisting of a component or a combination as described herein, can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. The component or combination can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the component or combination provided herein into preparations which can be used pharmaceutically.

[0175] The component or combination of the present disclosure can be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant), oral, by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.

[0176] In one embodiment, this technology relates to a composition comprising a component or a combination as described herein and a carrier.

[0177] In another embodiment, this technology relates to a pharmaceutical composition comprising a component or a combination as described herein and a pharmaceutically acceptable carrier.

[0178] In another embodiment, this technology relates to a pharmaceutical composition comprising a therapeutically effective amount of a component or a combination as described herein and a pharmaceutically acceptable carrier.

[0179] The pharmaceutical compositions for the administration of a component or a combination as disclosed herein can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy. The pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing thecompounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, each component provided herein is included in an amount sufficient to produce the desired effect. For example, pharmaceutical compositions of the present technology may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation. In one aspect, administration is intraperitoneal.

[0180] For topical administration, the component or the combination can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well-known in the art.

[0181] Systemic formulations include those designed for administration by injection (e.g, subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection) as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

[0182] Useful injectable preparations include sterile suspensions, solutions, or emulsions of the compounds provided herein in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. The formulations for injection can be presented in unit dosage form, e.g, in ampules or in multidose containers, and may contain added preservatives.

[0183] Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, and dextrose solution, before use. To this end, the component or the combination provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0184] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0185] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wettingagents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art with, for example, sugars, films, or enteric coatings.

[0186] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the combination of compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., corn starch or alginic acid); binding agents (e.g. starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). The tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques well known to the skilled artisan. The pharmaceutical compositions of the present technology may also be in the form of oil-in- water emulsions.

[0187] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.Dosages and Dosing Regimens

[0188] The appropriate amount and dosing regimen of the component or the combination, when present to be administered to the subject according to any of the methods disclosed herein, may be determined by one of ordinary skill in the art.

[0189] In some embodiments, the component or the combination as disclosed herein, may be administered to a subject in need thereof, either alone or as part of a pharmaceutically acceptable formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.

[0190] Administration of the component or the combination as disclosed herein may be effected by any method that enables delivery of the component or the combination to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration. Bolus doses can be used, or infusions over a period of 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, 120 or more minutes, or any intermediate time period can also be used, as can infusions lasting 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 16, 20, 24 or more hours or lasting for 1-7 days or more. Infusions can be administered by drip, continuous infusion, infusion pump, metering pump, depot formulation, or any other suitable means.

[0191] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0192] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure.

[0193] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present disclosure encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

[0194] Experimental

[0195] Example 1 -- Swell-and-click method for the covalent attachment of virus-like particles to polymer hydrogels

[0196] Materials and Methods

[0197] Expression and preparation of PhMV - VLPs

[0198] BL21 glycerol stocks containing pRSETa-PhMV coat protein (CP) and pET-PhMV CP previously reported

[0024] were inoculated into 50 mL of Lysogeny Broth (LB) containing 50 ug / mL of carbenicillin and 50 ug / ml kanamycin and the culture was incubated for 18 h (37 °C, 225 rpm). 10 ml of the preculture were then inoculated into 500 mL of Terrific Broth (TB) also containing carbenicillin and kanamycin 50 ug / ml and allowed to incubate (37°C, 250 rpm). When culture density reached an ODeoo-l (approximately 8 hours), protein expression was induced with 0.5 mM isopropyl-P-D-1 -thiogalactopyranoside (IPTG; Gold Biotechnology), and the cells were incubated overnight (30 °C, 250 rpm). The culture was then centrifuged (7500 x g, 10 min, 4 °C), the cell pellet was resuspended in 50 mM sodium citrate buffer (SCB) and the resulting suspension was sonicated (10 min, 30% amplitude, 5 sec ON, 2 sec OFF) and centrifuged (10000 x g, 10 min, 4 °C). The supernatant was collected and centrifuged (27000 x g, 30 min, 4 °C). The supernatant was then subjected to ultracentrifugation (35000 rpm, 3h, 4°C, 50.2 Ti rotor, Beckman Coulter). The resulting pellets were resuspended in SCB overnight. Insoluble material was removed by centrifugation (15000 x g, 10 min, 4 °C).Supernatant was extracted with 1 : 1 CHCh / BuOH and the aqueous layer was isolated by centrifugation (5000 x g, 10 min, 4 °C). The resulting solution was centrifuged (15000 x g, 10 min, 4 °C) to remove insoluble material. The suspension was then layered carefully onto a 10-40 % (w / v) linear sucrose gradient and ultracentrifuged (28000 rpm, 3h, 4°C, SW32 rotor, Beckman Coulter). The light scattering fractions were pooled, diluted in SCB 1 : 1, and concentrated by ultra-centrifugation (42000 rpm, 3h, 4°C, 50.2 Ti rotor, Beckman Coulter). The clear colorless pellet was finally resuspended in PBS pH 7.2 and stored at 4 °C. The VLP concentration was measured using a bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as a standard.

[0199] Bioconjugation of PhMV VLPs

[0200] General protocol: To a VLP solution in PBS (5 mg / ml), the corresponding bioconjugate reactant was added overnight at room temperature, followed by concentration with ultracentrifugation (52000 rpm, 70 min, 4°C, TLA 55 rotor, Beckman Coulter) over a 100 uL sucrose cushion (20% sucrose in PBS). The pellet was washed, resuspended, and centrifuged at 15.000 x g for 10 min and the supernatant collected.

[0201] PhMV-cy5.5: sulfo-Cyanine5.5 maleimide (5 eq / CP, Lumiprobe) was added to target inner cysteines (C75). Yields: 45%. PhMV-yne: sulfo dibenzocyclooctyne (DBCO)- PEG3-NHS ester (10 eq / CP, BroadPharm) and sulfo-Cyanine5.5 maleimide (5 eq / CP, Lumiprobe) were added one-pot to target external lysines (K62, K143, K153, and K166) and internal cysteines, respectively. Yields: 55%. PhMV-DBCO: sulfo-DBCO-PEG3-NHS ester (10 eq / CP, BroadPharm) was added to target external lysines. PhMV-(C)lick'. AC-PEG2K-N3 (120, 600, and 1200 eq / CP, Creative PEGworks) was added to PhMV-DBCO solution in PBS (1 mg / ml) following a strain-promoted azide-alkyne cycloaddition (SPAAC).

[0202] Structure of sulfo dibenzocyclooctyne (DBCO)-PEG3-NHS ester:

[0203] Characterization of PhMV VLPs

[0204] Size Exclusion Chromatography (SEC) was carried out using a Superose-6 Increase10 / 300 GL column (Pi = 5 MPa, AP = 5 MPa) on the Akta Pure system (Cytiva). The column was loaded with 250 pL sample (1.4 mg / mL) and the sample was run for 50 min at a flow rate of 0.5 mL / min in PBS buffer. Denatured protein subunits (5 p) were analyzed by polyacrylamide gels electrophoresis using 12% NuPAGE gels and l x MOPS buffer (Invitrogen). Samples were denatured by boiling in 1 : 10 reducing agent and 1 :4 SDS blue loading dye for 5 min. Intact PhMV VLPs (5 pg) were analyzed using 0.8% (w / v) native agarose gel electrophoresis in 0.1 M Tris-borate-EDTA (TBE) running buffer (pH 8.3). All gels were stained in Coomasie blue staining followed by photography under UV or far-red light in a FluorChem R system (Bio-Techne). Dynamic light scattering (DLS) analysis was performed in a Zetasizer Nano ZSP / Zen5600 instrument (Malvern Panalytical, Malvern, UK). All VLP samples were measured at a concentration of 0.4 mg / ml in PBS. Transmission electron microscopy (TEM) analysis was performed using a JEOL 1400 plus high-resolution EM operating at 80 kV and equipped with a bottom-mount Gatan One View (4k x 4k) camera. For imaging, samples were prepared by dip-casting VLP solutions (1 mg / ml) onto carbon-coated grids (Electron Microscopy Sciences) and washed with DI water (3x) prior to staining with 2% (w / v) uranyl acetate.

