Modified plant virus-like nanoparticles

Abstract

Provided herein is a plant virus-based nanocarrier system for the targeted delivery of small molecule drugs and. imaging reagents in vivo. The nanocarrier is an engineered virus like particle capsid protein (E-VLP-CP) comprising one or more internally cy stein e-mutated amino acids conjugated to a one or more thiol-reactive small molecules. Also provided herein is an engineered virus-like particle (E-VLP) comprising: a first capsid protein; a. second capsid protein; and a maleimide-PEGN-mal eimide linker internally cross-linking the first and the second capsid proteins to each other through internally surface exposed cysteines.

Description

[0001] Atty. Dkt. No. 114198-4960 MODIFIED PLANT VIRUS-LIKE NANOPARTICLES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C § 119(e) of U.S. Provisional Applications No. 63 / 514,018, filed on July 17, 2023, and Application No. 63 / 514,907, filed on July 21, 2023, the entire contents of each of which are incorporated herein by reference. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under CA253615 and CA202814 awarded by the National Institutes of Health (“NIH”) and under CHE2116298 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND There is significant optimism surrounding the use of nanomaterials for the improvement of disease diagnosis and treatment. Specifically, nanoparticles (NPs) have the potential to overcome traditional limitations of drug and gene delivery by improving the stability and pharmacokinetic properties of encapsulated cargo and circumvent biological barriers for targeted delivery. Examples of well-studied nanocarrier platforms are reviewed elsewhere, and include lipid-based NPs (e.g. liposomes), inorganic NPs (e.g. silica, gold, iron oxide NPs), and polymeric NPs (e.g. dendrimers, co-block polymers) as well a protein and virus-based NPs, each of which have their own advantages and disadvantages in terms of biocompatibility, pharmacokinetics, and ease of synthesis and small molecule encapsulation. All of these NP systems can be engineered to carry molecular cargo, including chemotherapeutics, immunomodulators, peptide / protein drugs, synthetic nucleic acids, as well as contrast agents. Barriers for the advancement of novel nanomaterials into clinical practice are numerous, and include concerns surrounding reproducible synthesis, scalable manufacturing, toxicity, and poor cargo loading. Additional concerns for the advancement of novel nanomaterials into clinical practice are stability. Stabilizing the VLP platform is crucial to ensure their structural integrity and functionality are maintained during synthesis (e.g., harsh synthesis conditions),

[0010] storage and shipping,

[0011] ,

[0012] and performance (e.g., heat- generating devices).

[0013] ,

[0014] 1 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 This disclosure addresses the limitation of prior nanoparticles and provides related advantages as well. In particular, this disclosure focuses on engineering virus-like nanoparticles, in particular to improve loading capabilities and / or increase stability of the virus-like nanoparticles. SUMMARY OF THE DISCLOSURE Virus-like particles (VLPs), like many biological molecules, can exhibit structural weakness, which may reduce shelf-life and functionality. VLPs can be used for therapeutic uses and also for agricultural uses, both of which may benefit from increased stability. Compared to other biologics, many VLPs have comparably good chemical, thermal and mechanical stability profiles – however there is a desire to increase the loading capacity of the VLPs and / or tailor the stability for downstream application as targeted delivery vehicles or components of a solid-state device. In this way, engineered VLPs (E-VLPs) are more robust than their wildtype counterparts. E-VLPs can be used as delivery vehicles for a variety of therapeutic agents, including small-molecule drugs, proteinaceous drugs, nucleic acids, and imaging agents, particularly for anticancer treatments and diagnostics. These engineered VLPs protect their payloads against degradation and clearance, thereby prolonging exposure to antigen-presenting cells (APCs). Additionally, E-VLPs can shield their cargo from the potentially damaging conditions of solid-state device manufacturing processes, such as heating and mechanical extrusion. Further, increased stability of engineered VLP can extend the systemic circulation time at physiological conditions, thereby enhancing the stimulation of the immune system to recognize and the activation level of T- cells. Engineered VLPs (E-VLPs) have applications in fields including drug delivery, vaccines, immunotherapy, imaging, sensors, battery electrodes, coatings, data storage, and catalysis. In one aspect, Applicant provides herein an engineering of an exemplary Physalis mottle virus-like nanoparticle (PhMV), to carry cargo and perform therapeutically and diagnostically-useful functions. Also provided herein are pluralities of the PhMV, compositions and kits comprising the PhMV, and methods of use. In addition, Applicant 2 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 provides methods for making and using nanoparticles having successfully loaded several cytotoxic agents into the nanoparticle, with demonstrated functionality in vivo. In one aspect, Applicant engineered an existing plant virus-like nanoparticle, the Physalis mottle virus-like nanoparticle (PhMV) and expanded its internal carrying capacity through rationally designed internal cysteine mutations (see e.g. FIGS. 4D and 6D). The disclosed cysteine-added mutants of PhMV display increased internal loading capacity of thiol-reactive small molecules, when compared head-to-head with native PhMV VLPs. The increased number of internal cysteine residues within the cysteine-added mutants of PhMV are shown to allow for increased internal binding / carrying of small molecule cargo (for example - chemotherapeutics, imaging agents) within the nanoparticle. The increased loading capacity can result in an additive, or even synergistic, advantage of the engineered nanoparticles delivering therapeutics such as a small molecule cargo to a cell. The disclosed engineered nanoparticles with increased internal loading capacity also can be used diagnostically as a multimodal imaging agent or therapeutically to deliver therapeutic agents. In another aspect, Applicant engineered virus-like particle (E-VLP) comprising: a first capsid protein; a second capsid protein; and a maleimide-PEGN-maleimide linker internally cross-linking the first and the second capsid proteins to each other through internally surface exposed cysteines, wherein the first and second capsid proteins are the same or different from each other, optionally wherein the maleimide-PEGN-maleimide linker has 1, 2, 4, or 8 branches. Also provided herein are pluralities of the E-VLP, compositions and kits comprising the E-VLP, and methods of use. The endoskeleton enhanced VLPs can be used as delivery vehicles of compounds including drugs and vaccines. The enhanced endoskeleton provides for a stronger particle in harsher conditions (for example temperature, pH, presence of surfactants and salts), as compared to a non-engineered VLP. The mechanical performance of the engineered VLPs is demonstrated by AFM single- molecule force spectroscopy techniques. Characterized by temperature dependent circular dichroism spectra, the melting temperature (Tm) is enhanced from 89.2 °C to > 100 °C. The rupture distance is 5-fold increased, while the rupture force is 1.7-fold increased. It still another aspect, Applicant provides a method of forming an engineered virus- like particle (E-VLP) comprising: combining a plurality of capsid proteins, and a multiarm 3 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 maleimide-PEGN-maleimide polymer to form the E-VLP, wherein each of the plurality of capsid proteins comprises internally surface exposed cysteine groups that react with a maleimide group of the multiarm maleimide-PEGN-maleimide polymer, and wherein the E- VLP comprises capsid proteins linked to one another by the multiarm maleimide-PEGN- maleimide polymer, optionally wherein the combining is conducted in a buffer solution, optionally wherein the combining is conducted from greater than 0⁰C to about 20⁰C. BRIEF DESCRIPTION OF THE DRAWINGS FIGURES 1A-1E: Structure-based design of cysteine-added PhMV VLPs. (FIG. 1A): PhMV VLPs (PDB 1QJZ) are composed of 180 identical coat proteins, which are arranged in asymmetric trimers. Cysteine-75 (C75) is located on the internal coat protein surface, along with A31 and S137 (see labels). The box surrounds the A chain of the asymmetric unit. (FIG. 1B): The side chain of C75 is oriented internally into the bulk protein. The internal-facing protein surface is displayed in mesh. (FIG. 1C): Distances of the alpha carbons (CD) of A31 and S137, relative to the CD of C75. (FIGS.1D-1E): Predicted orientation of the side chains of A31C and S137C mutants of PhMV CP. FIGURES 2A-2E: Purification of cysteine-added PhMV VLPs. (FIGS. 2A-2D): Characterization of PhMV-KB, PhMV Cys1, PhMV Cys2, and PhMV Cys1+2 VLPs using SDS-PAGE (FIG. 2A), native gel electrophoresis (FIG. 2B), size-exclusion chromatography (FIG. 2C), dynamic-light scattering (FIG. 2D), and transmission electron microscopy (FIG. 2E). Error bars represent the S.E.M. of five replicates. In FIG. 2C, Abs(280nm) is the lower line in each of PhMV-KB, PhMV Cys1, and PhMV Cys2. FIGURES 3A-3E: Cysteine-added PhMV VLPs show increased reactivity to thiol reactive small molecules. (FIG. 3A): DTNB (5,5’-dithio-bis-(2-nitrobenzoic acid) reacts with reduced cysteines to release 5-thio-2-nitrobenzoic acid, which is highly absorbent at 412 nm. (FIGS. 3B-3C): Quantification of free thiol concentration of native (FIG. 3B) or chemically denatured (FIG. 3C) PhMV-KB or cysteine-added VLPs using DTNB. (FIGS. 3D-3E): Incubation of PhMV-KB or cysteine-added VLPs with maleimide-Cy5 followed by separation by SDS-PAGE (FIG. 3D) and quantification of in gel fluorescence (FIG. 3E). In FIG. 3B, from top to bottom at 100μM are PhMV Cys1+2, PhMV Cys2, PhMV Cys1, and PhMV-KB. In FIG. 3C, from top to bottom at 100μM are PhMV Cys2, PhMV Cys1 / PhMV 4 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Cys1+2, and PhMV-KB. In FIG. 3E, from top to bottom at 10.00 eq / CP are PhMV Cys1+2, PhMV Cys2, PhMV Cys1, and PhMV-KB. FIGURES 4A-4F: Cysteine-added PhMV VLPs show increased aldoxorubicin loading capacity and resultant NPs display improved cytotoxicity. (FIG. 4A): Chemical components of aldoxorubicin. (FIGS. 4B-4C): Characterization of PhMV-KB-Aldox, PhMV Cys1-Aldox, PhMV Cys2-Aldox, and PhMV Cys1+2-Aldox VLPs by SDS-PAGE (FIG. 4B) and UV-Vis spectroscopy (FIG. 4C). (FIG. 4D): Calculation of doxorubicin per VLP based on particle absorbance at 480 nm. (FIGS.4E-4F): Dose-response curve (FIG. 4E) and calculated IC50 (FIG. 4F) of Aldox-conjugated PhMV VLPs in A2780 cells. Error bars represent the S.E.M. of three replicates (some error bars are smaller than the data point symbol). **: p<0.01, ***: p<0.001. In FIG. 4C, the highest curves are PhMV Cys2-Aldox and PhMV Cys1+2-Aldox, the middle curve is PhMV Cys1-Aldox, and the lowest curve is PhMV-KB-Aldox. In FIG. 4E, at the highest concentration the higher curves are PhMV Cys2-Aldox and PhMV Cys1+2 Aldox and the lower curves are PhMV Cys1-Aldox and PhMV-KB-Aldox. FIGURES 5A-5D: Cysteine-added PhMV VLPs show increased vcMMAE loading capacity and resultant NPs display improved cytotoxicity. (FIG. 5A): Chemical structure of valine-citrulline monomethyl auristatin E (vcMMAE). (FIG. 5B): Characterization of PhMV-KB-vcMMAE, PhMV Cys1-vcMMAE, PhMV Cys2-vcMMAE, and PhMV Cys1+2-vcMMAE VLPs by SDS-PAGE. (FIGS. 5C-5D): Dose-response curve (FIG. 5C) and calculated IC50 (FIG. 5D) of vcMMAE-conjugated PhMV VLPs in A2780 cells. Error bars represent the S.E.M. of three replicates (some error bars are smaller than the data point symbol). **: p<0.01. In FIG. 5C, at the highest concentration, PhMV-KB- vcMMAE has the highest curve and the other treatments end at the same point. The PhMV Cys 1+2-vcMMAE curve is the lowest overall. FIGURES 6A-6F: Cysteine-added PhMV VLPs show increased DOTA(Gd) loading capacity and resultant NPs display high R1 and R2 relaxivities. (FIG. 6A): Chemical structure of the gadolinium chelate maleimide-DOTA. (FIG. 6B): Characterization of PhMV-KB-DOTA(Gd), PhMV Cys1-DOTA(Gd), PhMV Cys2-DOTA(Gd), and PhMV Cys1+2-DOTA(Gd) VLPs by SDS-PAGE. (FIG. 6C): Calculation of number of Gd(III) ions per VLP based on ICP-MS data. (FIGS. 6D-6F): Summary data (FIG. 6D) and curves of 5 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 longitudinal (R1) (FIG. 6E) and transverse (R2) (FIG. 6F) relaxivities of PhMV-KB- DOTA(Gd), PhMV Cys1-DOTA(Gd), PhMV Cys2-DOTA(Gd), and PhMV Cys1+2- DOTA(Gd) VLPs at room temperature in a 3 T MR scanner. In FIG. 6C from right to left are PhMV-KB-DOTA(Gd), PhMV Cys1-DOTA(Gd), PhMV Cys2-DOTA(Gd), and PhMV Cys1+2-DOTA(Gd). In FIG. 6E at the highest mg / mL the highest curve is PhMV Cys1+2- DOTA(Gd), the second highest curve is PhMV Cys2-DOTA(Gd), and the bottom curve is PhMV-KB-DOTA(Gd). In FIG. 6F, at the highest mg / ML the highest curve is PhMV Cys1+2-DOTA(Gd), the second highest curve is PhMV Cys2-DOTA(Gd), and the bottom curve is PhMV-KB-DOTA(Gd). In FIGS. 6E and 6F PhMV-KB-DOTA(Gd) and PhMV Cys1-DOTA(Gd) nearly overlap. FIGURES 7A-7C: Vector maps for pRSETa-PhMV-HM, pRSETa-PhMV CP, and pET-PhMV CP. Vector maps for the plasmids used in this study, pRSETa-PhMV-HM (FIG. 7A), pRSETa-PhMV CP (FIG. 7B), and pET-PhMV CP (FIG. 7C). FIGURES 8A-8F: Purification and characterization of PhMV-HM and PhMV- KB VLPs. (FIG. 8A): SDS-PAGE of PhMV-KB VLPs at steps during purification. (FIGS. 8B-8F): Characterization of PhMV-HM and PhMV-KB VLPs using SDS-PAGE (FIG. 8B), native gel electrophoresis (FIG. 8C), size-exclusion chromatography (FIG. 8D), dynamic light scattering (DLS) (FIG. 8E), and transmission electron microscopy (FIG. 8F). Error bars represent the S.E.M. of three replicates. In FIG. 8D, the Abs (280nm) line is lower than the Abs(260nm) line. FIGURES 9A-9D: Characterization of aldoxorubicin-conjugated PhMV-KB and cysteine-added mutant VLPs. (FIGS. 9A-9B): Characterization of PhMV-KB-Aldox, PhMV Cys1-Aldox, PhMV-Cys2-Aldox, and PhMV Cys1+2-Aldox by native gel electrophoresis (FIG. 9A) and dynamic light scattering (FIG. 9B). Error bars represent the S.E.M. of five replicates (some error bars are smaller than the data point symbol). (FIGS. 9C-9D): Dose-response curve (FIG. 9C) and calculated IC50 (FIG. 9D) of Aldox- conjugated PhMV VLPs in A2780 cells as a function of delivered dose of aldoxorubicin. Error bars represent the S.E.M. of three replicates (some error bars are smaller than the data point symbol). In FIG. 9C, at the highest concentration the highest curve is PhMV Cys1- Aldox, and the other three nearly overlap. 6 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 FIGURES 10A-10D: Characterization of vcMMAE-conjugated PhMV-KB and cysteine-added mutant VLPs. (FIGS. 10A-10B): Characterization of PhMV-KB-vcMMAE, PhMV Cys1-vcMMAE, PhMV-Cys2-vcMMAE, and PhMV Cys1+2-vcMMAE by native gel electrophoresis (FIG. 10A) and dynamic light scattering (FIG. 10B). Error bars represent the S.E.M. of five replicates (some error bars are smaller than the data point symbol). (FIGS. 10C-10D): Dose-response curve (FIG. 10C) and calculated IC50 (FIG. 10D) of vcMMAE- conjugated PhMV VLPs in A2780 cells as a function of delivered dose of vcMMAE. Error bars represent the S.E.M. of three replicates (some error bars are smaller than the data point symbol). **: p<0.01. In FIG. 10C, at the highest concentration PhMV-KB-vcMMAE has the highest curve. While at that concentration the other treatments have approximately the same cell viability, PhMV Cys1-vcMMAE has the second highest curve overall, and PhMV Cys1+2-vcMMAE has the lowest curve overall. FIGURES 11A-11B: Characterization of DOTA(Gd)-conjugated PhMV-KB and cysteine-added mutant VLPs. (FIGS. 11A-11B): Characterization of PhMV-KB- DOTA(Gd), PhMV Cys1-DOTA(Gd), PhMV-Cys2-DOTA(Gd), and PhMV Cys1+2- DOTA(Gd) by native gel electrophoresis (FIG. 11A) and dynamic light scattering (FIG. 11B). Error bars represent the S.E.M. of five replicates (some error bars are smaller than the data point symbol). FIGURES 12A-12D: Characterization of magnetic resonance properties of DOTA(Gd)-conjugated PhMV-KB and cysteine-added mutant VLPs. (FIG. 12A): T1 and T2 mapping phantoms of a concentration series of PhMV-KB-DOTA(Gd), PhMV-Cys1- DOTA(Gd), PhMV-Cys2-DOTA(Gd), and PhMV Cys1+2-DOTA(Gd) obtained on a 3 T scanner at room temperature. (FIGS. 12B-12C): Curves of longitudinal (r1) (B) and transverse (r2) (C) relaxivity of PhMV-KB-DOTA(Gd), PhMV-Cys1-DOTA(Gd), PhMV- Cys2-DOTA(Gd), and PhMV Cys1+2-DOTA(Gd) at different concentrations of Gd(III) at room temperature in a 3 T scanner. (FIG. 12D): Chart of calculated r1and r2per Gd(III) for DOTA(Gd)-conjugated PhMV VLPs. In FIGS. 12B and 12C, PhMV Cys1+2-DOTA(Gd) has the longest curve, PhMV Cys2-DOTA(Gd) has the middle length curve, and PhMV- KB0DOTA(Gd) has the shortest curve. In each of FIG. 12B and 12C PhMV Cys1- DOTA(Gd) overlaps with, but it shorter than, the PhMV Cys2-DOTA(Gd) curve. 7 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 FIGURES 13A-13G: PhMV VLPs and STING agonist reaction scheme and characterization. (FIG. 13A): A thiol-reactive STING agonist (STG982-maleimide, Invivogen) was conjugated to the internal cavity of cysteine-added PhMV VLPs to create a potent anti-tumor immunotherapy delivery system (denoted PhMV-STG). (FIGS. 13B-13F): PhMV-STG conjugated particles were characterized via fast protein liquid chromatography (FIG. 13B), dynamic light scattering (FIG. 13C), SDS-PAGE denaturing gels (FIG. 13D), native agarose gel electrophoresis (FIG. 13E), and transmission electron microscopy (FIG. 13F). (FIG. 13G): STING agonist loading was quantified via tandem liquid chromatography- mass spectrometry. FIGURES 14A-14C: PhMV-STG particles show increased interferon response, prolonged tumor retention, and improved survival against intradermal tumors when compared to non-conjugated STING agonist. (FIG. 14A): PhMV-STG and free STG were incubated with THP-1 monocytes in vitro for 72 hours, then secreted interferon response was measured. From left to right are -Control (media), +Control (100 ng Poly [dA:dT] / Lyovec, Invivogen), PhMV (29μg), and 250, 125, 65.5, and 31.25ng of agonist. Left to right at 250, 125, 62.5, or 31.25 ng of agonist are STG 982 and PhMV-STG. Each bar represents the average and standard deviation of three replicates. (FIG. 14B): To model how long release of a thiol-maleimide conjugated drug may act in vivo, sulfo-Cy5.5-maleimide was conjugated to PhMV VLPs in place of the STING agonist. PhMV-Cy5.5 and free Cy5.5 were injected intratumorally in dermal B16F10 melanoma tumors and monitored via IVIS for fluorescence signal. Percent of the initial Cy5.5 fluorescent signal (taken minutes after injection) was measured and calculated at 1 h, 4 h, 24 h, 48 h, 96 h, and 168 h post intratumoral injection. Error bars signify the standard deviation of signals from 3 separate mice. Cy5.5 represents the lower curve. (FIG. 14C): PhMV-STG and free STG were tested as treatment against dermal B16F10, using PBS and non- conjugated PhMV VLPs as controls; the survival plot is shown here. At the same STG dose, PhMV-STG eliminated tumors in 4 / 6 mice, while free STG eliminated tumors in 2 / 6 mice. PhMV-STG had the highest probability of survival, followed by free STG. PBS and PhMV both had zero percent probability of survival by day 26, however the PBS curve is lower overall. Statistical significance was calculated using unpaired t-tests with **** = P < 0.0001, *** = P < 0.0002, ** = P < 0.0021, * = P < 0.0332, and ns = P < 0.1234. 8 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 FIGURES 15A-15D: (FIG. 15A): Chemical structure and projected 3D structure of the maleimide–PEG15–maleimide crosslinker (polymer model created by MolView.org). (FIG. 15B): Side view to show its helical conformation in water.[1](FIG. 15C): Structure of the Physalis mottle virus (PhMV) virus-like particle (VLP) and its coat protein (CP); the VLP has an icosahedral T = 3 symmetry and is assembled from 180 identical copies of a CP; three CP for a triangular unit with a-c (see the triangle in FIG. 15C) denoting their position; ‘a’ in clusters at the 5-fold axis while ‘b’ and ‘c’ are at the 2- and 3-fold axis. The internal solvent- exposed cysteine is highlighted in red. The outer size is estimated at ~30 nm (PhMV structure created by UCSF Chimera, PDB: 1E57). (FIG. 15D): Structural model of PhMV coat proteins with viral endoskeleton via our endoskeleton-armored strategy, highlighted by top and side view. Notably, the protein–PEG in this work is a model to show the idea and does not depict the realistic conformation. FIGURES 16A-16J: Characterization of Endoskeleton-Enhanced (EE-) PhMV. (FIG. 16A): Michael addition of the thiol to the maleimide results in the maleimide–thiol conjugate, a succinimide thioether. The reaction is fast, but the conjugate is relatively unstable and can undergo further reaction via one of two pathways: (i) it can undergo a retro- Michael conversion back to the original thiol and maleimide; (ii) it can undergo irreversible ring-opening hydrolysis to yield a stable hydrolysate (succinamic acid thioether) under alkaline condition, which prevents elimination of the maleimide–thiol conjugate and generates negatively charged / deprotonated carboxylate. This charge change serves as a proxy of the degree conjugation to the VLPs. (FIGS. 16B-16C): Electrophoretic mobility shift assay (EMSA) (FIG. 16B) and denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 16C) of PhMV and EE-PhMV under various eq. / Cys. The distance of negative shifting toward the anode positively correlates to the value of eq. / Cys. (FIG. 16D): Fast protein liquid chromatography (FPLC) data indicates that the endoskeleton leads to swelling of the VLPs, which agrees with (FIG. 16E) dynamic light scattering (DLS) data. FPLC Detection wavelength is 260 nm. eq / Cys 1000 has mean 36.38nm, eq. / Cys 100 has mean 35.55nm, and PhMV has mean 35.54nm. (FIG. 16F): TEM images demonstrate a good dispersity and integrity of native PhMV and EE-PhMV1000(eq. / Cys = 1000). (FIGS. 16G-16H): Schematic (FIG. 16G) and SDS-PAGE images (FIG. 16H) of maleimide–Cy5 competition assay. Framed by the white dashed box, the intense 9 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 fluorescence of EE-PhMV100(eq. / Cys = 100) indicates active thiol groups; the absence of fluorescence from EE-PhMV1000group indicates most reactive thiol groups are occupied by mal–PEG15–mal polymers. Complete gels are shown in supporting information. (FIG. 16I): Chemical structure of mal–PEG15–mal and methoxy-PEG17–maleimide (mPEG17–mal). (FIG. 16J): SDS-PAGE using mal–PEG15–mal and mPEG17–mal as crosslinkers. The results indicate the only the mal–PEG15–mal induces covalent linking within PhMV nanoparticles, thereby ruling out the possibility of physical entanglement. The ctrl lane represents EE- PhMV1000as a control. FIGURES 17A-17D: Generalization of the Endoskeleton-Enhanced Strategy was tested using Qβ VLPs. (FIG. 17A): Scheme of reduction of Qβ and re-bridging by mal– PEG15–mal of eq. / Cys ranging from 0–100. PDB: 1QBE. Ten equivalents of the reducing agent tris(2-carboxyethyl)phosphine (TCEP) were added to reduce disulfides, followed by re- bridging of mal–PEG15–mal crosslinker at 4°C for 16 hours (FIG. 17B): EMSA and denaturing SDS-PAGE of native Qβ, reduced Qβ, and reduced Qβ with eq. / Cys of 0–100. (FIGS. 17C-17D) DLS (FIG. 17C) and TEM analysis (FIG. 17D) of Qβ and EE-Qβ100nanohybrids demonstrate the monodispersity and structural integrity of Qβ VLPs and EE- Qβ100. In DLS analysis square is Qβ and circle is EE-Qβ100.FIGURES 18A-18G: Comprehensive stability assay of PhMV and EE-PhMV. (FIG. 18A): Turbidimetric assay (Abs600) and size measurement for determining the thermal- induced aggregation of PhMV (solid circle) and EE-PhMV (hollow square) in the range of 25–90 °C. Both groups appeared microscale aggregates at 90 °C. Samples at 4 °C are used as a negative control. Inset: photographs of samples after treatment of different temperatures for 15 min. Indicated on graphs whether the line is size or absorbance (abs600) (FIG. 18B): TEM images showing the thermal-induced morphological changes of PhMV and EE-PhMV over various temperatures. Circular dichroism (CD) spectra of (FIGS. 18C-18D) PhMV (FIG. 18C) and EE-PhMV (FIG. 18D) after thermal stressing at 4 °C (negative control) and 95 °C for 15 min. The content of β-structure is quantified at 218 nm, where indicated by dashed lines at 218 nm. ΘMRWrepresents the mean residue weight. (FIGS. 18E-18F): Temperature-dependent CD is conducted by monitoring the change of β-structure of PhMV (FIG. 18E) and EE-PhMV (FIG. 18F) ranging from 25–100 °C. (FIG. 18G): Size distribution under miscellaneous stressing conditions, including in presence of surfactant, 10 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 salt, pH, chemical denaturants, and organic solvents. DLS samples are triplicated and presented by intensity (%). At increasing concentrations under stressing conditions, the EE- PhMV (dashed lines, on the right side of each stressing condition) remained more uniform in size than PhMV (solid lines, on left side of each stressing condition). Both EE-PhMV and PhMV had more uniform size distribution at lower concentrations of the stressors. In FIG. 18G top and bottom, the solid line indicates PhMV, and the dotted line indicates EE-PhMV-1000. FIGURES 19A-19D: Mechanical performance of PhMV and EE-PhMV. (FIG. 19A): Schematic of single-molecule force spectroscopy (SMSF) study performed by AFM at the ‘force-extension’ operational mode. The exterior-exposed carboxylates were activated by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) crosslinker, followed by conjugation onto an amine-functionalized station and amine-functionalized AFM cantilever, respectively. Two critical parameters are schematized: rupture distance (Drup) and rupture force (Frup). (FIG. 19B): Representative force-extension curve of PhMV and EE-PhMV1000. (FIGS. 19C- 19D): Statistic mapping shows the two-dimensional distribution of rupture forces and distance of PhMV and EE-PhMV1000. The Drupand Frupare 6-fold and 1.9-fold increased. Number of events = 71. Statistical analysis: a p-value less than 0.05 is statistically significant. Independent samples t-test analysis reveals p < 0.001 for both rupture force and rupture distance between native PhMV and EE-PhMV1000. The statistical data is visualized via Seaborn.