[0205] Synthesis of Hydrogels (HGs) and VLP hydrogels (VHGs)

[0206] All reagents were purchased from Aldrich except otherwise stated. Hydrogels (HGs) were synthesized via free radical photopolymerization as follows: Polyethylene glycol methacrylate MW = 480 Da (PEGMA, 200 mg / ml, 417 mM), acrylamide (AAm, 150 mg / ml, 2110 mM), polyethylene glycol diacrylate MW = 700 Da (PEGDA, 2.5 mg / ml, 3.6 mM) and lithium aryl phospinate (LAP, 5 mg / ml, 17 mM) were dissolved in PBS followed by addition of Acrylate-PEG2K-azide MW = 2000 Da (AC-PEG2K-N3, 100 mg / ml, 48.3 mM, Creative PEG works) in PBS. 300 uL of yellowish mix were then casted into disk-shaped PDMS molds (8 mm diameter x 3 mm height, SYLGARD®184 kit) and cured for 10 min ( = 254 nm, 120,000 pj / cm2) in a UV reactor (CL-1000 254 UV crosslinker, UVP). HGs were then washed in MilliQ water, which was replenished (twice a day, four days), and dried gently at 30 °C until full dryness (xerogel). VLP-laden hydrogels (VHGs) were then designed following a swell- and-click approach. Namely, 300 uL PhMV-cy5.5 or PhMV-yne VLP solutions in PBS (1 mg / ml, 0.1 wt%) were added onto fully dried HG samples (~80 mg) in a sealed vial until full swelling uptake. After equilibration, the VHGs were gently dried to remove remnant water.Two xerogels were this way prepared, VLP -native hydrogel (VN-HG) and VLP-clicked hydrogel (VC-HG) from the PhMV-cy5.5 and PhMV-yne solutions, respectively.

[0207] Characterization of Hydrogels (HGs) and VLP hydrogels (VHGs)

[0208] Thermogravimetric analysis (TGA) was performed by analyzing sliced xerogel samples (25-40 mg), lyophilized VLPs (1.5-4 mg) or solid monomer powders (5-30 mg) on a Discovery SDT 650™ (TA Instruments™) simultaneous DSC / TGA. Samples were equilibrated at 50 °C, then ramped up at 10 °C / min until 1200 °C under N2 atmosphere. Mechanical properties of the materials were measured in a TA Instruments™ Discovery™ HR- 30 Hybrid Rheometer. Fully dried xerogels were swollen with 100 uL PBS to obtain disk samples of 9 mm diameter x 3 mm height after equilibration. Samples were then measured both for axial and frequency sweep tests. For axial compressive tests (co = 0 rad / s), samples were compressed with a stainless-steel geometry (8.0 mm parallel plate ETC stainless steel) at an axial force of 0.3N, gap speed of 100 pm / s and 75% initial strain. For frequency sweep studies, the same conditions were followed, however angular velocity was set to co = 1-250 rad / s. For both sets of experiments, 5 consecutive cycles were performed. Swelling degree (SD) studies were performed using a gravimetric method. Dried xerogel samples (~95 mg) were immersed in ultrapure water, then placed on filter paper to blot excess water and weighted at defined time intervals. The SD was calculated according to the formula: SD = (WT-WO) WOJ* 1OO, where WT and Wo are the weights of the swollen hydrogel at time T and at time T = 0, respectively. The porosity of the hydrogels was investigated using a Zeiss Sigma 500 scanning electron microscope (SEM) operated at 3 kV accelerating voltage with a SE2 Everhart Thornley and EDS 80 mm2 Oxford detector. Fully swollen hydrogels were freeze-dried and sliced prior to introduction in the vacuum chamber. Fourier transform infrared (FTIR) of xerogel samples (70-100 mg) was carried out in a Nicolet™ iS50 and 6700 spectrometer, fitted with Smart- iTR™ diamond Attenuated Total Reflectance (ATR) at a range of 500-4000 cm’1. Samples for each technique were measured in triplicate.

[0209] VLP loading and in vitro VLP release studies

[0210] VLP loading and release readings were quantified through UV / Vis absorbance spectroscopy using an Infinite® M Plex, multimode microplate reader. Cy5.5-labeled PhMV VLPs were measured at kmax = 675 nm followed by absorbance interpolation in freshly prepared calibration curves. For the loading studies: dried HG xerogels (Wo = 70-100 mg, SDmax= 0 wt%) were immersed in 4.95 mL PhMV-cy5.5 or PhMV-yne solutions in PBS at initialconcentrations (Ci) of Ci = 0.1, 0.5 and 0.75 mg / mL (0.01, 0.05 and 0.075 wt%, respectively) for 5 days until VHGs reached maximum swelling (Wo = 1500-2200 mg, SDmax = 1950-2500% wt%). The amount of loaded VLPs in the VHGs was then determined through UV / VIS detection as the amount not recovered in the PhMV-yne and PhMV-c5.5 loading solutions after hydrogel loading. Namely, VLP loading (%) = [(Mt - Mu) / Mt]*lQQ where Mt and Muare the total VLP added masses and the unloaded VLP masses in the loading media. For in vitro VLP release: HG xerogels (Wo = 70-100 mg, SDmax = 0 wt%) were immersed in 4.95 mL PhMV- cy5.5 or PhMV-yne solutions in PBS at Ci = 0.75 mg / mL until maximum swelling (Wo = 1950- 1990 mg, SDmax= 1950-1980 wt%). The fully loaded VHGs were then immersed in 15 ml PBS and incubated at 125 rpm for two weeks at room temperature and several months at 4°C. The cumulative release of VLPs from the VHGs was determined through UV / VIS detection of small aliquots (330 uL) at different timepoints, before pipetting back to the release medium. Namely, cumulative VLP release (%) = [(MR / (MI - MU)]* 100 where MR, Mt, and Muare the released VLP mass at each timepoint, the total VLP added masses and the unloaded VLP masses in the loading media, respectively.

[0211] Results and Discussion

[0212] Synthesis and characterization of PhMV VLPs

[0213] Physalis mottle virus (PhMV) was chosen as a model VLP. PhMV is a ~30 nm icosahedral plant virus with T= 3 symmetry, comprising 180 identical ~20 kDa coat protein (CP) subunits.

[0025] Homogeneous and monodisperse PhMV VLPs were obtained by CP expression in bacteria.

[0024] Applicant chose click chemistry for the covalent attachment of VLPs to hydrogels because this reaction is widely used for protein bioconjugation due to its efficiency, selectivity, and bio-orthogonality.

[0026] Although copper-based click chemistry is suitable for VLPs,

[0027] it depends on specific pH and catalytic conditions.

[0028] Therefore, Applicant used strain-promoted azide alkyne cycloaddition (SPAAC) as a simpler, catalyst- free click alternative. Applicant functionalized the VLPs with a ring-strained dibenzocyclooctyne (DBCO) group for that purpose. Applicant also conjugated the VLPs to a cyanine dye, allowing quantification during hydrogel loading and release. This dual modification was carried out in a one-pot process targeting different sites with spatial control (FIG. 1A). Specifically, sulfo-cyanine5.5 mal eimide and DBCO-PEG3-NHS ester were added to the VLP solution to target internal cysteines (C75) and surface-exposed lysines (K62, K143, K153 and K166), respectively.

[0029] The resulting alkynylated, cy5.5-labeled particle was named PhMV-yne. Applicant also synthesized an alkyne-free, cy5.5-labeled VLP as a comparative control, which was named PhMV-cy5.5. Both particles were produced with a yield of 45-55%.

[0214] Successful functionalization and particle integrity were demonstrated using a combination of techniques. Denaturing electrophoresis (FIG. IB) revealed the characteristic native CP band in PhMV (lane 1) at ~20 kDa. Functionalization of the VLP with sulfo-DBCO- PEG3-NHS (lane 2) resulted in a slightly higher molecular weight, shown as smearing above the CP band. Clear separation does not occur due to the low molecular weight of the linker (756.8 Da). The presence of fluorescent bands when gels were imaged under red light confirmed cy5.5 conjugation, which was quantified by UV / vis spectrophotometry. PhMV-yne (lane 3) displayed 50-55 cy5.5 dye molecules per VLP (29% total) and PhMV-cy5.5 (lane 4) displayed 70-75 per VLP (41% total).

[0215] Native electrophoresis also confirmed the bioconjugation of VLPs according to changes in particle mobility or fluorescence (FIG. 1C). Positively charged PhMV

[0030] (lane 1) migrates toward the cathode (-), whereas functionalization with sulfo-DBCO-PEGs-NHS (lane 2) creates a negative charge and causes migration toward the anode (+). This is typically observed when PhMV surface lysines are neutralized during NHS ester conjugation.

[0031] Conversely, cy5.5 labeling was confirmed by the presence of fluorescent bands in the PhMV- yne (lane 3) and PhMV-cy5.5 (lane 4) lanes, with the former also shifting mobility due to the successful alkynylation of lysines with DBCO. Both VLP solutions appeared bright blue due to dye labeling (FIG. ID). Size exclusion chromatography showed identical elution volumes for all VLPs, confirming similar particle sizes (FIG. 1C). The hydrodynamic radius was 32- 33 nm as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM) (FIGS. IF and 1G). DLS showed that the VLPs were monodisperse and homogeneous, and TEM imaging confirmed their uniform and icosahedral structure (FIG. 1H).

[0216] In these electrophoretic experiments, Applicant also shows the red fluorescence of the particle intensifies as the equivalent of red dye is increased (FIG. 17). This indicate the particle is successfully conjugating Cy5 dye to its interior. This is possible due to the VLP hosting cysteine amino acids inside the capsid protein that undergo Michael Addition with the maleimide dyes. VLP coat proteins with increased numbers of cysteines have increased loading capacity. A PhMV engineered variant with a greater number of cysteines than the wild-type PhMV (PhMV Cys 3x, with cysteines substituted at A31C and S137C in the PhMV coat protein) was more successful at conjugating the dye than the wild type variant “PhMV WT”, since it has more host cysteine amino acids per coat protein.

[0217] Synthesis of control hydrogels and VLP -laden hydrogels

[0218] The control hydrogel (HG) was synthesized by free-radical photopolymerization (FIG. 2). Briefly, acrylate-PEG2K-azide (AC-PEG2K-N3), polyethylene glycol methacrylate (PEGMA), acrylamide (AAm) and polyethylene glycol diacrylate (PEGDA), were dissolved with the photo-initiator lithium arylphosphonate. The yellow viscous mix was then cast into disk-shaped PDMS molds and cured in a UV reactor for 10 min. The resulting hydrogels (FIG. 2A) were washed in water for 4 days, with water replenished twice per day, to remove unreacted components and then gently dried to form HG xerogels consisting of a terpolymer network crosslinked with PEGDA and featuring pendant azide groups (FIG. 2).

[0219] The VLP-laden hydrogels (VHGs) were then synthesized using the swell-and-click method. A fully dried HG xerogel was swollen in a PhMV-yne solution (300 pL, 1 mg / mL, 0.1 wt%) until all the liquid had been taken up. During swelling, the alkynylated VLP underwent SPAAC with the azide pendant groups, producing a NLP-clicked hydrogel (VC-HG) in which the VLP was bound covalently to the polymer backbone of the HG (FIG. 2B). The VC-HG was then carefully dried to restore the xerogel state. As a comparative VHG control, an HG xerogel was also swollen in a PhMV-cy5.5 solution (300 pL, 1 mg / mL, 0.1 wt%) producing a VLP-native hydrogel (VN-HG, FIG. 2C) that was subsequently dried to give the corresponding xerogel in which the VLP is not covalently bonded to the HG scaffold.

[0220] Chemical reactions between VLPs and a swelling hydrogel have not been reported thus far. Accordingly, Applicant validated the swell-and-click approach by testing the SPAAC reaction between the alkylated VLPs, PhMV-yne (1 mg / mL, 0.1 wt%), and the azide monomer (AC-PEG2K-N3) at increasing concentrations (0, 10, 50, 100 mg / mL, the latter being the monomer concentration present during hydrogel synthesis). As a control, the same reaction was prepared using PhMV-cy5.5 (lacking the alkyne handle). As expected, electrophoresis showed no evidence of a click reaction between PhMV-cy5.5 and the azide monomer, regardless of the monomer concentration. Only PhMV CPs (~20 kDa) were detected by denaturing SDS-PAGE (FIG. 8A left). Conversely, two CP bands were observed by denaturing SDS-PAGE when PhMV-yne was mixed with the azide monomer (FIG. 8A, right). These represented the normal PhMV CP and a higher-molecular-weight version carrying the AC- PEG-N3 linker (MW 2 kDa). Comparative analysis of these bands using ImageJ software indicated that 230-270 lysines per VLP undergo SPAAC with the azide monomer, approximately one third of available lysines. The click reaction was also confirmed by native electrophoresis, where the clicked particles showed lower mobility than PhMV-yne due to thehigher radius after functionalization (FIG. 8B, right). Interestingly, this preliminary test showed that click chemistry between PhMV-yne and AC-PEG2K-N3 was successful even at 10-fold lower azide concentrations (10 mg / mL) than those used for the hydrogel synthesis (100 mg / mL). Changes in migration were not observed for PhMV-cy5.5 in native electrophoresis experiments because no reaction takes place between these particles and the AC-PEG2K-N3 monomer (FIG. 8B, left).

[0221] Characterization of control hydrogels and VLP-laden hydrogels

[0222] The simplest way to validate the swell-and click method is a VLP loading test, which determines the maximum efficiency of the hydrogels to incorporate VLPs during swelling. Previous reports show that hydrogels can react during swelling when the reactants are present in the same aqueous solution.

[0032] Success in the loading test is mainly determined by the chemical reactivity of the hydrogel and VLP, as well as VLP solubility and concentration in the swelling solution.

[0033]

[0223] Applicant immersed a fully dried HG xerogel in a large volume of VLP solution to cover several times the maximum swelling capacity (SDmax) of the HG (e.g., 5 mL for a xerogel with a mass of 70-100 mg). The PhMV-cy5.5 and PhMV-yne solutions were tested at initial concentrations of Ci = 0.1, 0.5 and 0.75 mg / mL (0.01, 0.05 and 0.075 wt%, respectively). Applicant then calculated SDmaxfor the hydrogel in the VLP solution and the VLP loading after the swelling process. The SDmaxis the percentage weight gain in water and VLP of a hydrogel after full swelling and is calculated using the equation S ) = [(Ws - Wo) / Wo]x100, where Ws and Wo are the weights of the fully swollen and fully dried hydrogels, respectively. VLP loading is calculated by UV / vis analysis (Xcy5 = 675 nm) of the VLP swelling solution using the formula VLP loading (%) = [(Mt - Mu) / Mt]*lQQ, where Mt and Mu are the total mass of VLPs added and the total unloaded VLP mass remaining in the supernatant, respectively. UV / VIS spectroscopic data can be found in FIG. 9.