[0052] FIGURE 20: Measuring the end-to-end distance between the sulfur atom of the cysteines on neighboring coat proteins. The range was 27.1–43.9 Å. The measurements were done by UCSF Chimera.

[0067] FIGURES 21A-21C: Maleimide–PEG with various n and fixed f = 2. (FIG. 21A): Molecular structure of maleimide–PEGn–maleimide molecules with different number (n) of ethylene glycol (EG) repeats, n = 6, 15, 43, or 115. (FIGS. 21B-21C): Native agarose gel and denaturing SDS-PAGE analysis of PhMV and EE-PhMV with different eq. / CP. Gels were stained by Coomassie Blue. L refers to the ladder of molecular weights. EMSA and SDS- PAGE indicate that the linear mal–PEG15–mal crosslinker (n = 15) is the most efficient based on the following reasonings: mal–PEGn–mal linkers with n = 115 have a Flory radius of 60.3 Å, this explains that no reactivity was evident per EMSA (Table 3, FIG. 21B). The high 11 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 molecular weight polymers are excluded from the VLPs per size-exclusive gating effect. Shortening the length of the polymer enables diffusion and interior access. The shorter linear mal–PEGn–mal crosslinkers with n = 6, 15, and 43, respectively, have smaller Flory radii (< 33.3 Å), and each resulted in successful conjugation. While the mal–PEG43–mal crosslinker (n = 43, Flory radius = 33.3 Å) is small enough to access VLP’s interior, the longer ethylene glycol chain with a counter length of 120 Å may cause steric hindrance yielding inefficient conjugation and linking between cysteines. This explains why the reaction of mal–PEG43– mal saturates at a lower degree of linking (eq. / Cys = 100) compared with that of mal– PEG15–mal (eq. / Cys = 1000), which has a counter length of 41.7 Å (FIG. 21C). Also, when the polymer is too short, linking is not effective. The shortest linear crosslinker (n = 6, contour length = 16.7 Å) is shorter than the shortest inter-cysteine distance (27.1 Å). Only a negligible degree of inter-CP linking is observed on SDS-PAGE (FIG. 21B). The fact that linking was observed, may be explained by the fact that the VLPs are not static objects, yet dynamic structures; the distance measurements between neighboring cysteines thus is simply an approximation. FIGURES 22A-22B: Maleimide–PEG with various n and f = 4 or 8. (FIG. 22A): Molecular structure of 4-arm PEGn–maleimide with different number (n) of ethylene glycol (EG) repeats, n = 8 or 25, and 8-arm PEG(25)–maleimide molecules. (FIG. 22B): Native agarose gel and denaturing SDS-PAGE analysis of PhMV and EE-PhMV with different eq. / CP. Gels were stained by Coomassie Blue. L refers to the ladder of molecular weights. The MP15M lane refers to the control of mal–PEG15–mal with eq. / Cys = 1000. FIGURES 23A-23B: Integrity of nucleic acid encapsulated in PhMV and EE- PhMV. The PhMV and EE-PhMV nanohybrids with various eq. / Cys were analyzed into EMSA. The gel was pre-stained by GelRed (×1) for the detection of nucleic acid and imaged under UV light. Coomassie blue staining was added as a control. The well-defined shape of bands suggests the internal linking neither eject nor damage the nucleic acid contents. It is noted that the introduction of mal–PEG15–mal does not lead to ejection of the nucleic acid contents within the EE-PhMV nanohybrids – while devoid of their genome, the VLPs carry random host RNA (as seen in FIGS. 23A-23B). This may lead to the opportunity for endoskeleton-armored VLPs to carry genetic cargo, e.g., nucleic acid therapeutics, or enable 12 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 the possibility of internal cargo loading by electrostatic attraction. These lanes come from the same gels. FIGURES 24A-24B: Complete gel of FIG. 16H. (FIGS. 24A-24B): Indicated by the black and white boxes, the lanes are selected and put together to show the maleimide–Cy5 competition assay. All lanes were from the same samples and underwent the same operating parameters. Other lanes outside of the frames are either not relevant or overloaded. The contrast was adjusted by ImageJ.

[0036] FIGURE 25: Quantifying the degree of linking. From FIG. 15C, the signals from the remaining monomeric bands at ~20k Da were extracted and subtracted by the total amount of protein. Analysis was done by ImageJ.

[0036] For the selected synthesis condition (i.e., eq. / Cys = 1000), the ratio of monomer to total amount was estimated as 14.4%, compared with the non-conjugated PhMV. FIGURES 26A-26B: Full FPLC profiles of PhMV and EE-PhMV1000: crosslinkers contained within VLPs. In this measurement, a 25-mL Superose-6 increase 10 / 300 GL column was used. The single and co-eluted peak at ~12 mL indicates the integrity of both PhMV and EE-PhMV1000VLP. The absence of other obvious peaks suggests that the mal–PEG15–mal crosslinkers were securely conjugated or contained within the VLPs. The crosslinkers strongly have strong absorbance at 280 nm (data not shown). FIGURES 27A-27C: Generalization of Endoskeleton-Enhanced Strategy, testbed: Qβ VLP. (FIG. 27A-27C): The disulfide bonds of Qβ VLPs were reduced by TECP first, leaving reactive cysteines on interfaces for upcoming linking. Mal–PEGn–mal crosslinker with n = 6, 15, and 43 with eq. / Cys = 1–100 was then added (FIG. 27A), followed by (FIG. 27B) native gel electrophoresis and (FIG. 27C) SDS-PAGE. Both groups show similar trend: on native gel, as the eq. / Cys increases, the EE-Qβ nanohybrids gain more negativity, and therefore more mobility toward anode; on SDS-PAGE, both groups develop oligomers from lighter weight to heavier weight, showing the same trend as EE-PhMV. Therefore, we believe the disulfide bonds from VLPs can be utilized as target functional group by the viral endoskeleton strategy. The reduced-Qβ VLPs show less electrophoretic mobility in EMSA compared to the native Qβ VLPs, which can be explained by the reduced structural integrity. Nevertheless, once the cysteine residues are connected again, now with 13 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 the mal–PEG15–mal crosslinker, stability and electrophoretic mobility is restored as function of eq. / Cys. The results were reproducible using mal–PEGn–mal crosslinker with n = 6, 15, and 43 with eq. / Cys = 1–100 (FIG. 25). The mal–PEG15–mal (eq. / Cys = 100) is selected to present the change of mobility on EMSA and the laddering effect on SDS-PAGE over eq. / Cys. DLS and TEM demonstrate that EE-Qβ (with eq. / Cys = 100) maintains the monodispersity and integrity of native Qβ. TCEP was used as a reducing agent, because it does not interfere with maleimide chemistry as compared to the more commonly used beta- mercaptoethanol (BME) and dithiothreitol (DTT).