[0224] FIG. 3A shows the maximum swelling capacity (SDmax) of the HG in each VLP solution. The SDmaxwas roughly the same for both VLPs, which means the hydrogel always incorporates the same mass of water regardless of the VLP type. However, the SDmaxdeclined slightly as the VLP concentration increased SDmax= 2480 ± 28%, 2124 ± 8.0% and 1954 ± 1.5 wt% for PhMV-cy5.5 at Ci = 0.1, 0.5 and 0.75 mg / mL, respectively; SDmax= 2408 ± 75%, 2136 ± 24% and 1979 ± 87 wt% for PhMV-yne at Ci = 0.1, 0.5 and 0.75 mg / mL, respectively).This typical trend is observed in hydrogel swelling studies whereby increasing the concentration of the swelling solution leads to lower swelling ratios.

[0034]

[0225] Conversely, the loading percentage clearly differed between the two VLPs (FIG. 3B) The clickable alkyne-modified VLP achieved higher loading values of 46.2%, 56.2% and 34.7% at Ci = 0.1, 0.5 and 0.75 mg / mL, respectively, compared to the PhMV-cy5.5 control with values of 33.3%, 42.2% and 15.5% at the same initial concentrations. At Ci = 0.75 mg / mL, the loading value of PhMV-yne was more than double that of PhMV-cy5.5, whereas the difference was only 1.3-1.4-fold at lower VLP concentrations. This suggests VLP loading is a function of both the VLP chemistry and Ci. The higher VLP loading observed for PhMV-yne indicates that the particles undergo a click reaction with the hydrogel during swelling, even at VLP concentrations as low as 0.1 mg / mL (0.01 wt%), confirming the suitability of the swell- and-click approach.

[0226] Thermogravimetric analysis

[0227] Thermogravimetric analysis (TGA) measures the change in mass of a sample over time as the temperature changes. Given that the thermal stability of a material strongly depends on its chemical structure, Applicant can infer the presence of new chemical bonds by thermal decomposition. Furthermore, differential thermograms (DTGs) reveal subtle differences between TGA profiles. Applicant therefore prepared DTGs for the monomers, hydrogel and VLPs to identify the chemical nature of each temperature loss (FIG. 4) and then analyzed the differential thermograms of the VLP hydrogels (FIG. 4). The full range thermograms (TGs) and differential thermograms of the former and latter groups can be found in FIGS. 10 and 11, respectively. Further, each weight loss and temperature are summarized in Table 1.

[0228] Table 1. Summary of weight (wt%) and temperature (°C) for each TG loss.

[0229] The differential thermograms of the hydrogel and its monomeric components AAm and AC-PEG2K-N3 were plotted in the range 250-450 °C (FIG. 4A) along with the corresponding thermograms (FIG. 10). The hydrogel showed a shoulder-like loss at 295 °C that matches the AAm monomer and this reflects intramolecular decomposition reactions in the polyacrylamide backbone.

[0035] A second loss at 390 °C was attributed to the AC-PEG2K- N3 monomer as well as the C-C main backbone in AAm. [36,37] The presence of both monomers in the hydrogel backbone confirmed the structure of the hydrogel (FIG. 4A).

[0230] The differential thermograms of the VLPs were plotted in the range 150-450 °C (FIG. 4B). The differential thermogram of PhMV showed losses at 220 and 285 °C. The loss at 220 °C was barely apparent for PhMV-cy5.5, indicating it is caused by thermal decomposition of the free cysteine residues in PhMV. These residues are covalently conjugated to the mal eimide dye in PhMV-cy5.5, which improves their thermal stability and reduces the magnitude of the loss. The second loss at 285 °C was partially quenched in PhMV-DBCO, indicating this loss is caused by the thermal decomposition of free lysine residues. Their thermal stability increases when they are conjugated to NHS esters, also reducing the peak loss. As expected, the cysteine loss at 220 °C in PhMV-DBCO is unchanged because no cysteines were conjugated in this VLP.

[0231] Further evidence of VLP conjugation can be observed in the high-temperature region (FIG. 10) The loss at 880 °C in PhMV, probably caused by decomposition of the protein backbone,

[0038] notably increases in PhMV-DBCO and PhMV-cy5.5. This accelerated thermal decomposition in the conjugated VLPs is consistent with a higher number of ester and thiosuccinimide bonds (the end products of lysine and cysteine conjugation, respectively) in the polypeptide backbone, which decompose in the high-temperature range.

[0232] The differential thermograms of the VLP -native hydrogel, hydrogel and PhMV- cy5.5 (FIG. 4C) show that the VLP-native hydrogel (the result of incorporating PhMV-cy5.5 into the HG) shares a very similar thermal profile with the precursor hydrogel, with two main losses at 295 and 390 °C. The 390 °C loss in VLP-native hydrogel can be attributed solely to the hydrogel polymer backbone, although the 295 °C loss can be deconvoluted into the AAm loss corresponding to the amide groups in the hydrogel and the loss attributed to the lysineresidues in the PhMV-cy5.5 particle at 285 °C. Due to the co-decomposition of both groups at this temperature, the loss at 295 °C is greater in VLP-native hydrogel than in hydrogel and PhMV-cy5.5 alone.

[0233] FIG. 4D shows the differential thermograms of the VC-HG (the result of incorporating PhMV-yne into the hydrogel), hydrogel, PhMV-yne (including the free alkyne bond) and PhMV-C(lick), wherein the alkyne bond has undergone SPAAC with an azide monomer. Interestingly, the loss at 295 °C is partially quenched in the VLP-clicked hydrogel (VC-HG) compared to the VLP-native hydrogel (VN-HG), which indicates the VC-HG is thermally more stable than the VLP-native hydrogel at this temperature. Given the same AAm contribution in both hydrogels, this improvement in thermal stability can only be attributed to difference in the VLP. In the VC-HG, the alkynylated VLP can exist either as a non-clicked VLP or clicked to the azide pendant groups. By comparing the thermal profiles of these VLPs, it is clear the lysine loss in PhMV-C(lick) is lower than in PhMV-yne, which indicates the clicked PhMV is thermally more stable than its non-clicked counterpart. The reduced loss at 295 °C for the VLP-clicked hydrogel compared to the VLP-native hydrogel therefore indicates that PhMV exists in the clicked state inside the hydrogel, which increases the thermal stability of the lysines and reduces the overall signal loss of VLP-clicked hydrogel at this temperature. These findings support the use of the swell-and-click approach disclosed herein.

[0234] Mechanical Properties

[0235] As previously stated, the PhMV-yne VLP is highly multivalent, with 230-270 clicked junctions per particle and thus has a high potential to crosslink the polymeric network. Applicant therefore compared the mechanical and rheological properties of the hydrogel and VLP -laden hydrogels in compressive strength and frequency sweep tests. These provide insight into the stiffness and viscoelasticity of the materials, both of which are influenced by the degree of crosslinking. [39,40]

[0236] Compressive strength studies involved the application of a uniaxial load to cylindrical hydrogel samples -prepared by swelling the corresponding xerogels in 100 uL PBS- causing them to shorten and spread laterally (FIG. 5A). Hydrogel disk samples were compressed in this manner until a = 75% strain (i.e., 75% sample height) for five consecutive cycles. After compression, the hydrogel and VLP-laden hydrogel samples fully regained their original shape without breaking, thus proving the mechanical integrity of both hydrogel andVLP-laden hydrogels. The average compressive cycles were then plotted as a stress-strain curve (FIG. 5B)

[0237] The compressive stress at maximum strain (a = 70%) of the VLP-laden hydrogels was somewhat lower than that of the control hydrogel, reaching values of 66, 59 and 54 kPa for the hydrogel, VLP-native hydrogel and VLP-clicked hydrogel, respectively (FIG. 5B). In the a = 0-10% region, the Young’s modulus (E) can be extracted from the slope of the stressstrain curve (FIG. 5B inset). This was similar for the hydrogel (16.3 ± 5.68 kPa) and VLP- native hydrogel (15.97 ± 4.89 kPa), but lower for the VLP-clicked hydrogel (12.57 ± 6.02 kPa). These results show that the incorporation of VLPs into the hydrogel produces more elastic, low-moduli materials. Typically, hydrogels with lower moduli and higher elasticities translate into less compact, loosely crosslinked networks.

[0041] In the VLP-laden hydrogel systems, despite the multivalent crosslinking sites of the alkynylated VLP, the latter does not appear to crosslink the hydrogel network at this VLP concentrations (1 mg / mL, 0.1 wt%). Rather, without wishing to be bound by any particular theory, Applicant hypothesizes the particle covalently functionalizes the hydrogel network without network stiffening.

[0238] Rheological analysis under the same compressive conditions was followed by a frequency sweep in the range co = 0-100 rad / s. Applicant plotted the storage modulus (G') and loss modulus (G") against the oscillation frequency (FIG. 12A). Throughout the frequency range, G' > G" with no crossover point for all three hydrogels, the typical correlation observed for solid-like, fully crosslinked hydrogels.

[0042] The phase angle or loss tangent (5) increased from 5° to 25° for all samples throughout the frequency range, which indicates that all hydrogels transition slightly from elastic to somewhat viscoelastic materials (FIG. 12B). The hydrogel and VLP-laden hydrogels both behave as similarly crosslinked, solid-like materials that retain their integrity even following high stress.

[0239] The rheological and mechanical tests indicated that the incorporation of the VLPs has a low mechanical impact on the hydrogel, probably reflecting the post-synthesis swell-and- click method. Because the VLPs are swollen and clicked into the hydrogel only after the latter has been crosslinked by PEGDA and the degree of crosslinking has been fixed during synthesis, the VLPs do not appear to contribute significantly to further crosslinking at this VLP concentrations (1 mg / mL, 0.1 wt%), despite their multivalency. This contrasts with previous in situ VLP crosslinking methods, where the covalent binding of VLPs in the hydrogel increased the mechanical strength of the materials, [14, 15, 19] ultimately limiting the number ofVLPs that can be loaded and thus potentially interfering with the ability to match the required therapeutic window in clinical settings.

[0240] Swelling and porosity studies

[0241] The swelling degree (SD) of a hydrogel is defined as its capacity to incorporate water within its porous structure.

[0043] SD and microporosity are two interconnected parameters, along with the mechanical properties, which can indicate whether VLPs exert any crosslinking effect. Typically, hydrogels with a high degree of crosslinking result in more compact networks with smaller pores, reducing the swelling degree.

[0044] Applicant investigated the swelling degree of the hydrogels over time using a gravimetric method. First, Applicant immersed the fully dried hydrogel, VLP-clicked hydrogel and VLP-native hydrogel xerogels in water and measured their weights at different time points, according to the formula SD = [(WT - Wo) / Wo] x 100, where WT and Wo are the weights of the swollen hydrogel at time T and time T = 0, respectively. The swelling degree values are plotted in FIG. 6.

[0242] Burst swelling was observed for all hydrogels during the first day, but a plateau was reached on the third day, with slightly higher swelling degree values for the HG (1800 ± 16 wt%), followed by the VN-HG (1760 ± 15 wt%) and VC-HG (1700 ± 58 wt%). The lower swelling degree value for the VLP-clicked hydrogel suggests the alkynylated particle exerts some crosslinking effect in the hydrogel, albeit to a negligible extent given the small difference compared to the VLP-native hydrogel. A similar conclusion can be drawn from the maximum swelling (SDmaxwhere hydrogel xerogels became swollen in the PhMMV-cy5.5 and PhMV- yne solutions at increasing concentrations (FIG. 3A). There was no decrease in SDmaxvalues over the entire VLP concentration range (0.1-0.75 mg / ml) when the hydrogel was swollen in the PhMV-yne solution compared to PhMV-cy5.5, indicating that the alkynylated VLP, despite its multivalency, does not increase the degree of hydrogel crosslinking.

[0243] These phenomena can be explained by the swell-and-click approach Applicant used to incorporate the VLPs, which takes place once the crosslinking degree of the hydrogel has been fixed during synthesis, and therefore determines beforehand both the mechanical and swelling properties of the materials.

[0244] The porosity of the maximally swollen hydrogels was determined by scanning electron microscopy (SEM). All three hydrogels formed a microporous network with pore sizes in the same range: 251.3 ± 137.8 pm for hydrogel, 200.8 ± 109.9 pm for VLP-native hydrogel and 226.2 ± 116.2 pm for VLP-clicked hydrogel. The morphology of the pores washeterogeneous, as typically observed for free-radical polymerized hydrogels.

[0045] In agreement with the swelling degree data and mechanical properties, the similar pore dimensions and morphologies imply similar degrees of crosslinking independent of VLP functionalization. These results confirm that the swell-and-click post-synthesis approach has advantages over in situ VLP crosslinking methods where the VLPs are incorporated during polymer synthesis because the swell-and-click method did not affect significantly hydrogel properties such as swellability, porosity or mechanical consistency at the current studied VLP range (0.1-1 mg / mL, 0.01-0.1 wt%). It is thus possible that a greater quantity of VLP cargo was covalently attached to the hydrogel without over-stiffening the network or compromising the hydrophilicity and pore sizes.

[0245] FITR spectroscopy

[0246] FTIR spectroscopy of all three samples hydrogel, VLP-native hydrogel and VLP- clicked hydrogel revealed that the most intense bands can be attributed to C-C (2874 cm'1) and C-0 stretch (1088 cm'1) from the PEG moiety,

[0046] which is abundant in both the PEGMA and AC-PEG-N3 monomers (FIG. 13A). Carbonyl C=O strong bands were also detected for acrylate esters (1727 cm'1) and acrylamide (1660 cm'1), as well as the N-H stretching for the latter (3450-3170 cm'1).

[0047] Intense free azide N=N=N bands (here, 2101 cm'1) are reported to disappear following successful click chemistry.

[0048] However, in the swell-and-click system, the N3 stretch was too weak to reveal any proof of such reaction. This reflects the PEG-rich composition of the AC-PEG-N3 commercial monomer used in the hydrogels, which dwarfed the C=O and N=N=N bands, as seen in the spectra for the AC-PEG-N3 monomer (MW 2 kDa) and even at lower PEG weights (MW 1 kDa) (FIG. 13B).

[0247] In vitro VLP release experiments

[0248] VLPs are used to develop vaccines targeting chronic diseases and cancer due to their potent immunogenicity.

[0049] However, long-term treatment is a challenge where repeated administration is required.

[0050] Applicant therefore tested out VLP-laden hydrogels as proof- of-concept long-term reservoirs to release VLPs in a sustained manner. In the VLP-laden hydrogel systems, the robust triazole bond binding the VLP to the hydrogel is not broken or hydrolyzed,

[0051] but the ester bonds in the PEG backbone gradually break under mildly alkaline conditions.

[0052] PEGylated VLPs would therefore be released from the hydrogel as the latter slowly degrades over time, allowing the sustained release of the VLPs over a period of months.

[0249] Hydrogel xerogels (70-100 mg, SDmax = 0 wt%) were swollen in 5 ml of the PhMV- yne or PhMV-cy5.5 solutions at a fixed concentration (Ci= 0.75 mg / ml, 0.075 wt%) until they reached the maximum swollen state (Wo = 1950-1990 mg, SD max = 1950-1980 wt%) (FIG. 14), following the same protocol used for VLP loading (FIG. 3). A baseline control HG xerogel was swollen in PBS. All maximum-swollen hydrogels were then immersed in fresh PBS under stirring and protected from light. At specific time points, an aliquot was withdrawn for UV / vis spectrophotometry (Lvs.s = 675 nm) and the aliquot was returned to the supernatant to keep the final volume constant.