[0068] Indicated by the black and white boxes, the lanes are selected and put together to show EMSA and denaturing SDS-PAGE of native Qβ, reduced Qβ, and reduced Qβ with eq. / Cys of 0–100. FIGURES 28A-28B: Temperature-dependent CD spectra of PhMV (FIG. 28A) and EE-PhMV (FIG. 28B) between 25 °C and 95 °C. The full spectrum was measured with a temperature interval of 10 °C. Each interval experiences 10 min incubation before spectrum measurement, and then temperature rise for next round. The changes of characteristic peaks at 218 nm agree well with the time-dependent CD spectra in FIGS. 18E-18F. FIGURES 29A-29C: Demonstrating the abailability of exposed carboxylate groups on PhMV. (FIG. 29A): The chemical structureof amine–Cy5. (FIG. 29B): Scheme of conjugation of PhMV VLP with amine–Cy5. (FIG. 29C): The EMSA and SDS-PAGE analysis of the resulitng PhMV–Cy5. The 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with eq. / Cys = 2, and N-Hydroxysuccinimide (NHS) with eq. / Cys =8, and amine–Cy5 with eq. / Cys = 4 were used. The fluorescent signals demonstrated the successsful loading of amine–Cy5 fluorephores onto the CP, proving the feasibiliity of upcoming amine–AFM cantilever experiments. FIGURE 30: Conjugation scheme of PhMV or EE-PhMV1000 NP for SMFS test. PhMV or EE-PhMV1000exhibits five exterior-exposed carboxylates (namely D58, D83, E39, E47, and C-terminus). These carboxylates were activated by the addition of 1-ethyl-3-(3- dimethyl aminopropyl)carbodiimide (EDC) crosslinker to yield o-acylisourea active ester for following conjugations. First, the activated particles were immobilized on an amine- functionalized station via EDC chemistry, followed by PBS washing to remove free reagents. Then, the immobilized particles were conjugated onto an amine-functionalized AFM 14 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 cantilever tip by conjugating the o-acylisourea active ester and the amine from cantilever tip. The force and distance are monitored between VLP and the cantilever. FIGURES 31A-31B: Comparing the force spectra of PhMV and EE-PhMV1000. (FIG. 31A): The location of the five pulling events shown in panel b relative to the overall distribution for each particle. (FIG. 31B): Five spectra on the left side are selected from non- modified PhMV, with a rupture distance of 0–100 nm; while those on the right side are from EE-PhMV1000, with a rupture distance of 0–500 nm. The pulling events of EE-PhMV1000feature a sawtooth-like pattern as compared to the smooth and shorter-distance pulling of non-modified PhMV. FIGURES 32A-32B: (FIG. 32A) Proposed force-driven unfolding process of CPs of non-modified PhMV and EE-PhMV1000. The dashed lines represent the macromolecular interactions between CPs, which is a balance between short-range, predominantly hydrophobic attraction and long-range electrostatic repulsion.42, 43The blue solid lines represent the covalently crosslinked mal̽PEG15̽mal polymers before and after being stretched. The VLPs have 180 CPs assembled in a three-dimensional icosahedral architecture. The interactions with other CPs in a three-dimensional network are omitted for clarity. (FIG. 32B): The force spectrum of non-modified PhMV and EE-PhMV1000. The PhMV shows a short-distance and smooth force curve, which indicates no unfolding. The characteristic sawtooth-like curve of EE-PhMV1000indicates unfolding CPs. FIGURES 33A-33B: Statistic histogram analysis of mechanical performance of PhMV and EE-PhMV. Histograms of rupture distance (left panel) and rupture force (right panel) for PhMV (upper in each panel) and EE-PhMV (lower in each panel) in PBS. The green curves describe the distribution of the pulling events (mean ± sd). N = 71. FIGURES 34A-34B: Force spectra of multiple-pulling events. Representative force-extension curves of PhMV (FIG. 34A) and EE-PhMV1000(FIG. 34B) VLP involving multiple-pulling. Multi-pulling event is characterized by an obvious drop whose slope equals to spring constant between two pulling (raises) events, as labelled by arrows with notes. This is unwanted, and possibly due to one AFM attaching to multiple viruses or multiple attachment points on the same viruses, and thereby were excluded from the measurement of mechanical performance. 15 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 DETAILED DESCRIPTION Definitions As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting or separating or both limiting and separating the subject matter described. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entireties to more fully describe the state of the art to which this invention pertains. In some aspect, technical publications are referenced by a number making reference to a publication, the full bibliographic citation for the publication is found immediately preceding the claims. 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, 2ndedition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. 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)). As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Alternatively, “consisting essentially of” active agents intends that only the recited active agents are the sole active agents to achieve one or more diagnostic or therapeutic uses. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace 16 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology. “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. 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”). All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. 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. As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (–) 15%, 10%, 5%, 3%, 2%, or 1 %. “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. 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. 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 17 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 by a method 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. In some embodiments, a subject is a human. A “composition” as used herein, refers to an active agent, such as a compound as disclosed herein and a carrier, inert or active. The carrier can be, without limitation, solid such as a bead or resin, or liquid, such as phosphate buffered saline. 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 / antibody 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; disaccharides, 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. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. 18 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. The compositions used in accordance with the disclosure 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, i.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 are 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. Administration or treatment in “combination” refers to administering two agents such that their pharmacological effects are manifest at the same time. Combination does not 19 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 require administration at the same time or substantially the same time, although combination can include such administrations. 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. 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 (i.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. In one aspect, treatment excludes prophylaxis. 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 20 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 disease, a status of being suspect of having such disease, or a status of at high risk of having such disease. 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. As used herein, the term “plant virus” includes viruses that infect plants or plant systems, e.g., leaves, root and / or stems. Plant viruses can be stably stored (and are stable without cold chain requirements). Plant viruses do not infect or replicate in mammalian cells, thus adding another layer of safety compared to oncolytic viral therapies. Non-limiting examples include tobacco mosaic virus (TMV), cytoplasmic type citrus leprosis virus, tobacco mold green mottle virus (TMGMV), physalis mottle virus like particle (PhMV), cowpea chlorotic mottle virus (CCMV), and cowpea mosaic virus (CPMV). Method of replicating and producing virus for therapeutic application are known in the art and described in WO 2022 / 221692, published October 20, 2022, and incorporated herein by reference. Virus-like Particles (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). 21 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 As used herein, the term “plant virus nanoparticle,” “plant viral nanoparticle” or “VLP” refers to a non-replicating, viral shell, derived from one or more plant 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. As used herein, “capsid protein” and “coat protein” are used interchangeably. 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). As used herein, VLP intends naturally occurring (wild-type or native) VLP and engineered VLP, unless explicitly stated otherwise. In some embodiments, the plant virus nanoparticle is a virus belonging to the order Picornavirales (i.e. a plant picornavirus). A plant picornavirus is a virus belonging to the family Secoviridae, which together with mammalian picornaviruses belong to the order of the Picornavirales. Plant picornaviruses are relatively small, nonenveloped, positive- stranded RNA viruses with an icosahedral capsid. In some embodiments, the virus particles are selected from the Comovirinae virus subfamily. The Comovirinae subfamily comprises over 60 species across at least 3 genera (Comovirus, Fabavirus, Nepovirus). Some Picornavirales have a monopartite genome, while others have a bipartite genome. Exemplary plant viruses from the Comovirinae subfamily include, but are not limited to Broad bean wilt virus 1, Tobacco ringspot virus, Cowpea severe mosaic virus, Turnip ringspot virus, Broad bean wilt virus 2, Bean pod mottle virus (BPMV), Potato virus B, 22 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Tomato ringspot virus, and Rice tungro spherical virus. Additional information about plant picornaviruses can be found in Zell et al (2017) Journal of General Virology, 98: 2421–2422. In a further embodiment, the virus particles are from the genus Comovirus. A preferred example of a comovirus is the cowpea mosaic virus particles. Other suitable plant virus includes, but is not limited to bean pod mottle virus (BPMV), cowpea severe mosaic virus or rice tungro spherical virus. In some embodiments, the plant virus nanoparticle or 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). Cowpea mosaic virus (CPMV) is a VLP and a plant-infecting member of the order Picornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied and a positive-sense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsidated. CPMV has an icosahedral capsid structure, which is ~30 nm in diameter and is formed from 60 copies each of a Large (L) and Small (S) coat protein. These two coat proteins are processed from a single RNA-2-encoded precursor polyprotein (VP60) by the action of the 24 K viral proteinase which is encoded by RNA-1. Thus capsid assembly, as well as viral infection, is dependent on the presence of both genomic segments in an infected plant cell. In some embodiments, the VLP particles have been treated, prepared and / or inactivated by methods known in the art. 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 comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 460-833): 23 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 MEQNLFALSLDDTSSVRGSLLDTKFAQTRVLLSKAMAGGDVLLDEYLYDV VNGQDFRATVAFLRTHVITGKIKVTATTNISDNSGCCLMLAINSGVRGKYSTDVYTIC SQDSMTWNPGCKKNFSFTFNPNPCGDSWSAEMISRSRVRMTVICVSGWTLSPTTDVI AKLDWSIVNEKCEPTIYHLADCQNWLPLNRWMGKLTFPQGVTSEVRRMPLSIGGGA GATQAFLANMPNSWISMWRYFRGELHFEVTKMSSPYIKATVTFLIAFGNLSDAFGFY ESFPHRIVQFAEVEEKCTLVFSQQEFVTAWSTQVNPRTTLEADGCPYLYAIIHDSTTG TISGDFNLGVKLVGIKDFCGIGSNPGIDGSRLLGAIAQ (SEQ ID NO: 1), or an equivalent thereof. 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): GPVCAEASDVYSPCMIASTPPAPFSDVTAVTFDLINGKITPVGDDNWNTHIY NPPIMNVLRTAAWKSGTIHVQLNVRGAGVKRADWDGQVFVYLRQSMNPESYDART FVISQPGSAMLNFSFDIIGPNSGFEFAESPWANQTTWYLECVATNPRQIQQFEVNMRF DPNFRVAGNILMPPFPLSTETPPL (SEQ ID NO: 2), or an equivalent thereof. Cowpea mosaic virus (CPMV) is a plant-infecting member of the order Picornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied and a positive-sense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsidated. CPMV has an icosahedral capsid structure, which is ~30 nm in diameter and is formed from 60 copies each of a Large (L) and Small (S) coat protein. These two coat proteins are processed from a single RNA-2-encoded precursor polyprotein (VP60) by the action of the 24 K viral proteinase which is encoded by RNA-1. Thus capsid assembly, as well as viral infection, is dependent on the presence of both genomic segments in an infected plant cell. The terms “CPMV” “CPMV virus” or “CPMV particles” are used interchangeably, referring to a CPMV comprising, or alternatively consisting essentially of, or yet consisting 24 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 of a capsid and an RNA genome (which is also referred to herein as a viral genome) encapsidated in the capsid. In some embodiments, the CPMV particles have been treated, prepared and / or inactivated by a method as disclosed herein. In some embodiments, the CPMV particle further comprises a heterologous RNA, which is heterologous to (i.e., not naturally presented in) a native CPMV free of any human intervention. In some embodiments, the plant virus nanoparticle or 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, Car1 (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). In some instances, the plant virus nanoparticle or 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 wild-type CCMV capsid, optionally expressed by Car1, 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 comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03601: MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGYS VSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSC VTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAALT EGDVIVHLEVEHVRPTFDDSFTPVY (SEQ ID NO: 3), or an equivalent thereof. 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). 25 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 In some embodiments, the plant virus nanoparticle or VLP is derived from Physalis mottle virus (PhMV). Physalis mottle virus (PhMV) is a +ssRNA virus from the family Tymoviridae that forms a ~30 nm-sized icosahedral capsid from 180 identical capsid proteins CPs, and can be recombinantly expressed and purified as a homogenous and stable VLP. In some instances, the plant virus nanoparticle or 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 protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P36351: MDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQVSLQTADP ITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVYGGQSF VLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTASIQISG RIRLSKPMLIAN (SEQ ID NO: 4), or an equivalent of each thereof. In some cases, the gene encoding the PhMV coat protein comprises or consists essentially of, or yet further consists of, the sequence as set forth in NC_003634.1:5958- 6524 CP [organism=Physalis mottle virus] [NCBI GeneID=940246]: ATGGACTCTTCGGAAGTTGTCAAAGTCAAGCAGGCCTCCATCCCCGCCCCTGGCT CCATTCTCTCCCAGCCCAACACAGAACAATCACCTGCCATAGTTCTCCCTTTTCA GTTTGAAGCCACTACTTTCGGCACCGCTGAAACCGCAGCCCAAGTCTCTCTCCAG ACTGCCGACCCCATTACCAAACTGACCGCCCCCTACCGACATGCTCAGATCGTCG AGTGCAAAGCTATCCTCACTCCAACTGATCTTGCTGTCTCCAATCCCCTCACAGT CTACCTAGCATGGGTCCCCGCCAACTCCCCTGCCACTCCGACTCAAATACTGCGA GTCTACGGCGGTCAGTCTTTTGTTCTTGGCGGCGCCATCTCAGCCGCCAAAACCA TTGAGGTCCCCCTCAATCTTGACTCTGTCAACCGCATGTTGAAAGACAGCGTGAC CTACACTGACACCCCCAAGCTCCTTGCCTACTCAAGAGCCCCCACCAACCCCTCG AAAATCCCAACCGCTAGTATTCAGATCAGCGGTCGCATTCGGCTCTCCAAGCCAA TGCTGATAGCCAACTAA (SEQ ID NO: 5), or an equivalent thereof. In some embodiments, the plant virus nanoparticle or VLP is derived from Sesbania mosaic virus (SeMV). SeMV is a positive stranded RNA virus that belongs to the genus 26 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Sobemovirus. In some instances, the virus or 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, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the SeMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q9EB06: MAKRLSKQQLAKAIANTLETPPQPKAGRRRNRRRQRSAVQQLQPTQAGISM APSAQGAMVRIRNPAVSSSRGGITVLTHSELSAEIGVTDSIVVSSELVMPYTVGTWLR GVAANWSKYSWLSVRYTYIPSCPSSTAGSIHMGFQYDMADTVPVSVNQLSNLRGYV SGQVWSGSAGLCFINGTRCSDTSTAISTTLDVSKLGKKWYPYKTSADYATAVGVDV NIATPLVPARLVIALLDGSSSTAVAAGRIYCTYTIQMIEPTASALNN (SEQ ID NO: 6), or an equivalent thereof. 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. 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 Å resolution. Inside the capsid helix, near the core, is the coiled RNA molecule, which is made up of 6,395 ±10 nucleotides. 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: 27 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVWKPSPQ VTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLDATRRV DDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT (SEQ ID NO: 7), or an equivalent thereof. In some embodiments, the plant virus is Potato Virus X (PVX). PVX belongs to the Potexvirus genus of the family Flexivirida. PVX virions are described as flexible, thread-like bodies measuring approximately 500-515 nanometers in length and 13-15 nanometers in diameter. Each viral particle comprises around 1300-1350 helically folded identical CP subunits enclosing a 6.4 knt viral RNA, with each turn of the primary helix consisting of 8.9 CP subunits. In some cases, the coat protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProt ID P17782: MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAIVASNAVATNED LSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLA AAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVT NPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRITGTT TAEAVVTLPPP (SEQ ID NO: 8) In a further aspect, the plant virus is cytoplasmic type citrus leprosis virus. Citrus leprosis viruses belong to multiple genera: Cilevirus (bipartite positive-sense single-stranded RNA [ssRNA], belonging to the family Kitaviridae), Higrevirus (tripartite ssRNA, belonging to the family Kitaviridae), and Dichorhavirus (bipartite negative-sense ssRNA, belonging to the family Rhabdoviridae, order Mononegavirales). Both cileviruses and higreviruses can occur in the cytoplasm. See Ramos-González et al., 2017. Phytopathology 107(8): 963-976. In some cases, the citrus leprosis Cilevirus capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in UniProtKB A0A2S1GUH8: MSNIVSFSLSNPSPALIAEIMDAISRHGLNVPAGLAQAPVQRQRQVRQPQIPPPQPRQA PRQRVNPPARAAPQQAQNRPAAPPQIGLPIRRVAVAGGSVGSVEHQEYYRSLPGYSK TYGCTKYNPNTPYTIVGFKLSEPSEELAVVDAKDLKASFKRRLKSLGFPSCGTDSIIV AHEYPDHYIAVIFPGAPYQLPVECPKDKVASPEDAKKVALAGCIRDINSVTDVRGILP VSYLELERLGMPPPLVLLPDDDDDQQVDEQEE (SEQ ID NO: 9), or an equivalent thereof. 28 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 In some embodiments, the VLP is derived from a bacteriophage. In some embodiments the bacteriophage is Qβ (Qbeta or alternatively QB). In some embodiments, the coat protein is a wild-type bacteriophage Qβ coat protein. Qβ is a member of the Fiersviridae family, of the Norzivirales order. See genome.jp / virushostdb / 2789016, last accessed July 1, 2024. Bacteriophage Qβ coat protein self-assembles to form an icosahedral capsid with a T=3 symmetry, about 26 nm in diameter, and consisting of 89 capsid proteins dimers (178 capsid proteins). It is also involved in viral genome encapsidation through the interaction between a capsid protein dimer and the multiple packaging signals present in the RNA genome. Binding of the capsid proteins to the viral RNA induces a conformational change required for efficient T=3 shell formation. Additionally, it acts as a translational repressor of viral replicase synthesis late in infection. This latter function is the result of capsid protein interaction with an RNA hairpin which contains the replicase ribosome-binding site. See, for example, Gorzelnik et al., Proc Natl Acad Sci U S A. 2016 Oct 11;113(41):11519-11524; Basnak et al., J Mol Biol. 2010 Feb 5;395(5):924-36; and Lim et al., J Biol Chem. 1996 Dec 13;271(50):31839-45. In further embodiments, the coat protein is modified, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some embodiments, a bacteriophage Qβ coat protein comprises, or alternatively consists essentially of, or yet further consists of the sequence as set forth in the UniProtKB ID P03615: MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQP SRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTEL AALLASPLLIDAIDQLNPAY (SEQ ID NO: 10), or an equivalent thereof. In other embodiments, the bacteriophage is P22 (family Podoviridae) or MS2 (family Fiersviridae). P22 and MS2 each have an icosahedral plasmid. In some embodiments, a bacteriophage P22 major capsid protein comprises, or alternatively consists essentially of, or yet further consists of the sequence as set forth in the UniProtKB ID P26747: MALNEGQIVTLAVDEIIETISAITPMAQKAKKYTPPAASMQRSSNTIWMPVEQESPTQ EGWDLTDKATGLLELNVAVNMGEPDNDFFQLRADDLRDETAYRRRIQSAARKLAN NVELKVANMAAEMGSLVITSPDAIGTNTADAWNFVADAEEIMFSRELNRDMGTSYF 29 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 FNPQDYKKAGYDLTKRDIFGRIPEEAYRDGTIQRQVAGFDDVLRSPKLPVLTKSTAT GITVSGAQSFKPVAWQLDNDGNKVNVDNRFATVTLSATTGMKRGDKISFAGVKFLG QMAKNVLAQDATFSVVRVVDGTHVEITPKPVALDDVSLSPEQRAYANVNTSLADA MAVNILNVKDARTNVFWADDAIRIVSQPIPANHELFAGMKTTSFSIPDVGLNGIFATQ GDISTLSGLCRIALWYGVNATRPEAIGVGLPGQTA (SEQ ID NO: 11), or an equivalent thereof. In some embodiments, a bacteriophage MS2 capsid protein comprises, or alternatively consists essentially of, or yet further consists of the sequence as set forth in the UniProtKB ID P03612: MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQN RKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLL KDGNPIPSAIAANSGIY (SEQ ID NO: 12), or an equivalent thereof. In some embodiments, the VLP is derived from a mammalian virus. In some embodiments, the mammalian virus is hepatis B virus (HBV, family Hepadnaviridae). HPV is icosahedral in shape. In some embodiments, a HPV coat protein comprises, or alternatively consists essentially of, or yet further consists of the sequence as set forth in the UniProtKB ID Q81110: LHHISPSPARSQSEGPIFSSWWLQFRNSKPCCDYCLTHIVNLLEDWGPCTEHGEHNIRI PRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGSTHVSWPKFAVPNLQSLTNLL SSNLSWLSLDVSAAFYHIPLHPAAMPHLLVGSSGLPRYVARLSSTSRNINHQHGTMQ DLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILGFRKIPMGGGLSPFLLAQFTSAICS VVRRAFPHCLAFSYMDDVVLGAKSVQHLESLFTSITNFLLSLGIHLNGRLVFQTSTRH GDESFCSQSSGILSRSPVGPCVRSQLKQSRLGLQPQQGSLARGKSGRSGSIRARVPPTT RRSFGVEPSGSGHIDNRASSTSSCLHQSAVRKTAYSHLSTSKRQSSSGHAV (SEQ ID NO: 13), or an equivalent thereof. The virus can be obtained according to various methods known to those skilled in the art. The virus can be obtained according to various methods known to those skilled in the art. In embodiments where plant virus particles are used, the virus particles can be obtained from the extract of a plant infected by the plant virus or using the method disclosed herein. For example, cowpea mosaic virus can be grown in black eyed pea plants, which can be infected within 10 days of sowing seeds. Plants can be infected by, for example, coating the 30 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 leaves with a liquid containing the virus, and then rubbing the leaves, preferably in the presence of an abrasive powder which wounds the leaf surface to allow penetration of the leaf and infection of the plant. Within a week or two after infection, leaves are harvested and viral nanoparticles are extracted. In the case of cowpea mosaic virus, 100 mg of virus can be obtained from as few as 50 plants. Procedures for obtaining plant picornavirus particles using extraction of an infected plant are known to those skilled in the art. See Wellink J., Meth Mol Biol, 8, 205- 209 (1998). Procedures are also available for obtaining virus-like particles. Saunders et al., Virology, 393(2):329-37 (2009). The disclosures of both of these references are incorporated herein by reference. In some embodiments, the engineered VLP has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm. 