[0250] The cumulative release of VLPs from the hydrogels is plotted in FIG. 7. Nearly 100% of the PhMV-cy5.5 VLPs were released from the VLP- native hydrogel in a burst process after the first 24 h, whereas only 49.6% of the PhMV-yne VLPs were released from the VLP- clicked hydrogel, plateauing afterwards (FIG. 7A). This sharp difference further suggests covalent attachment between the alkynylated VLPs and the hydrogel, and thus corroborates the success of the swell-and-click approach. However, about half of the alkyne VLP cargo was burst-released during the first day, and these are probably alkynylated VLPs that did not click successfully during the VLP loading step, but instead simply diffused into the hydrogel. Given that PhMV-yne loading at Ci = 0.75 mg / ml was 34.7%, and the released VLPs represented 49.6% of that load, the actual load of clicked PhMV-yne VLPs in the hydrogel can be calculated after release as CVLPS = [34.7% x (100 - 49.6%)] / 100 = 17.5%. It follows that 17.5% of all the PhMV-yne particles added during the loading step remained clicked to the hydrogel even after 2 weeks. After that time and having reached a steady release profile, all released PhMV-yne VLPs are presumed to be in equilibrium with the release medium, and thus the hydrogels were immersed again in fresh buffer to encourage the release of the remaining loaded VLPs and study the month-term release (FIG. 7B). After this additional buffer immersion (t = / i months), a milder -10% burst VLP release was observed for VLP-clicked hydrogel, which gradually increased until reaching 63% release of PhMV-yne VLPs after 2 months. These findings also highlight the suitability of the swell-and-click approach to successfully fabricate, for the first time, long-term VLP release reservoirs.

[0251] The successful click of the VLPs significantly reduced the burst release during the early hours of the experiment (FIG. 15), with > 30% of the PhMV-cy5.5 VLPs released from the VLP-native hydrogel after the first hour and 65% after the seventh hour, in contrast to only -16% and -37% of the PhMV-yne VLPs released from the VLP-clicked hydrogel at the same time points. While burst release often leads to premature cargo loss and unwanted sideeffects,

[0053] in immunotherapy / vaccine development the initial burst release is viewed as a positive trait - it mimics the “prime” while the sustained release then mimics the multiple “boosts” but built into a single formulation and administration. The present VLP-clicked hydrogel therefore achieves both, burst release to kick-start the immune system followed by slow-release for sustained efficacy in the time-scale of weeks to months, which is favorable for the desired application.

[0252] Finally, Applicant investigated the structural integrity of the VLPs after release from the hydrogels. Applicant used UV / vis spectrophotometry, which has a low detection limit suitable for highly diluted particles. In the 195-295 nm range, the spectra of the released PhMV-cy5.5 and PhMV-yne VLPs were similar to their counterparts prior to loading and distinct from the profiles of denatured VLPs (FIGS. 16A and 16B). This confirmed that the released VLPs from the hydrogels were not denatured and retained full structural integrity. In the 530-730 nm range, the UV / vis spectra of the conjugated cy5.5 dye in the VLPs (keys.5 = 675 nm) was also preserved after release (FIGS. 16C and 16D), which confirmed that conjugated cy5.5 is not chemically modified after leaving the hydrogel and the UV / vis absorbance readings are therefore reliable. The swell-and-click approach thus allows the covalent bonding of VLPs to a hydrogel without altering the consistency and properties of the scaffold and facilitating the slow release of the cargo. Previously reported in situ VLP crosslinking methods required a minimum viral concentration of 5-10 mg / ml (0.5-1 wt%) for the hydrogel to gelate consistently. [14, 15, 19] In the swell-and-click approach, hydrogel gelation is independent of VLP functionalization, and the minimum VLP load that can be covalently attached to the hydrogel is dependent only on the detection limit of the instrumentation used to quantify particle loading and release, which in this study was as low as 0.1 mg / ml (0.01 wt%).

[0253] Conclusions

[0254] For the first time, Applicant achieved the covalent binding of VLPs to a hydrogel by exploiting its swellability using a novel swell-and-click approach, and thus circumventing the need for high viral loadings to achieve hydrogel gelation. In one aspect, azide- functionalized, mechanically stable hydrogels readily swell in an aqueous VLP solution, during which alkyne VLPs bind to the hydrogel scaffold via copper-free click chemistry. Applicant confirmed covalent functionalization of the VLPs with loadings as low as 0.1-1 mg / mL (0.01- 0.1 wt%) and no apparent changes in the hydrophilicity, mechanical stability, viscoelasticity or microporosity of the hydrogels despite the potential for VLPs to form additional crosslinks. This implies the VLPs do not likely contribute to further crosslinking, which instead is mainlyattributed to PEGDA. Further, the swell-and-click protocol more than doubled the VLP loadings in the hydrogel, and sequentially combined an initial burst VLP release followed by a month-sustained release, both of which are desired for cancer immunotherapy treatment. Applicant’s novel methodology outperforms the previous synthetic approaches in that VLP loadings and hydrogel mechanical stability can now be controlled independently, leading to mechanically stable, long-term release VLP reservoirs. This promising swell-and-click approach facilitates the scalability of the fabrication process and moves a significant step forward towards clinical translation of long-term VLP vaccination in cancer disease.

[0255] Example 2 — Controlled release of anti-cancer drug-loaded virus-like particles (VLPs)

[0256] The layered system is presented as follows: the DOX anticancer drug is, first, chemically (covalently) bound inside of the VLP via an imino bond. Imino bonds are usually labile bonds that cleave with relative ease under mild acidic conditions. Then, the DOX-loaded VLP is infused (not chemically bound) inside the hydrogel. The hydrogel can then then implanted next to the target tissue.

[0257] Due to the VLP being entangled within the intricate polymer mesh of the hydrogel, the VLP will be slowly released out onto the media. This means the delivery of the drug-loaded nanocarrier to the tumor site will be extended, which will ensure a steady, long-term anticancer treatment. Once the DOX-loaded VLP arrives to the tumor microenvironment (TME), the acidic pH of that environment will trigger the cleavage of the imino bond, which will deliver the anticancer drug to the tumor site and attack the cancer cells. The overall system operates as an extended source of anticancer virus particles with target-specificity over the TME.

[0258] PhMV WT and PhMV Cys 3x (engineered PhMV variant with three cysteines) conjugated aldoxorubicin via maleimide chemistry, which was confirmed with intensifying fluorescence in FIG. 18A. Further, FPLC shows co-elution of VLP, Cy5 dye and aldoxorubicin and no elution of free conjugates (FIG. 18C). Thus, Applicant provides a means of successful conjugation of small drugs, such as acid-cleavable doxorubicin, inside VLPs. Further, VLPs can be easily loaded within the hydrogels by a simple swelling process (see e.g. FIG. 18B).

[0259] Experimental — A DOX-loaded VLP is loaded onto the hydrogel, but is not chemically bound to it to the hydrogel. The hydrogel is infused inside the hydrophilic network,which means the release of the VLP from the hydrogel is a simple function of time as the particle diffuses out from the hydrogel onto the medium. D

[0260] Doxorubicin (DOX), is chemically attached to the VLP, specifically to its inner cavity, i.e., inside the capsid protein, through an imino bond (FIG. 18C). The bond is a dynamic covalent bond, which means that, under normal pH conditions (neutral pH), the bond will remain unbroken, whereas under acidic conditions, the bond will be cleaved, and the DOX drug will be released (Small Sci. 2023, 3, 2300067). By tuning the degree of DOX conjugation to the VLPs, the total drug loading into the hydrogels can be customized. The meshed structure that comprises the hydrogel entraps the VLPs and slows down their release, thus incrementing the long-lasting anti-cancer effect of the drug payload.

[0261] Applicant sought to evaluate the release of the chemotherapeutic drug doxorubicin (DOX) within a double-encapsulated system: the VLPs chemically bind the DOX to its internal cavity via an acid-cleavable bond and the DOX-loaded VLPs are then encapsulated within a hydrogel (HG) (FIG. 18B). DOX / VLP-loaded hydrogels will thus operate as a layered-release system whereby the HG slows down the release of the VLP carriers and the VLPs then selectively deliver the DOX cargo to an acidic environment, such as the tumor microenvironment.