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. 31 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 “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 of the 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. “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. 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. 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 32 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 by a method 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. In some embodiments, a subject is a human. Modes For Carrying Out the Disclosure Virus-based NPs are protein-based nanostructures which make promising nanocarriers based on their high degree of structural uniformity, biocompatibility, and ease of synthesis in biological systems and manipulation by means of chemical conjugation, self-assembly, and genetic engineering.(15)Virus-like particles (VLPs) are proteinaceous NPs derived from the coat proteins (CPs) of viral capsids that lack internal genetic material and are therefore noninfectious.(16)Extensively studied VLPs are derived from the bacteriophages QE, P22, and MS2, the mammalian hepatitis B virus (HBV), and plant viruses tobacco mosaic virus (TMV), cowpea chlorotic mottle virus (CCMV), and potato virus X (PVX) – amongst others. These virus-based NPs can be synthesized by recombinant expression of their CPs through fermentation or plant molecular farming and manipulated through chemical biology approaches to carry a diverse array of molecular cargo. Although noncovalent loading of small molecules does not require modification of the cargo, covalent methods of cargo loading are often preferred as they are less prone to non-specific cargo release.(16)Additionally, the use of enzymatic- or pH-labile linkers, which are widespread in antibody- drug conjugates,(18)can increase specificity of molecular cargo release at the tissue of interest.(19),(20)Provided herein are engineered virus-like particles and methods of use thereof. According to one aspect, the VLP capsid protein is modified to include additional cysteine residues. This modified VLP capsid protein is referred to as an E-VLP-CP. Cysteine is of interest because it can bind to maleimides under the proper physiologic conditions.(22),(27)33 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 According to another aspect, the VLP is modified to include maleimide-PEGN- maleimide linker internally cross-linking capsid proteins of the VLP. Methods to Prepare Cysteine-Mutated Engineered VLP-CP (E-VLP-CP) Provided herein is a method to prepare an engineered virus like particle capsid protein (VLP-CP) conjugated to a thiol reactive small molecule, the method comprising the steps of preparing a cysteine-mutated VLP CP by mutating one or more internal amino acids of a wild-type VLP-CP; contacting the cysteine-mutated VLP CP with one or more thiol reactive molecules to prepare a thiol-containing VLP CP; and contacting one or more maleimide- containing small molecules with the thiol containing VLP CP, thereby preparing the engineered VLP-CP. In one aspect the VLP CP is mutated to replace the one or more internal amino acids with a cysteine. In one aspect, the method further comprises added to the VLP- CP with a carrier, such as a pharmaceutically acceptable carrier. The wild-type VLP-CP may be selected from a plant virus, an animal virus, or a bacteriophage. In one aspect, the wild-type VLP-CP is selected from a CP from a plant virus selected from the Tymoviridae, Secoviridae, Bromoviridae, Virgaviridae, Kitaviridae, and Alphaflexiviridae families. In one aspect, the wild-type VLP-CP is selected from a CP from a virus selected from a bacteriophages Qbeta, P22, and MS2, cytoplasmic type citrus leprosis virus, the mammalian hepatitis B virus (HBV), and plant viruses Tobacco mosaic virus (TMV), Cowpea chlorotic mottle virus (CCMV), Potato virus X (PVX) and Physalis mottle virus (PhMV). In one aspect, the one or more internal amino acids are surface exposed and project internally. In one aspect, the one or more internal amino acids has an alpha carbon (Cα) which is at least 7 Å from any other cysteine, or amino acid to be mutated to a cysteine. In this way, the amino acids will not form intramolecular disulfide bridges. In one aspect of this method, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. In a further aspect, the one or more internal amino acids are selected from A31C and S137C, wherein the wild-type VLP-CP comprises the sequence 34 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 according to SEQ ID NO: 4, or an equivalent thereof. See FIG. 1 and FIG. 2. The internal amino acids selected to be mutated will vary based on the wild-type VLP-CP used. The thiol reactive small molecules can be diagnostic or therapeutic small molecules. In some embodiments, the thiol reactive small molecules include a maleimide moiety. In one embodiment, the one or more thiol reactive small molecules are anti-cancer agents, for example chemotherapeutic agents. In another embodiment, the one or more thiol reactive small molecules are selected from the group of aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid- gadolinium (DOTA(Gd)), or STG982-maleimide. See FIGS. 4-6, and 13. In yet another embodiment, the thiol-reactive small molecules are toll-like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists). For example, the TLR 7 / 8 agonist 3M-012 and TLR9 agonist Class B CpG are crosslinked via a thiol-maleimide.(46)In another embodiment of the disclosed method, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C, and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. In another aspect, the wild-type VLP- CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. In another embodiment of the disclosed method, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C, and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof, and wherein the one or more thiol reactive small molecules is aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’- (1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982-maleimide. The thiol reactive small molecules can be diagnostic or therapeutic small molecules. In some embodiments, the thiol reactive small molecules include a maleimide moiety. In one 35 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 embodiment, the one or more thiol reactive small molecules are anti-cancer agents, for example chemotherapeutic agents. In another aspect, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4 or an equivalent thereof, and the thiol reactive small molecule is selected from aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane-1,4,7,10- tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), orSTG982-maleimide. In yet another embodiment, the thiol-reactive small molecules are toll-like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists). For example, the TLR 7 / 8 agonist 3M-012 and TLR9 agonist Class B CpG are crosslinked via a thiol-maleimide.(46)Engineered Cysteine-Mutated -VLP-CP Provided herein is an engineered virus like particle capsid protein (E-VLP-CP) comprising one or more internally cysteine-mutated amino acids in a wild-type VLP-CP conjugated to a one or more thiol-reactive small molecules. Also provided is a composition comprising the E-VLP-CP with a carrier, such as a pharmaceutically acceptable carrier. The wild-type VLP-CP may be selected from a plant virus, an animal virus, or a bacteriophage. In one aspect, the wild-type VLP-CP is selected from a CP from a plant virus selected from the Tymoviridae, Secoviridae, Bromoviridae, Virgaviridae, Kitaviridae, and Alphaflexiviridae families. In one aspect, the E-VLP-CP the wild-type VLP-CP is selected from a CP from a virus selected from a bacteriophages Qbeta, P22, and MS2, the mammalian hepatitis B virus (HBV), and plant viruses Tobacco mosaic virus (TMV), cs, cytoplasmic type citrus leprosis virus, Cowpea chlorotic mottle virus (CCMV), Potato virus X (PVX) or Physalis mottle virus (PhMV). In one aspect, the diameter of the wild-type VLP-CP is selected from: about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, 36 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm. See e.g. FIG. 11. In one aspect, the wild-type VLP-CP is icosahedral. In one aspect, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C, and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. See FIGS. 1A-1E. In a further aspect, the one or more internal amino acids are selected from A31C and S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. The internal amino acids selected to be mutated will vary based on the wild-type VLP-CP used. The thiol reactive small molecules can be diagnostic or therapeutic small molecules. In some embodiments, the thiol reactive small molecules include a maleimide moiety. The thiol reactive small molecules can be anti-cancer agents, for example chemotherapeutic agents. In another embodiment of the E-VLP-CP, the one or more thiol reactive small molecules are selected from the group of aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982-maleimide. In yet another embodiment, the thiol-reactive small molecules are toll-like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists). In another embodiment of the disclosed method, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, or PhMV A31C, and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. In another aspect, the wild-type VLP- CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C or PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. In another embodiment of the cysteine-mutated E-VLP-CP, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C, and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof, the thiol reactive small 37 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 molecule is selected from aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’- (1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), orSTG982-maleimide. See FIGS. 4A-4F, FIG. 13A. In another aspect of the cysteine-mutated E-VLP-CP, the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C and PhMV S137C, and wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof, and the thiol reactive small molecule is selected from aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’-(1,4,7,10- tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982- maleimide. The thiol reactive small molecules can be diagnostic or therapeutic small molecules. In another aspect of the cysteine-mutated E-VLP-CP, the wild-type VLP-CP is a cytoplasmic type citrus leprosis virus cytoplasmic type citrus leprosis virus, see e.g., Ortega- Rivera O.A. et al. (2023) Production of cytoplasmic type citrus leprosis virus-like particles by plant molecular farming. Virology https: / / doi.org / 10.1016 / j.virol.2022.11.004. In another aspect, the cytoplasmic type citrus leprosis virus and the one or more thiol reactive small molecules are selected from the group of aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982-maleimide. The thiol reactive small molecules can be diagnostic or therapeutic small molecules. In another embodiment of the E-VLP-CP, the one or more thiol reactive small molecules are selected from the group of aldoxorubicin, monomethyl auristatin E (MMAE), or 2,2’,2’’,2’’’- (1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)). The thiol reactive small molecules can be diagnostic or therapeutic small molecules In another embodiment of the E-VLP-CP, the one or more thiol reactive small molecules are selected from the group of aldoxorubicin, monomethyl auristatin E (MMAE), 2,2’,2’’,2’’’- (1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982-maleimide. The thiol reactive small molecules can be diagnostic or therapeutic small molecules. 38 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 In vitro and in vivo uses of Engineered Cysteine-Mutated VLP-CP The cysteine-mutated E-VLP-CP of this disclosure can be used in a method to deliver a thiol-reactive small molecule to a cell or tissue, comprising contacting the cell or tissue with a VLP comprising the cysteine mutated E-VLP-CP conjugated to one or more thiol-reactive small molecules as disclosed herein. The contacting can be is in vitro or in vivo. The cell can be a prokaryotic or a eukaryotic cell, and can be of any species, an animal, a mammal, a murine, a canine, a feline, or a human, for example. In some embodiments, the cell is a cancer cell, optionally an ovarian cancer cell or a melanoma cell. In one aspect, the cancer cell line is A2780 or B16F10. See FIGS. 10C-10D, and FIG. 14C. In another aspect, the cancer cell line is selected from mammalian cancer cell lines, for example breast cancer cells (e.g. MDA-MB-231 and MCF-7), ovarian cancer cells (e.g. A2780 and OVCAR3), melanoma cancer cells (e.g. SK-MEL-3, and SH-4, and B16F10), or cell lines from other cancers. Cancer cell lines are commercially available, for example from the American Type Culture Collection (ATCC). The E-VLP-CP can also be used to deliver a small molecule to a subject, comprising administering to the subject the E-VLP-CP as disclosed herein. The subject can be an animal or a mammal, such as a human patient, or a murine, feline, canine, equine, for example. Administration can be local to the site of action or systemic. In one aspect, provided is a kit for use in a method as disclosed herein. The method comprises, consists essentially of, or consists of (a) an E-VLP-CP and an optional instruction for use. Engineered VLP (E-VLP) comprising maleimide-PEGN-maleimide internal cross-linker In one aspect, provided herein is an engineered virus-like particle (E-VLP) comprising internal-crosslinkers, the E-VLP comprising: at least a first and a second capsid protein; and a maleimide-PEGN-maleimide linker internally cross-linking the first and the second capsid proteins to each other through internally surface exposed cysteines. In one aspect, the N in the PEGNrepresents an integer in a range selected from: 1 to about 125, 1 to about 115, 1 to about 100, 1 to about 50, 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to 39 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 about 20; about 5 to about 15; about 5 to about 10; or about 15. See e.g. FIG. 15A, FIG. 21A, and FIG. 22A. In another aspect, the first and second capsid proteins of the E-VLP are the same or different from each other. In another aspect, the capsid proteins of the E-VLP are from a plant Picornavirus nanoparticle, for example a Comovirus nanoparticle, a Tymovirus nanoparticle or other plant virus with an icosahedral shaped plasmid. In some embodiments the capsid proteins of the E- VLP are selected from a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic virus nanoparticle, a bean pod mottle viral nanoparticle, a rice tungro spherical viral nanoparticle, or a Physalis mottle virus (PhMV). In one aspect, the capsid proteins of the E-VLP are from a bacteriophage, optionally an icosahedral shaped bacteriophage, for example Qβ. In another aspect, the first and / or the capsid proteins of the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers are PhMV capsid proteins. In a further aspect, the first and / or the capsid proteins of the E-VLP are PhMV capsid proteins and wherein the N in the PEGNrepresents an integer in a range selected from: 1 to about 125, 1 to about 115, 1 to about 100, 1 to about 50, 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15, and the first and / or the second capsid proteins are from PhMV. In a further aspect, the first and / or the capsid proteins of the E-VLP are PhMV capsid proteins and wherein the N in the PEGNrepresents an integer in a range selected from about 10 to about 20; or about 15, and the first and / or the second capsid proteins. In one aspect, in the E-VLPs provided herein the maleimide-PEGN-maleimide linker has a molecular weight in a range selected from: about 0.5k Da to about 15k Da, from about 0.5k Da to about 10k Da, from about 0.5k Da to about 5k Da, from about 0.5k Da to about 2.5k Da, from about 0.5k Da to about 1k Da, or about 1k Da (see e.g. FIG. 16C, FIG. 16J, and FIGS. 21B-21C). 40 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 In yet another aspect, the maleimide-PEGN-maleimide linker in the E-VLPs provided herein has 1, 4, or 8 branches (see e.g. FIG. 21A and FIG. 22A). In yet another aspect, the ratio of maleimide-PEGN-maleimide linkers to internally surface exposed cysteines of the VLPs is selected from: about 1 to about 2000, about 10 to about 2000, about 10 to about 1500, about 10 to about 1000, about 10 to about 500, about 10 to about 100, or about 100, or about 1000 (see e.g. FIG. 16B). Virus-like particles (VLPs), like many biological molecules, can exhibit structural weakness, which may reduce shelf-life and functionality. VLPs can be used for therapeutic uses and also for agricultural uses, both of which benefit from increased stability. Thus, the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers is stronger than a non-engineered control VLP and more resistant to rupturing. For example, in one aspect the change in tip-substrate separation at which the force returns to zero following an event selected from rupture of the E-VLP, detachment of the E-VLP, or other scission event of the E-VLP (DRUP) is selected from: about 1 nm to 500 nm, about 10 nm to about 400 nm, about 30 nm to about 300 nm, about 40 nm to about 200 nm, about 50 to about 100 nm, about 10 to about 100 nm, about 50 nm, or about 100 nm. Alternatively, the DRUPof the E- VLP is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, or about 100-fold higher than the DRUPof a non-engineered control VLP. In yet another aspect, the force required to rupture (FRUP) the E-VLP is about 1-fold, about 2- fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold higher than the FRUPrequired to rupture a non-engineered control VLP. See e.g. FIGS. 19A-19D, FIG. 31, and FIGS. 33A-33B. Further provided is a plurality of E-VLPs as described herein, wherein the capsids of the E-VLP are the same or different from each other. In a further aspect, provided is a substantially homogenous plurality of E-VLPs as described herein, wherein in a further aspect, the capsids of the E-VLP are the same or different from each other. As used herein the term “substantially homogenous” intends a plurality wherein at least about 70%, or about 80%, or about 85%, or about 90%, or about 95% or about 98% of the plurality are identical to each other. 41 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 In some embodiments, the E-VLP has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm. In some embodiments, the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers has increased stability in the presence of chemical denaturants: surfactants, salt, low pH, high pH, chemical denaturants, and organic solvents than a non-engineered VLP comprised of the same viral capsid proteins (see e.g. FIG. 18G, Table 6). Non-engineered VLPs can stretch out in the presence of chemical denaturants as compared to an engineered VLP. In some embodiments, the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers has increased stability at higher temperatures as compared to a non-engineered VLP of the same viral capsid proteins (see FIGS. 18A-18B, 18E-18F). In one aspect, the E-VLP comprising maleimide-PEGN-maleimide internal cross- linkers is comprised of the cysteine-mutated E-VLP-CPs disclosed herein. Methods of Use of the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers Also provided is a method to deliver an E-VLP comprising maleimide-PEGN- maleimide internal cross-linkers to a cell or tissue, comprising contacting the cell or tissue with the E-VLP with one or more of: the E-VLP, the plurality, or the composition of this disclosure, thereby delivering the E-VLP to the cell or tissue. The contacting is in vitro, ex vivo, or in vivo, and / or the cell or tissue is animal or plant cell or tissue. In another aspect, the capsid proteins of the E-VLP are from a plant Picornavirus nanoparticle, for example a Comovirus nanoparticle, a Tymovirus nanoparticle or other plant virus with an icosahedral shaped plasmid. In some embodiments the capsid proteins of the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers are selected from a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic virus nanoparticle, a bean pod mottle viral nanoparticle, a rice tungro 42 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 spherical viral nanoparticle, or a Physalis mottle virus (PhMV). In one aspect, the capsid proteins of the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers are from a bacteriophage, optionally an icosahedral shaped bacteriophage, for example Qβ. In another aspect, the first and / or the capsid proteins of the E-VLP for use in this method are PhMV capsid proteins. In a further aspect, the first and / or the capsid proteins of the E-VLP are PhMV capsid proteins and wherein the N in the PEGNrepresents an integer in a range selected from: 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15, and the first and / or the second capsid proteins are from PhMV. In a further aspect, the first and / or the capsid proteins of the E-VLP are PhMV capsid proteins and wherein the N in the PEGNrepresents an integer in a range selected from about 10 to about 20; or about 15, and the first and / or the second capsid proteins. In some aspects, the E-VLP comprising maleimide-PEGN-maleimide internal cross- linkers are used as drug delivery vehicles. Thus, in one aspect, the E-VLP further comprises a therapeutic agent such as a small molecule, an antibody fragment or a therapeutic nucleic acid, e.g., mRNA. In a further aspect, the contacting is in vitro or in vivo, and the cell or tissue is an animal cell or tissue, that can be selected from canine, feline, equine, bovine, rat, simian or human. In a further aspect, the cell or tissue is a plant cell or tissue and the contacting comprising depositing the E-VLP on one or more of the stem, roots or leaves of the plant. In another aspect, the capsid proteins of the E-VLP are from a plant Picornavirus nanoparticle, for example a Comovirus nanoparticle, a Tymovirus nanoparticle or other plant virus with an icosahedral shaped plasmid. In some embodiments the capsid proteins of the E-VLP are selected from a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic virus nanoparticle, a bean pod mottle viral nanoparticle, a rice tungro spherical viral nanoparticle, or a Physalis mottle virus (PhMV). In one aspect, the capsid proteins of the E-VLP are from a bacteriophage, optionally and icosahedral shaped bacteriophage, for example Qβ. 43 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 In another aspect, the first and / or the capsid proteins of the E-VLP are PhMV capsid proteins. In a further aspect, the first and / or the capsid proteins of the E-VLP are PhMV capsid proteins and wherein the N in the PEGNrepresents an integer in a range selected from: 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15, and the first and / or the second capsid proteins are from PhMV. In a further aspect, the first and / or the capsid proteins of the E-VLP are PhMV capsid proteins and wherein the N in the PEGNrepresents an integer in a range selected from about 10 to about 20; or about 15, and the first and / or the second capsid proteins. In another aspect, provided is a method for administrating an E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers to a subject in need thereof, the method comprising, or consisting essentially of, or further consisting of administering to the subject an E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers of this disclosure, thereby administering the E-VLP to the subject. In some aspects, the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers is a drug delivery vehicle. In some aspects, the drug is a thiol-reactive small molecule. The subject can be an animal or mammal, e.g., a human patient. As is apparent to the skilled artisan, in one aspect, an effective amount or a therapeutically effective amount is administered which is determined by the treating physician or veterinarian. When administered to an animal, the methods provide an animal model to test combination therapy or personalized therapies. For example, the animal can be inoculated with tumor cells from a human patient. Alternatively, when the subject is a non-human animal, the methods provide veterinarian therapies. When the subject is a human patient, the methods provide methods to achieve one or more of the disclosed therapeutic benefits. Also provided are kits comprising one or more of the E-VLP comprising maleimide- PEGN-maleimide internal cross-linkers, the plurality, or the composition as disclosed herein, and instructions for use in the disclosed methods. 44 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Methods of Making the E-VLPs comprising maleimide-PEGN-maleimide internal cross-linkers In further embodiments, methods are provided for forming the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers as described herein. In one aspect, the E- VLP) comprises, or consists essentially of, or consists of at least a first and a second capsid protein; and a maleimide-PEGN-maleimide linker internally cross-linking the first and the second capsid proteins to each other through internally surface exposed cysteines. In one aspect, the N in the PEGNrepresents an integer in a range selected from: 1 to about 125, 1 to about 115, 1 to about 100, 1 to about 50, 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15. See e.g. FIG. 15A, FIG. 17A, and FIG. 22A. In another aspect, the first and second capsid proteins of the E-VLP comprising maleimide-PEGN-maleimide internal cross-linkers are the same or different from each other. In another aspect, the capsid proteins of the E-VLP are from a plant Picornavirus nanoparticle, for example a Comovirus nanoparticle, a Tymovirus nanoparticle or other plant virus with an icosahedral shaped plasmid. In some embodiments the capsid proteins of the E- VLP are selected from a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic virus nanoparticle, a bean pod mottle viral nanoparticle, a rice tungro spherical viral nanoparticle, or a Physalis mottle virus (PhMV). In one aspect, the capsid proteins of the E-VLP are from a bacteriophage, optionally an icosahedral shaped bacteriophage, for example Qβ. The methods include linking a plurality (at least 2) of capsid proteins, each having internal surface exposed cysteine groups, with a multiarm maleimide-PEGN-maleimide polymer. The linking proceeds by reaction of the thiol groups in the cysteine groups with the maleimide groups of the polymer, which, upon hydrolysis forms a succinamic acid thioether group. The multiarm maleimide-PEGN-maleimide polymer may have at least two maleimide groups on the polyethylene glycol backbone, in some embodiments. In other embodiments, there may be more than two maleimide groups. Illustrative examples having 4 and 8 maleimide groups are shown in FIG. 22A. 45 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 The methods of forming E-VLP materials include combining a plurality of capsid proteins, and a multiarm maleimide-PEGN-maleimide polymer to form the E-VLP, wherein each of the plurality of capsid proteins comprises internally surface exposed cysteine groups that react with a maleimide group of the multiarm maleimide-PEGN-maleimide polymer, and wherein the E-VLP comprises capsid proteins linked to one another by the multiarm maleimide-PEGN-maleimide polymer. In some embodiments, the multiarm maleimide- PEGN-maleimide polymer comprises two or more maleimide arms on the PEGNgroup. In other embodiments, the multiarm maleimide-PEGN-maleimide polymer comprises 2, 4, or 8 maleimide arms (also referred to herein as maleimide branch) on the PEGNgroup. In some embodiments the N in the PEGNrepresents an integer in a range selected from: 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15. In some embodiments, conjugation of the E-VLPs comprising maleimide-PEGN- maleimide internal cross-linkers is optimized with an eq. / Cys of about 1000. In any of the above embodiments of the methods, the combining may be conducted in a buffer solution. The temperature and light exposure of the linking reaction should be controlled, with the temperature being from greater than 0⁰C (avoiding freezing) to about 20⁰C. Further, the combining may be conducted by incubating the plurality of capsid proteins in the buffer solution, and the multiarm maleimide-PEGN-maleimide polymer at greater than 0⁰C to about 20⁰C for about 1 hour to about 72 hours. In some embodiments, the temperature is less than 10⁰C, or less than 5⁰C. The formed E-LVPs may then be collected via filtration or centrifugation. The formed E-LVPs may be in pellet form. In some embodiments, the methods to form the E-VLP comprising maleimide-PEGN- maleimide internal cross-linkers may be applied to the cysteine-mutated E-VLP-CP, wherein the capsid proteins used are the E-VLP-CPs. The resulting engineered virus-like particle would thereby have cysteine-mutated capsid proteins linked together with at least one maleimide-PEGN-maleimide polymer cross-linker. An E-VLP-CP with more cysteine residues could have more maleimide-PEGN-maleimide polymer crosslinkers, compared to a wildtype VLP-CP with fewer cysteine residues. 46 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Compositions 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. The component or combination of the present disclosure can be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal 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. In one embodiment, this technology relates to a composition comprising a component or a combination as described herein and a carrier. In another embodiment, this technology relates to a pharmaceutical composition comprising a component or a combination as described herein and a pharmaceutically acceptable carrier. 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. 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 the compounds 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 47 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 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. 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. 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. 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. 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. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art. 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 48 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 wetting agents (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. 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. 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, cremophoreTM, 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 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 49 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 herein, may be determined by one of ordinary skill in the art. 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. 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, 1416, 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. 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. 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 50 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 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. 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. Experimental Methods Example 1 An ongoing challenge in precision medicine is the efficient delivery of therapeutics to tissues / organs of interest. Nanoparticle delivery systems have the potential to overcome traditional limitations of drug and gene delivery through improved pharmacokinetics, tissue targeting, and stability of encapsulated cargo. Physalis mottle virus (PhMV)-like nanoparticles are a promising nanocarrier platform which can be chemically targeted on the exterior and interior surface through reactive amino acids. Cargo-loading to the internal cavity is achieved with thiol-reactive small molecules. However, the internal loading capacity of these nanoparticles is limited by presence of a single reactive cysteine (C75) per coat protein with low inherent reactivity. Here, Applicant uses structure-based design to engineer cysteine-added mutants of PhMV VLPs that display increased reactivity towards thiol- 51 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 reactive small molecules. Specifically, the A31C and S137C mutants show greater than 10- fold increased rate of reactivity towards thiol reactive small molecules and PhMV Cys1 (A31C), PhMV Cys2 (S137C), and PhMV Cys1+2 (double mutant) VLPs display up to three- fold increased internal loading of the small molecule chemotherapeutics aldoxorubicin and vcMMAE and up to four-fold increased internal loading of the MRI imaging reagent DOTA(Gd). These results further improve upon a promising plant virus-based nanocarrier system for use in targeted delivery of small molecule drugs and imaging reagents in vivo. Results and Discussion Preparation and characterization of PhMV VLPs. Applicant’s prior work with PhMV VLPs used a gene construct with an N-terminal hexahistadine tag and enterokinase cleavage site (FIG. 7A).(27)This form of PhMV VLP is herein referred to as PhMV-HM (HM = Hema Masarapu, who generated and provided this vector). Applicant re-designed the expression system to (1) remove the tag, which is not used for VLP purification and (2) Applicant sought to increase expression yield. Applicant established a dual expression vector system to generate untagged PhMV VLPs (herein referred to as PhMV-KB) using the carbenicillin-resistance plasmid pRSETa and kanamycin- resistance plasmid pET in E. coli (FIG. 7). This system improved VLP yield approximately five-fold to ~250 mg of VLP per liter of culture. Approximately 50% of the total bacterial lysate proteins were found to be recombinant PhMV CP (FIG. 8A). As expected, SDS-PAGE of PhMV-HM VLPs shows a single band at ~25 kDa corresponding to N-terminal tagged PhMV CP, while PhMV-KB VLPs shows a single band at ~20 kDa, corresponding to the native PhMV CP (FIG. 8B). PhMV-HM and PhMV-KB VLP preparations are both monodisperse, homogenous, and have similar whole particle characteristics by native gel electrophoresis, size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy (FIGS. 8C-8F), consistent with prior literature.(21)Structure-based design of cysteine-added PhMV VLPs. The thiol of the single cysteine (C75) of the PhMV CP, which is oriented internally inside the VLP (FIG. 1A), is accessible to alkylation with maleimides under physiologic conditions.(22),(27)Despite a theoretical maximum of 180 possible binding sites per VLP, prior studies suggest a maximum of 130-150 maleimide-based small molecules can be bound per 52 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 VLP using excess small molecules under optimal conditions,(29),(30)likely because it is partially buried within the bulk protein (FIG. 1B). Without wishing to be bound by any particular theory, Applicant hypothesized that further internal-facing amino acids of the PhMV coat protein could be mutated to cysteine to increase the loading capacity of PhMV VLPs without significantly altering the VLP superstructure or changing its surface characteristics. As such, Applicant identified alanine-31 and serine-137 as two potential residues, since both side chains are surface exposed and project internally, and do not participate in the electrostatic interactions between coat proteins that maintain PhMV coat integrity.(28)Additionally, the alpha carbon (CD) for both amino acids is located greater than 12 Å from C75 and each other (FIG. 1C), so are unlikely to form intramolecular disulfide bridges, which are rarely observed with CD distances beyond 7.0 Structural modeling of the A31C and S137C mutants predicts these side chains will be highly solvent exposed, and therefore highly accessible to chemical modification (FIGS. 1D-1E). Plasmids encoding the cysteine-added mutant PhMV CPs were generated through site directed mutagenesis and PhMV Cys1 (A31C), PhMV Cys2 (S137C), and PhMV Cys1+2 (A31C and S137C) VLPs were expressed and purified similarly to PhMV VLPs. Cysteine- added CPs had similar migratory characteristics to the native CP on SDS-PAGE (FIG. 2A). It should be noted that the VLPs, while devoid of genomic RNA, carry host RNA and differences in RNA affinity was noted. PhMV-KB and PhMV Cys1 VLPs showed similar RNA binding and migration by native gel electrophoresis, while the PhMV Cys2 and PhMV Cys1+2 VLPs shows mildly increased migration toward the cathode (FIG. 2B). PhMV Cys1+2 shows reduced RNA carrying, suggestive of reduced RNA affinity. The A260 / A280 ratio of PhMV Cys1 is 1.42, nearly identical to that of native PhMV (1.41), while PhMV Cys2 and PhMV Cys1+2 have an A260 / A280 of 1.23 and 0.96, respectively, again suggestive of reduced RNA carrying. Without wishing to be bound by any particular theory, Applicant hypothesizes that the reduced RNA-binding affinity of the PhMV Cys2 and Cys1+2 mutants is due to disruption of native CP-RNA interactions. All PhMV VLPs showed similar elution profiles on size exclusion chromatography, indicating the formation of intact VLPs, and notably without significant free coat protein or evidence of aggregation (FIG. 2C). All VLPs were homogenous by dynamic light scattering and showed characteristic ~30 nm diameter on transmission electron microscopy (FIGS. 2D-2E). 53 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Characterization of the cysteine reactivity of cysteine-added PhMV VLPs. Applicant next quantified the concentration of free thiols in PhMV-KB and cysteine- added mutant VLPs using DTNB (5,5’-dithio-bis-(2-nitrobenzoic acid), known as Ellman’s reagent), which reacts quickly with free thiols to generate the fluorescent species 5-thio-2- nitrobenzoic acid, which is highly colored and can be assayed by UV-Vis spectrophotometer at 412 nm and quantified by comparison to a standard curve generated with L-cysteine (FIG. 3A). Applicant observed a greater than 10-fold increase in the concentration of free thiols in PhMV VLP cysteine mutants as compared to PhMV-KB (FIG. 3B). This difference was substantially decreased after denaturation of the VLPs with SDS (FIG. 3C), suggesting the differences in DTNB-reactivity between the native C75 of PhMV and mutant C31 and C137 was due to differences in solvent exposure and / or local environment, which can dramatically alter cysteine pKaand therefore reactivity,(32)and not due to differences in oxidation. Applicant next assessed the reactivity of PhMV-KB and cysteine mutant VLPs to maleimide-Cy5. Maleimides are commonly used thiol-reactive warheads for the bioconjugation of small molecules to macromolecules. While Applicant typically performs bioconjugation reactions between the native cysteines of PhMV VLPs and maleimide- containing small molecules for at least 16 hours to increase modification efficiency, Applicant performed this experiment for only 6 hours to maximize potential differences in reactivity between PhMV-KB and the Cys1, Cys2, and Cys1+2 mutants. After reactions were quenched, VLPs were denatured and separated by SDS-PAGE to avoid potential internal fluorescent quenching within the VLP.(27)Applicant observed a marked difference in the amount of modification of native PhMV coat proteins as compared to cysteine-added mutants, as indicated by markedly increased fluorescent signal (FIG. 3D). Quantitatively, PhMV Cys1, PhMV Cys2, and PhMV Cys1+2 CPs showed 17-, 26-, and 34-fold increased fluorescent labeling, respectively, as compared to PhMV-KB (FIG. 3E), consistent with results with DTNB. Overall, these results indicate that the Cys1, Cys2, and Cys1+2 mutants of PhMV have markedly increased reactivity to cysteine-reactive small molecules. Characterization of the aldoxorubicin loading capacity of cysteine-added PhMV VLPs. 54 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Applicant has previously shown that the chemotherapeutic doxorubicin can be chemically conjugated to PhMV VLPs through reaction with aldoxorubicin,30which contains a cysteine-reactive maleimide coupled to doxorubicin through an acid-labile hydrazone linker (FIG. 4A).(19)Doxorubicin remains covalently associated with these VLPs at physiologic pH but is released in the acidic tumor microenvironment or after endocytosis into the acidic endolysosomal compartment.