[0262] Example 3 - Controlled degradation of virus-based hydrogels

[0263] Applicant sought to investigate the VLP as a released macromolecule, rather than a carrier of another therapeutic agent. The purpose of this project is to harness the imino group to covalently (but reversibly) attach the VLP to the hydrogel and then study the cleavability of the VLP from such hydrogel under chemical stimulus (either acidic pH and / or small molecules containing primary amine groups).

[0264] This release system is presented as follows: first, a VLP is conjugated to a double reactive handle that contains an aldehyde group in one end and an alkyne group in the opposite end. The VLP reacts with the aldehyde end via imino bond to render a VLP containing a cleavable alkyne group. The VLP is now clicked to the hydrogel through the remaining alkyne end following Applicant’s swell-and-click methodology (Materials Today Chemistry 38 (2024) 102100).

[0265] Degradation of the VLP -bound hydrogel network can be triggered by chemical cues which cleave the imino reversible bond. Chemical cue can include protons, small molecules (e.g., amino-based small molecules such as lysine, pyridoxal phosphate, or aniline), acidic pH, or hydrogen peroxide which cleave the imino reversible bond. The VLP release rate from the hydrogel can thus be tuned by controlling exposure to these chemical stimuli. For example, the acidity of the environment can be altered to affect the release rate of the hydrogel. Release rate can also be affected by the concentration of chemical stimulus which can break the imine bond (e.g. concentration of proton, concentration of hydrogen peroxide, concentration of amine compounds) or the pore and mesh size of the hydrogels.

[0266] In some aspects, this system could operate as a long-term implantable VLP reservoir that would be surgically placed next to the tumor. Due to the reversible covalent nature of the imino bond, the VLP will be released selectively upon contact of the hydrogel with the acidic conditions of the TME. Without wishing to be bound by any particular theory, Applicant expects the release of the VLP to be slower due to the imino bond as compared to a VLP that is merely infused or imbedded in the hydrogel. The inclusion of the imino bond in the hybrid virus hydrogel, connecting the VLP to the hydrogel, operates as long-term release formulation of VLPs with target-specificity over the TME, appropriate for cancer immunotherapy.

[0267] Finally, in some aspects, imino bonds may be used to both conjugate the therapeutic drug agent (e.g. DOX) to the VLP and conjugate the VLP to the hydrogel. In this respect, Applicant provides a system whereby, under the proper cue (for example, acidic pH and / or small molecules containing a primary amine group), both the DOX drug would be cleaved from the VLP and simultaneously the VLP would be cleaved from the hydrogel.

[0268] Successful imino bond formation', agarose electrophoresis showed successful conjugation of the VLP to the “clickable” handle through imino bond formation under basic catalytic conditions (FIG. 19). Due to the dynamic covalent nature of the imino bond, the latter will be cleaved under specific chemical cues.

[0269] Successful “click-ability” after conjugation through imino handle: After attachment through the imino handle, Applicant demonstrated the VLP is “clickable” from the alkyne end though copper-free “click” chemistry. This demonstrates the PhMV-DBCO particle can be conjugated to the hydrogel for further degradation studies. FPLC studies also confirmed stability of the PhMV-DBCO particle (FIG. 19).

[0270] Embodiments1. A method of preparing a hybrid virus hydrogel, the method comprising contacting a virus-like particle (VLP) or derivative thereof comprising an alkyne moiety with a swelled hydrogel comprising an azide group.2. The method of embodiment 1, wherein the alkyne moiety is part of a strained ring of the VLP.3. The method of embodiment 1, wherein the alkyne moiety is a ring-strained dibenzocyclooctyne (DBCO).4. The method of embodiment 1 or 2, wherein the VLP is selected from the group of Physalis mottle virus (PhMV), Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), Cowpea chlorotic mosaic virus (CCMV).5. The method of any of embodiments 1-4, wherein the VLP diameter is about 10 nm to about 100 nm, or from about 20 nm to about 80 nm, or from about 30 nm to about 60 nm, or from about 30 nm to about 40 nm, or from about 30 nm to about 35 nm, or about 33 nm.6. The method of any of embodiments 1-5, wherein the VLP is detectably labeled.7. The method of any one of embodiments 1-6, further comprising drying the swelled hydrogel to remove water and form a VLP xerogel.8. The method of any of embodiments 1-7, wherein the swelled hydrogel comprises the reaction product of a cross-linking agent and at least one azide-containing monomer or oligomer, a polyalkylene glycol, and a (meth)acrylate.9. The method of any of embodiments 1-7, wherein the swelled hydrogel comprises the reaction product of an acrylate-polyethylene glycol azide, a polyethylene glycol methacrylate, an acrylamide, and polyethylene glycol diacrylate.10. A hybrid virus hydrogel prepared by the method of any of embodiments 1 -9 or the VLP xerogel of any of embodiments 7-9.11. A hybrid virus hydrogel comprising a polymeric network with a covalently bound VLP, wherein the polymeric network comprises a swelled hydrogel comprising an azide group.12. The hybrid virus hydrogel of embodiment 11, wherein the VLP is bound to the polymeric network via an azide-yne linkage.13. The hybrid virus hydrogel of embodiment 11 or 12, wherein the VLP comprises a DBCO moiety.14. The hybrid virus hydrogel of embodiments 8-13, wherein the VLP further comprises a therapeutic drug agent, optionally wherein the therapeutic drug agent is a chemotherapeutic agent.15. The hybrid virus hydrogel of any of embodiments 11-14, wherein the VLP concentration is approximately 0.01-1 wt% of the swelled hydrogel, approximately 0.01-0.5 wt% of the swelled hydrogel, approximately 0.01-0.25 wt% of the swelled hydrogel, or approximately 0.01-0.1 wt% of the swelled hydrogel.16. The hybrid virus hydrogel of any of embodiments 11-14 wherein the swelled hydrogel has at least a, 30%, at least a 40%, or at least a 50% increased VLP loading value compared to a swelled hydrogel in which the VLP is not bound to the polymeric network via the adize-yne linkage.17. A composition comprising the hybrid virus hydrogel of any of embodiments 11-16, and a carrier, optionally a pharmaceutically acceptable carrier.18. The composition of embodiment 17, further comprising a stabilizer or a preservative.19. A method to deliver a VLP to a cell or tissue in need thereof, comprising contacting the cell or tissue with an effective amount of the hybrid virus hydrogel of any of embodiments 11- 16, or the composition of embodiment 17 or 18.20. The method of embodiment 19, wherein the contacting is in vitro or in vivo.21. A method to administer a VLP to a subject in need thereof, comprising administering to the subject an effective amount of the hybrid virus hydrogel of any of embodiment 11-16, or the composition of embodiment 17 or 18.22. The method of embodiment 21, wherein the subject is a mammal, optionally a human patient.23. The method of embodiment 21 or 22, wherein the method induces an immune response in the subj ect.24. A method to deliver a VLP to a cell or tissue in need thereof, comprising contacting the cell or tissue with an effective amount of the hybrid virus hydrogel of any of embodiments 11- 16, or the composition of embodiment 17 or 18, wherein the VLP is released over a time periodto the cell or tissue at an amount less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% than the amount of a corresponding hybrid virus hydrogel wherein the VLP is not covalently bound to the hydrogel.25. The method of embodiment 24, wherein the time period is up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, or up to eight weeks, or greater than eight weeks.26. The method of embodiment 24 or 25, wherein the VLP further comprises a therapeutic drug agent, optionally wherein the therapeutic drug agent is conjugated to the VLP via an imino bond.27. A method to deliver a VLP subject in need thereof, comprising administering to the subject in need with an effective amount of the hybrid VLP of any of embodiments 11-16, or the composition of embodiment 17 or 18, wherein the VLP is released over a time period to the cell or tissue at an amount less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% than the amount of a corresponding hybrid virus hydrogel wherein the VLP is not covalently bound to the hydrogel, optionally wherein the subject in need thereof is a mammal, further optionally wherein the subject in need thereof is a human.28. The method of embodiment 27, wherein the time period is up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, or up to eight weeks.29. The method of embodiment 27, wherein the time period is greater than eight weeks.30. The method of embodiment 27 or 28, wherein the VLP further comprises a therapeutic drug agent, optionally wherein the therapeutic drug agent is conjugated to the VLP via an imino bond.31. The method of embodiment 30, wherein the therapeutic drug agent is a chemotherapeutic drug agent.32. The method of any of embodiments 27-31, wherein the hybrid virus hydrogel or composition is implanted in the subject in need thereof, wherein the implantation is adjacent to a tumor in the subject in need thereof.33. The method of any of embodiments 27-32, wherein the subject in need thereof has a tumor.34. The method of embodiment 33, wherein the tumor is a solid tumor selected from bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer.35. A hybrid virus hydrogel comprising a polymeric network with a covalently bound VLP: a) the VLP comprising an alkyne moiety and at least one imino group; and b) the polymeric network comprising a swelled hydrogel comprising an azide group, wherein the VLP is conjugated to the polymeric network via the imino group, and optionally wherein the VLP further comprises a therapeutic drug agent.36. The hybrid virus hydrogel of claim 35, wherein the therapeutic drug agent is a chemotherapeutic agent.37. The hybrid virus hydrogel of embodiment 26, wherein the therapeutic drug agent is conjugated to the VLP via a second imino group.