(33)Applicant purified aldoxorubicin-conjugated PhMV-KB and cysteine mutants, PhMV-KB-Aldox, PhMV Cys1-Aldox, PhMV Cys2-Aldox, and PhMV Cys1+2-Aldox, and ensured removal of excess small molecules by ultracentrifugation over a sucrose cushion. As expected, Applicant observed covalent modification of native PhMV and cysteine-added CPs with aldoxorubicin, as illustrated by increased coat protein fluorescence at 488 nm (FIG. 4B). All aldoxorubicin-conjugated VLPs maintained structural integrity and were homogenous and monodisperse (FIGS. 9A-9B). The amount of covalently bound doxorubicin, as measured by UV-Vis spectrometry, was significantly increased in all cysteine mutant VLPs as compared to PhMV-KB; the average number of doxorubicin molecules per VLP was 83, 184, 235, and 238 for PhMV-KB-Aldox, PhMV Cys1-Aldox, PhMV Cys2- Aldox, and PhMV Cys1+2-Aldox, respectively (FIGS. 4C-4D). To assess if the doxorubicin within these PhMV VLPs remained functional, Applicant determined the cytotoxicity for each VLP in A2780 ovarian cancer cells. All Aldox-VLPs show dose-dependent cytotoxicity (FIG. 4E). PhMV Cys2-Aldox and PhMV Cys1+2-Aldox had significantly decreased IC50 values of 11.2 μg / mL and 9.88 μg / mL, respectively, as compared to PhMV-KB-Aldox, which had an IC50 of 36.8 μg / mL (FIG. 4F). When normalized to the delivered aldoxorubicin concentration, there is no significant difference in cytotoxicity between aldoxorubicin-conjugated PhMV VLPs (FIGS. 9C-9D), which suggests that observed differences in cytotoxicity at a given VLP concentration is due to the variation in doxorubicin delivered per VLP, and not an altered mechanism of cytotoxicity upon delivery. Characterization of the MMAE loading capacity of cysteine-added PhMV VLPs. Monomethyl auristatin E (MMAE), a derivative of the anti-tubulin cytotoxin auristatin E,(34)is widely used in antibody drug conjugates. It is often conjugated to tumor- targeting antibodies as vcMMAE which contains a reactive maleimide and a valine-citrulline (vc) linker (FIG. 5A), which highly stable in serum but is rapidly cleaved in the endolysosomal compartment to expose the toxic payload.(20)Applicant have previously 55 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 shown that vcMMAE can be chemically conjugated to the native cysteines of the PVX and engineered thiols of TMV for tumor delivery.(35),(36)Applicant purified vcMMAE-conjugated PhMV-KB and cysteine-added mutants, PhMV-KB-vcMMAE, PhMV Cys1-vcMMAE, PhMV Cys2-vcMMAE, and PhMV Cys1+2-vcMMAE, and ensured removal of excess small molecules by ultracentrifugation over a sucrose cushion. vcMMAE has a molecular weight of 1.3 kDa, so covalent adducts with the PhMV coat protein can be observed by gel shift on SDS-PAGE. While Applicant observed minimal modification of the native PhMV CP, approximately one-half of the PhMV Cys1 and PhMV Cys2 CPs show bioconjugation to vcMMAE, and greater than two-thirds of the PhMV Cys1+2 CPs are single, doubly, or triply modified (FIG. 5B). When quantified by densitometry, this corresponds to approximately 43, 118, 109, and 202 vcMMAE molecules covalently bound to PhMV-KB-vcMMAE, PhMV Cys1-vcMMAE, PhMV Cys2-vcMMAE, and PhMV Cys1+2-vcMMAE VLPs, respectively. All vcMMAE-conjugated VLPs maintained structural integrity and were homogenous and monodisperse (FIGS. 10A-10B). To assess if MMAE bound to PhMV VLPs could be appropriately released for cytotoxic effect, Applicant determined the cytotoxicity for each PhMV-vcMMAE VLP in A2780 cells. All vcMMAE-VLPs show dose dependent cytotoxicity (FIG. 5C). PhMV Cys1-vcMMAE, PhMV Cys2-vcMMAE, and PhMV Cys1+2- vcMMAE had significantly decreased IC50 values of 49.2 μg / mL, 33.0 μg / mL, and 13.4 μg / mL, respectively, as compared to PhMV-KB-vcMMAE, which had an IC50 of 871 μg / mL (FIG. 5D). When normalized to the delivered vcMMAE concentration, there is a similar trend in cytotoxicity between vcMMAE-conjugated PhMV VLPs (FIGS. 10C-10D), with IC50 values of 1.62 μM, 999 nM, and 754 nM of vcMMAE delivered by PhMV Cys1- vcMMAE, PhMV Cys2-vcMMAE, and PhMV Cys1+2-vcMMAE, respectively, compared to 10.5 μM for PhMV-KB-vcMMAE. This suggests that there may be an additive advantage with delivery of a larger number of vcMMAE molecules per NP. Characterization of the Gd(III) loading capacity of cysteine-added PhMV VLPs. Gadolinium (Gd), an element of the lanthanide series that is highly paramagnetic due to its seven unpaired electrons, is commonly used as an magnetic resonance imaging (MRI) contrast agent.(37)Owing to its toxicity in a free state, Gd(III) is administered bound to a metal chelate, often diethylenentriamene pentaacetate (DTPA) or 2,2’,2’’,2’’’-(1,4,7,10- tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA).(38)Applicant has previously 56 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 shown that maleimide-functionalized DOTA (FIG. 6A) can be conjugated to PhMV VLPs through reactive thiols to generate a PhMV-based T1MR contrast agent.22Here Applicant conjugated DOTA in the presence of Gd; Applicant purified DOTA(Gd)-conjugated PhMV- KB and cysteine-added mutants, PhMV-KB-DOTA(Gd), PhMV Cys1-DOTA(Gd), PhMV Cys2-DOTA(Gd), and PhMV Cys1+2-DOTA(Gd), and ensured removal of excess Gd(III) ions by buffer exchange chromatography, ultracentrifugation, and dialysis. Maleimide-DOTA has a molecular weight of 786 Da, so a small gel shift is observed on SDS-PAGE after covalent modification of the PhMV coat protein. It is difficult to quantify DOTA-binding by this method, but PhMV Cys1+2 CPs clearly show a more substantial gel shift than the single mutants PhMV Cys1 and PhMV Cys2, or native PhMV CPs (FIG. 6B). All DOTA(Gd)- conjugated VLPs maintained structural integrity and were homogenous and monodisperse (FIGS. 11A-11B). The number of Gd(III) ions per VLP, as measured by ICP-MS, was significantly increased in all cysteine-added PhMV VLPs as compared to PhMV-KB; the average number of Gd(III) ions per VLP was 26.8, 52.6, and 87.7 for PhMV Cys1- DOTA(Gd), PhMV Cys2-DOTA(Gd), and PhMV Cys1+2-DOTA(Gd), respectively, compared to 19.1 for PhMV-DOTA(Gd) (FIG. 6C). Future studies exploring preformation and purification of the Mal-DOTA(Gd) complex prior to reaction with PhMV VLPs could be performed to further optimize Gd-loading efficiency. To assess if the increased Gd(III) ion loading translated to improved T1-weighted MR contrast attributes, Applicant next determined the T1and T2of a concentration series of the DOTA(Gd)-conjugated VLPs at 3.0 T (FIG. 12A). Owing to their similar Gd(III) ion loading, the R1and R2of PhMV-KB-DOTA(Gd) and PhMV Cys1-DOTA(Gd) were not significantly different, whereas PhMV Cys2-DOTA(Gd) and PhMV Cys1+2-DOTA(Gd) showed 1.90 and 2.98-fold increased R1values, respectively (FIGS. 6D-6F), indicated improved T1-weighted MR signal per VLP. The relatively high R2 / R1ratio of these VLPs (~5.0) is expected based on the high magnetic field strength used for these experiments, but suggests that further optimization of Gd(III) loading may be required to maximize their T1- weighted MR signal.(39)R1-relaxivity is also typically determined per Gd(III) ion to allow for comparison across monomeric vs multivalent systems,(40)and was calculated for all DOTA(Gd)-conjugated PhMV VLPs (FIGS. 12B-12D). The r1of PhMV-KB-DOTA(Gd) was 10.0 mM-1s-1per Gd(III) ion, consistent with MR theory and prior results that high 57 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 molecular weight complexes show increased r1-relaxivity due to slowed molecular tumbling (WR).(37),(41)The DOTA(Gd)-conjugated PhMV cysteine mutants displayed decreased r1 of 7.17 mM-1s-1, 6.91 mM-1s-1, and 6.49 mM-1s-1per Gd(III) for PhMV Cys1-DOTA(Gd), PhMV Cys2-DOTA(Gd), and PhMV Cys1+2-DOTA(Gd), respectively. Since no difference in total internal hydration is expected across these different VLPs, this observation may be due to increased water residency time (Wm) (i.e., decreased rates of exchange of inner shell and bulk water molecules) in PhMV VLPs with higher total Gd(III) ion count or increased coordination of other ligands such as phosphate or endogenous RNA.(41)Nevertheless, these results demonstrate that the increased Gd(III) ion loading capacity of cysteine-added PhMV VLPs correlate with overall improved T1-MR contrast agent properties. Conclusions Applicant has used structure-based design to rationally design a series of cysteine- added mutants of PhMV VLPs to increase the internal covalent loading capacity. Applicant shows that these mutants show increased reactivity towards thiol-reactive small molecules, including the chemotherapeutics aldoxorubicin and vcMMAE, and the MRI contrast agent DOTA(Gd). These cysteine-added mutants improve upon the PhMV VLP technology, which shows promise as a platform for the targeted delivery of small molecule drugs and imaging reagents in vivo. Materials and Methods Preparation of PhMV VLPs and cysteine-added mutants. PhMV-HM VLPs were prepared by expressing the CP in BL21(DE3), as previously described.(27)To prepare native PhMV VLPs (referred to as PhMV-KB VLPs), the PhMV CP gene (NCBI Gene ID 940246 (SEQ ID NO: 5; amino acid sequence corresponds to SEQ ID NO: 4) was cloned into pRSETa and pET vectors using established methods (FIG. 7).(42)PhMV VLPs were then expressed in BL21(DE3) with minimal modifications to the method previous described.(21),(27)The key difference between PhMV-HM and PhMV-KB is that the HM expression construct introduces a cleavable N-terminal hexahistadine tag, while PhMV- KB does not (HM = Hema Masarapu, who generated and provided this vector). Briefly, BL21(DE3) were double transformed with pRSETa-PhMV CP (carbenicillin resistant) and pET-PhMV CP (kanamycin resistant) to increase the plasmid copy number per cell. A single 58 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 colony was isolated and used to inoculate 50 mL of Luria Broth (LB, Sigma) supplemented with carbenicillin at 50 μg / mL and kanamycin at 50 μg / mL, which was grown for 18 hours at 37⁰C. This was used at a 1:100 dilution to inoculate 1 L of terrific broth (TB, Sigma Aldrich) supplemented with carbenicillin and kanamycin. Cultures were grown to OD600~ 1.0 and induced with 0.5 mM IPTG (Sigma) at 30⁰C overnight. Cultures were then pelleted at 7,500 x g for 10 min at 4⁰C, lysed by sonication in 50 mM sodium citrate pH 5.5 (SCB) and clarified at 30,000 x g for 30 min at 4⁰C. Lysates were then ultracentrifuged at an average of 111,818 x g for 3 hours at 4⁰C. Pellets were resuspended overnight in SCB and extracted with 0.5 volumes of 1:1 n-BuOH:CHCl3. The aqueous layer was isolated, layered onto a 10-40% linear sucrose gradient and separated by ultracentrifugation at an average of 103,613 x g for 3 hours at 4 ºC. The light scattering zone was collected, diluted with SCB, and centrifuged at an average of 161,018 x g for 3 hours at 4⁰C. The final pellet was resuspended in SCB supplemented with 0.5 mM TCEP (Sigma Aldrich) to yield pure VLPs, which were concentrated to greater than 10 mg / mL and stored at 4⁰C. Protein concentration was determined by BCA Assay (Thermo Fisher) using bovine serum albumin (BSA) as a standard. Yields were typically 250 mg of VLP per 1 L of bacterial culture. Plasmids for the expression of cysteine-added mutants of PhMV (PhMV Cys1 (A31C), PhMV Cys2 (S137C), PhMV Cys3 (A31C S137C)) were generated by polymerase chain reaction (PCR)-based site- directed mutagenesis using a high-fidelity polymerase (Q5, New England Biolabs [NEB]), and the mutagenesis primers listed in Table 1. Crude PCR reactions were digested with Dpn- I (NEB), purified to remove free nucleotides, and used to transform DH5D (NEB). Single colonies were isolated and amplified and vector integrity was confirmed by DNA sequencing. PhMV Cys1, PhMV Cys2, and PhMV Cys1+2 VLPs were purified similarly to PhMV-KB VLPs. Table 1. Site-directed mutagenesis primers. 59 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 VLP characterization. VLPs were characterized by SDS-PAGE (5 μg VLP on a 12% Bis-Tris, NuPAGE, Thermo Fisher), native gel electrophoresis (10 μg VLP on a 0.8% agarose in TBE), UV-Vis (Nanodrop 200 spectrophotometer, ThermoFisher), size exclusion (Superose 6 Increase 10 / 300 GL column at 0.5 mL / min on an AKTA FPLC, GE), dynamic light scattering (Zetasizer Nano ZSP / Sen5600, Malvern Panalytical), and transmission electron microscopy with 400-mesh hexagonal copper grids using UAc-negative-staining (2% w / v) and a FEI TecnaiSpirit G2 BioTWIN TEM at 80 kV for image acquisition, as applicable. The concentration of PhMV-bound doxorubicin was determined by UV-Vis spectroscopy using the extinction coefficient H(DOX, 488 nm) = 11,500 1. The concentration of Gd(III) ions was determined by inductively coupled plasma-mass spectrometry (ICP-MS). Briefly, 10 μL of sample was diluted in 490 μL concentrated nitric acid and 500 μL distilled water. The sample was then heated to 95⁰C for 10 min then added to 9 mL distilled water prior to analysis using an iCAP RQ system (Thermo Fisher). Samples were compared to four standard concentrations of Gd(III) (Sigma-Aldrich). All measurements were completed in duplicate. Structural analysis. Structural analysis of PhMV VLPs was performed using UCSF Chimera(43)using datasets PDB 1QJZ and PDB 1E57. The Multiscale models function was used for generation of the complete VLP. Modeling of cysteine-added mutants was performed with the rotamers tool using the Dunbrack 2010 rotamer library,(44)selecting the highest probability cysteine rotamer. Cysteine reactivity studies using DTNB and maleimide-Cy5. 60 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Free thiol concentrations of PhMV-KB and cysteine-added mutant VLPs were determined using DTNB (5,5’-dithio-bis-(2-nitrobenzoic acid), Ellman’s reagent, Thermo Fisher). Serial dilutions of PhMV-KB or cysteine-added mutant VLPs (100 μM -> 0.78 μM CP) were made in 10 mM KP pH 8.0. DTNB was then added to a final concentration of 200 μM and the reaction mixture was incubated at room temperature for 15 minutes. The resulting solution was then analyzed on a plate reader (Tecan) for absorbance at 412 nm. Free thiol concentrations were determined by comparison with a standard curve generated using L- cysteine (Sigma Aldrich). Denaturation studies were performed by heating of VLPs at 95⁰C for 5 min in 1% sodium dodecyl sulfate (SDS, Sigma-Aldrich), followed by serial dilution, reaction with DTNB, and analysis on a plate reader, as above. PhMV-KB or cysteine-added mutant VLPs were incubated with serial dilutions of maleimide-sulfoCy5 (10 eq / CP -> 0.01 eq / CP; LumiProbe) in 10 mM KP pH 7.5 at room temperature for 6 hours. Samples were then quenched with 50 mM dithiothreitol (DTT, Gold Bio), denatured at 95⁰C in SDS-loading buffer and separated by SDS-PAGE. In-gel fluorescence was measured using a FluorChem R system (ProteinSimple), then gels were stained with Coomassie Blue. Quantification was performed using FIJI (NIH).(45)Bioconjugation reactions with active ingredients. PhMV-KB or cysteine-added mutant VLPs were incubated with aldoxorubicin (MedChem Express) or valine-citrulline monomethyl auristatin E (vcMMAE; MedChem Express) at 3 molar equivalents per CP (eq / CP) in 10 mM potassium phosphate (KP) pH 7.5, overnight at room temperature. The resulting product was purified by ultracentrifugation (121,139 x g, 70 min) over a sucrose cushion (20% sucrose). The pellet was resuspended in PBS and resulting drug-laden VLPs were concentrated to 5-10 mg / mL and stored at 4⁰C. DOTA(Gd)-conjugated PhMV VLPs were generated by incubation of PhMV-KB or cysteine- added mutant VLPs with 3 eq / CP 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-acetic acid-10- maleimidoethylacetamide (Mal-DOTA, Macrocyclics) and 10 eq / CP GdCl3in TBS (25 mM Tris pH 7.5, 150 mM NaCl) at room temperature overnight. VLPs were then purified by buffer exchange (PD-10 column), ultracentrifugation, as above, and dialyzed into PBS to remove unbound Gd(III). Final DOTA(Gd)-laden VLPs were concentrated to 5-10 mg / mL and stored at 4⁰C. 61 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Cytotoxicity assay. A2780 cells were cultured in RPMI media (Corning) supplemented with 10% fetal bovine serum (FBS, R&D Systems) and 1% penicillin-streptomycin (Cytiva) at 37ºC in a 5% CO2humidified incubator. A2780 cells were plated at 3,000 cells / well in 96 well plates and grown overnight. Serial dilutions of Aldox- or vcMMAE-conjugated PhMV VLPs were added directly to the media and cells were incubated for an additional 72 hours. Cells were then analyzed using a CellTiter Glo assay (Promega) according to the manufacture’s protocol. All conditions were performed in triplicate. Curves were fit using a least-squares methods (Prism, GraphPad). MR relaxivity studies. The magnetic resonance transverse and longitudinal relaxivities of DOTA(Gd)- conjugated PhMV-KB and cysteine-added mutant VLPs were determined on a preclinical 3.0 T scanner (BioSpec 3T, Bruker) using an 82 mm inner diameter transmit-receive volume coil. T1 mapping was performed using a variable repetition time (TR) rapid acquisition with relaxation enhancement (RARE) sequence with 10 TRs ranging from 390 – 6990 ms and echo time (TE) 7 ms. T2 mapping was performed using a Carr-Purcell-Meiboom-Gill (CPMG) sequence with TR 9853 ms and 20 TEs ranging from 30-600 ms. T1 and T2 values were calculated using the Image Sequence Analysis tool on ParaVision 360 v3.3 (Bruker) and values plotted using Prism (GraphPad). R1and R2were calculated using a linear fit (least- squares). Example 2 PhMV-STING Modified Plant Virus-Like Nanoparticles Applicants have produced an immune-stimulating drug nanocarrier by employing cysteine-added mutant Physalis mottle virus-like particles (PhMV VLPs) to internally load a stimulator of interferon genes (STING) agonist via thiol-maleimide chemistry. FIG. 13 shows the reaction scheme (A) and characterization of the resulting conjugated nanoparticles (FIGS. 13 B-G). Through fast protein liquid chromatography (FPLC, FIG.13B), dynamic light scattering (DLS, FIG. 13C), and transmission electron microscopy (TEM, FIG. 13F) Applicant saw that loading the STING agonist to the internal cavity did not alter the size or polydispersity of PhMV VLPs and maintained the characteristic ~30 nm diameter of native 62 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 PhMV. In an SDS-PAGE denaturing gel (FIG. 13D), Applicant found that migration of STG- conjugated coat proteins (CPs) was slightly delayed in comparison to non-conjugated CPs (20 kDa), with a faint band emerging at ~21 kDa, correlating to the added 1283 Da mass of the STG moiety. Native agarose gels (FIG. 13E) show that PhMV VLPs maintain some host RNA from the bacterial expression process, which is not decreased by the addition of STG. Additionally, migration through the gel is minimally slowed for PhMV-STG, but overall still shows a positively charged particle moving toward the anode. Loading of the STING agonist in PhMV VLPs was quantified through tandem liquid chromatography-mass spectrometry (LC-MS), with the largest population of CPs remaining unconjugated and ~24% of all available cysteines being addressed. To evaluate the activity of PhMV-STG nanoparticles, Applicant first tested them in a THP-1 monocyte reporter cell assay (FIG. 14A). Over a 72-hour period of THP-1 cell stimulation by either free STG or PhMV-STG, both induced interferon response, but with PhMV-STG inducing a significantly higher response. Without wishing to be bound by any particular theory, Applicant hypothesizes this is due to the faster degradation of free STG, while PhMV protects STG from degradation and releases it slowly to produce continual STING agonism over a longer period. To evaluate intratumoral release of a thiol-maleimide conjugated molecule from PhMV VLPs, Applicant replaced the STING agonist with a sulfo- Cy5.5-maleimide fluorophore. Applicant then injected either PhMV-Cy5.5 or free Cy5.5 into a dermal B16F10 melanoma tumor and observed the fluorescent signal over time via an in vivo imaging system (IVIS, FIG. 14B). Compared to the initial fluorescence, PhMV-Cy5.5 showed a sustained signal over 168 hours (7 days), while free Cy5.5 decreased significantly within the first 48 hours. Finally, Applicant tested PhMV-STG and free STG as intratumoral immunotherapies against dermal B16F10 melanoma (FIG. 14C). At the same concentration of STG (20 μg), both PhMV-STG and free STG were able to eradicate tumors, with PhMV- STG outperforming free STG with 4 / 6 eliminated tumors from PhMV-STG and 2 / 6 from free STG. Other potential small molecule immunodrugs that could benefit from PhMV VLP internal conjugation are toll-like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists). 63 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Example 3 Many virus-like particles (VLPs) have good chemical, thermal, and mechanical stability compared to other biologics. However, stability needs improvements to enable VLP- based materials for translation and commercialization. Applicant developed an endoskeleton- armored strategy to empower VLP stability. Specifically, the VLPs of physalis mottle virus (PhMV) and Qbeta (Qβ) were used to demonstrate this concept. Applicant built an internal polymer ‘backbone’ using maleimide–PEG15–maleimide crosslinker to covalently interlink viral coat proteins inside the capsid cavity, while the native VLPs are held together only by non-covalent bonding between subunits. Endoskeleton-armored VLPs exhibited significantly improved thermal stability (95°C for 15 min), increased resistance to denaturants (i.e., surfactants, pHs, chemical denaturants, and organic solvent), and enhanced mechanical performance. Single-molecule force spectroscopy demonstrated a 6-fold increase in rupture distance and a 1.9-fold increase in rupture force of endoskeleton-armored PhMV. Overall, this endoskeleton-armored strategy provides more opportunities for materials development and applications. As described herein, the EE-VLP (for example, EE-PhMV or EE-Qβ) of Example 3 is an engineered virus-like particle (E-VLP). The EE-VLP of Example 3 is an E-VLP that includes at least one maleimide-PEGN-maleimide linker internally cross-linking the first and the second capsid proteins to each other through internally surface exposed cysteines. VLPs can be engineered in other aspects. Experimental Discussion Virus-like nanoparticles (VLPs) are nanomaterials based on the ordered and self- assembling capsids of viruses that lack their infectious genetic material.[2]VLPs are an attractive nanotechnology platform for various discoveries and applications in virology,[3]vaccination,[4], [5]bionanotechnology,[6], [7]and material science.[8], [9]Stabilizing the VLP platform is crucial to ensure their structural integrity and functionality are maintained during synthesis (e.g., harsh synthesis conditions),