[0271] Equivalents

[0272] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

[0273] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5’ to 3’ direction.

[0274] The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and describedor portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure.

[0275] Thus, it should be understood that although the present disclosure has been specifically disclosed by specific embodiments and optional features, modification, improvement and variation of the embodiments therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of particular embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

[0276] The scoped of the disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0277] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that embodiments of the disclosure may also thereby be described in terms of any individual member or subgroup of members of the Markush group.

[0278] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

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Claims

WHAT IS CLAIMED IS:

1. A method of preparing a hybrid virus hydrogel, the method comprising contacting a virus-like particle (VLP) or derivative thereof comprising an alkyne moiety with a swelled hydrogel comprising an azide group.

2. The method of claim 1, wherein the alkyne moiety is part of a strained ring of the VLP, optionally wherein the alkyne moiety is a ring-strained dibenzo-cyclooctyne.

3. The method of claim 1 or 2, wherein the VLP is selected from the group of Physalis mottle virus (PhMV), Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), Cowpea chlorotic mosaic virus (CCMV), optionally wherein the VLP diameter is about 10 nm to about 100 nm, or from about 20 nm to about 80 nm, or from about 30 nm to about 60 nm, or from about 30 nm to about 40 nm, or from about 30 nm to about 35 nm, or about 33 nm.

4. The method of any of claims 1-3, wherein the VLP is detectably labeled.

5. The method of any one of claims 1-4, further comprising drying the swelled hydrogel to remove water and form a VLP xerogel.

6. The method of any of claims 1-5, wherein the swelled hydrogel comprises the reaction product of a cross-linking agent and at least one azide-containing monomer or oligomer, a polyalkylene glycol, and a (meth)acrylate.

7. The method of any of claims 1-5, wherein the swelled hydrogel comprises the reaction product of an acrylate-polyethylene glycol azide, a polyethylene glycol methacrylate, an acrylamide, and polyethylene glycol diacrylate.

8. A hybrid virus hydrogel or virus hydrogel prepared by the method of any of claims 1- 7 or the VLP xerogel of any of claims 5-7.

9. A hybrid virus hydrogel or virus hydrogel comprising a polymeric network with a covalently bound VLP.

10. The hybrid virus hydrogel of claim 9, wherein the VLP is bound to the polymeric network via an azide-yne linkage.

11. The hybrid virus hydrogel of any of claims 8-10, wherein the VLP further comprises a therapeutic drug agent, optionally wherein the therapeutic drug agent is a chemotherapeutic agent.

12. The hybrid virus hydrogel of any of claims 8-11, wherein the VLP concentration is approximately 0.01-1 wt% of the swelled hydrogel, approximately 0.01-0.5 wt% of the swelled hydrogel, approximately 0.01-0.25 wt% of the swelled hydrogel, or approximately 0.01-0.1 wt% of the swelled hydrogel.

13. The hybrid virus hydrogel of any of claims 8-11 wherein the hydrogel has at least a, 30%, at least a 40%, or at least a 50% increased VLP loading value compared to a swelled hydrogel in which the VLP is not bound to the polymeric network via the adize-yne linkage.

14. A composition comprising the hybrid virus hydrogel of any of claims 8-13, and a carrier, optionally a pharmaceutically acceptable carrier.

15. The composition of claim 14, further comprising a stabilizer or a preservative.

16. A method to deliver a VLP to a cell or tissue in need thereof, comprising contacting the cell or tissue with an effective amount of the hybrid virus hydrogel of any of claims 8-13, or the composition of claim 14 or 15, optionally wherein the contacting is in vitro or in vivo.

17. A method to administer a VLP to a subject in need thereof, comprising administering to the subject an effective amount of the hybrid virus hydrogel of any of claims 8-13, or the composition of claim 14 or 15, wherein the subject is a mammal, optionally a human patient.

18. The method of claim 17, wherein the method induces an immune response in the subject.

19. A method to deliver a VLP to a cell or tissue in need thereof, comprising contacting the cell or tissue with an effective amount of the hybrid virus hydrogel of any of claims 8-13, or the composition of claim 14 or 15, wherein the VLP is released over a time period to the cell or tissue at an amount less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% than the amount released in a corresponding hybrid virus hydrogel wherein the VLP is not covalently bound to the hydrogel, optionally wherein the time period is up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, or up to eight weeks, or greater than eight weeks.

20. A method to deliver a VLP subject in need thereof, comprising administering to the subject in need with an effective amount of the hybrid virus hydrogel of any of embodiments 8-13, or the composition of embodiment 14 or 15, wherein the VLP is released over a time period to the cell or tissue at an amount less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% than the amount released a corresponding hybrid virus hydrogel whereinthe VLP is not covalently bound to the hydrogel, optionally wherein the subject in need thereof is a mammal, further optionally wherein the subject in need thereof is a human, optionally wherein the time period is up to one day, up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, or up to eight weeks, or greater than eight weeks.

21. The method of claim 20, wherein the VLP further comprises a therapeutic drug agent, optionally wherein the therapeutic drug agent is conjugated to the VLP via an imino bond.

22. The method of claim 21 , wherein the therapeutic drug agent is a chemotherapeutic drug agent.

23. The method of any of claims 20-22, wherein the hybrid virus hydrogel or composition is implanted in the subject in need thereof, wherein the implantation is adjacent to a tumor in the subject in need thereof.

24. The method of any of claims 20-23, wherein the subject in need thereof has a tumor, optionally wherein the tumor is a solid tumor selected from bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer.

25. A hybrid virus hydrogel comprising a polymeric network with a covalently bound VLP: a) the VLP comprising an alkyne moiety and at least one imino group; and b) the polymeric network comprising a swelled hydrogel comprising an azide group, wherein the VLP is conjugated to the polymeric network via the imino group, and optionally wherein the VLP further comprises a therapeutic drug agent.

26. The hybrid virus hydrogel of embodiment 25, wherein the therapeutic drug agent is a chemotherapeutic agent.

27. The hybrid virus hydrogel of claim 26, wherein the therapeutic drug agent is conjugated to the VLP via a second imino group.

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