[0010] storage and shipping,

[0011] ,

[0012] and performance (e.g., heat-generating devices).

[0013] ,

[0014] Researchers have developed strategies for multiple- point polymer conjugation to the VLP’s exterior, but these particles’ external chemical functionalities are therefore occupied.15Alternatively, the interior of VLPs can be polymer- filled via a step-wise approach or atom-transfer radical polymerization.[16-18]Still, these 64 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 studies mainly focused on improving the loading capabilities instead of stability (mini survey, Table 2). As a result, the performance of VLPs stabilized by internal polymeric crosslinkers has yet to be fully understood—the community still lacks a simple yet versatile strategy to stabilize VLPs. Table 2. A mini-survey of viruses, virus-like particles, and protein cages stabilized by synthetic materials. Substrate(s) Synthetic materials Synthesized Stressor(s) Ref. interfaces Physalis mottle Bi-valent PEG Internal Heating; surfactant, extreme Applicant’s virus-like particles, interfaces ionic strength, pHs, Urea, and Disclosure Qβ virus-like between CPs guanidinium chloride; particle methanol; mechanical pulling (through AFM). Tobacco mosaic ZIF-8 External Heating, methanol,

[0014] virus guanidinium chloride, and ethyl acetate. Brome mosaic virus Fe3+–TA External Dehydration

[0063] Tobacco mosaic Gd(DOTA)-azide; External Heating (through laser)

[0064] virus polydopamine Heat shock protein Tripropargyl amine; Internal Heating (100 and 120 ℃)

[0017] 2-Azido-1- azidomethyl- ethylamine Qβ virus-like Poly(2-oxazoline)s External Heating (100 ℃)

[0015] particle Bacteriophage P22 2-aminoethyl Internal Heating (max.85 ℃)

[0016] virus-like particle methacrylate Abbreviation: ZIF-8, zeolitic imidazolate framework-8; Gd, gadolinium; DOTA, monoamide-1,4,7,10- tetraazacyclododecane-N-N′-N″-N′′′-tetraacetic acid; TA, Tannic acid. Here, Applicant focused on VLPs derived from physalis mottle virus (PhMV) and the bacteriophage Qβ. PhMV VLP is derived from +ssRNA virus from the family Tymoviridae, forms a ~30 nm-sized icosahedral capsid from 180 identical CPs, and can be recombinantly expressed and purified as a homogenous and stable VLP.

[0020] The PhMV CP contains one solvent-exposed cysteine residue (C75), internally oriented within the VLP and amenable to 65 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 alkylation with thiol-reactive small molecules.

[0021] Qβ VLPs also form T3 icosahedrons assembled from 180 identical CPs to measure 28 nm in diameter.

[0022] The Qβ VLP has two cysteine residues (C74 and C80) per CP that form inter-subunit disulfide bonds and link individual CP into hexametric or pentameric CPs.

[0023] The disulfide bonds can be reduced and repurposed for chemical conjugation.

[0024] ,

[0025] In addition, VLPs feature pores through which cargo can diffuse to access the interior

[0026] . Although VLPs are not static objects, yet dynamic structures, the pore sizes could be estimated. For examples, the PhMV VLP possesses pores diameters of 1.0–1.4 nm,

[0020] and the Qβ VLPs possesses large pores with either 1.5 or 3.0 nm.

[0027] Without being bound by theory, Applicant sought to stabilize VLPs. Specifically, Applicant built an internal VLP ‘backbone’ through covalent inter-CP linking using maleimide–PEGn–maleimide bi-valent crosslinkers, denoted as mal–PEGn–mal (FIG. 15). A combination of gel electrophoresis (native and denaturing), fast protein liquid chromatography (FPLC), dynamic light scattering (DLS), and transmission electron microscopy (TEM) was used to assay and validate the structural properties of the endoskeleton-armored VLPs. Chemical competition assays provided insights into the chemical conjugation efficiency. Endoskeleton-enhanced (EE-) VLPs were exposed to various stressors – i.e., surfactant, extreme pHs, chemical denaturants, organic solvent, and mechanical stress – and characterized by turbidimetric assay, TEM, DLS, and temperature- dependent circular dichroism (CD) spectroscopy. Finally, the mechanical performance was mapped by single-molecule force spectroscopy. Synthesis and Characterization of Endoskeleton-Enhanced (EE-) VLPs First, Applicant tested mal–PEGn–mal crosslinkers of various molecular weights (0.6– 5k Da) and number of branches (f = 1, 4, and 8). To achieve a high degree of linking, Applicant needed to define a suitable size cut-off (i.e., Flory radius) for the polymer to enter the porous VLP capsid and access the internal cysteine side chains. Further, it was important to moderate contour length enabling access to neighboring cysteine residues while avoiding masking effect, i.e., if the polymer chain is too long, the reactive groups may become buried due to the extra steric hindrance.

[0032] The end-to-end distance between the sulfur atom of the cysteines on neighboring coat proteins is 27.1–43.9 Å (FIG. 20). To determine an optimized synthesis, Applicant first tested mal–PEGn–mal crosslinkers with different number of 66 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 ethylene glycol repeats (n = 6, 15, 43, and 115, where f is fixed at 1), number of branches (f = 1, linear; f = 4, and 8, multi-arm), and equivalents of crosslinker per cysteine (denoted as eq. / Cys, i.e., the ratio of the number of mal–PEGn–mal crosslinkers to the number of cysteines) (Table 3). Upon successful coupling of mal–PEGn–mal to the VLPs, the succinimide thioether intermediate undergoes ring-opening hydrolysis to add a formal negative charge (FIG. 16A).

[0033] ,

[0034] The adduct also adds molecular weight. Therefore, Applicant used a combination of electrophoretic mobility shift assay (EMSA) and denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis (FIGS. 16B-16C, FIGS. 21-24) to analyze the cross-linked products. The combination of methods has been validated as reliable to monitor molecular weight and charge changes for modified VLPs.

[0016] ,

[0025] ,

[0035] EMSA and SDS-PAGE indicate that the shorter mal–PEGn–mal crosslinkers (n = 6, 15, and 43, f = 1) resulted in successful linking. When n = 15 is used, the linking is the most efficient due to its relatively small Flory radius (17.6 Å) and moderate contour length (41.7 Å) (Table 3, FIG. 21). Notably, the longest crosslinker (n = 115, Flory radius = 60.3 Å) is excluded from the VLPs by the gating effect of size-exclusive pores between CPs. The crosslinker with n = 43 yielded inefficient labeling, despite being short enough to enter the VLPs (Flory radius = 33.3 Å); its long ethylene glycol chain (contour length = 120 Å) may cause steric hindrance yielding inefficient conjugation. Lastly, when the crosslinker is too short (n = 6, contour length = 16.7 Å) to reach neighboring cysteine, the linking is proven not effective in EMSA. Then, Applicant tested multi-branched mal–PEGn–mal crosslinkers (f = 4, n = 8; f = 4, n = 25; and f = 8, n = 25) but found that even when the crosslinkers are small enough (Flory radii < 36.6 Å) to enter VLPs, the multi-branched mal–PEGn–mal crosslinkers are less reactive compared to their linear counterparts (FIG. 22). The increased rigidity and masking effects can explain this reduced reactivity. Next, the synthesis is optimized with eq. / Cys ranging from 0–2000 by EMSA and SDS-PAGE analysis. Conjugation is most effective when eq. / Cys = 1000. EMSA shows a characteristic sigmoid curve (FIG. 16B) over eq. / Cys, plateauing at eq. / Cys = 1000. This is consistent with SDS-PAGE analysis showing an increase in the molecular weight of the CP (19.98k Da) to dimers, multimers, and finally, a higher molecular weight cross-linked CP assembly that lacks electrophoretic mobility (MW > 198k Da), plateaued at eq. / Cys = 1000, 67 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 (FIG. 16C). The percentage of remaining monomeric bands (i.e., unreacted CP and CP– PEG15–mal) at ~20k Da decreases from 100% (eq. / Cys = 0) to 14.4% (eq. / Cys = 1000), processed by ImageJ (FIG. 25).

[0036] Notably, the band laddering observed in SDS-PAGE cannot be interpreted as the true molecular weight of the CP–polymers – this is because the EE-VLPs (especially eq. / Cys > 50) are so stable that the standard denaturation process (1% w / w SDS, 95 °C for 5 min) may not be sufficient to denature the VLPs (see FIG. 18). Nevertheless, the SDS-PAGE analysis agrees with EMSA and provides insights into the degree of linking of the EE-VLPs. In summary, the linear mal–PEG15–mal telechelic crosslinker (n = 15, f = 1, and eq. / Cys = 1000) was adopted for synthesis of EE-VLPs for further characterization. After optimizing synthesis conditions, Applicant analyzed and compared the VLP nanoparticle characteristics of native PhMV, EE-PhMV100, and EE-PhMV1000. In FPLC, the EE-PhMV1000, EE-PhMV100, and non-modified PhMV elute at 12.1, 12.4, and 12.9 mL, respectively (FIG. 16E). The crosslinkers are contained securely within the VLPs (FIG. 26). The shoulder at ~10-mL elution volume may represent a small fraction (less than 5% of the total VLP fraction) of particle doublets or aggregates, possibly due to ultracentrifugation and purification.

[0017] The DLS results are consistent with FPLC. The hydrodynamic sizes of non- modified PhMV and EE-PhMV1000are measured by DLS to be 35.54 ± 8.87 nm and 36.38 ± 9.35 nm in hydrodynamic diameters with polydispersity index (PDI) reported, respectively (FIG. 16D, Table 4). This ~2.4% size increase may be attributed to PEG crosslinkers protruding through the pores; this has been observed with other VLP–polymer complexes.

[0018] ,

[0037] Lastly, TEM images confirmed good dispersity and morphologic integrity of EE-PhMV VLPs (FIG. 16F). Together, the data suggest that mal–PEG15–mal crosslinking of the VLPs does not alter their structural conformation, even at the high degree of internal linking obtained at eq. / Cys = 1000. To determine the degree of conjugation of EE-PhMV, Applicant used a double- conjugation reaction competition assay with maleimide–Cyanine5 (mal–Cy5, MW = 605.8 Da). Incomplete cysteine conjugation leaves unoccupied side chains for further reaction with mal–Cy5, resulting in fluorescent CP detectable on SDS-PAGE. While there is some fluorescent signal observed for the Cy5-labeled EE-PhMV100, no fluorescence was observed with EE-PhMV1000, suggesting that a complete reaction with the mal–PEG15–mal linker was 68 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 indeed achieved (FIGS. 16G-16H, FIG. 24, Table 5). In addition to the fluorescent CP, higher molecular weight dimers and multimers are apparent. Multimers are often observed when viral CPs are labeled with polymers, because CP–polymer conjugates tend to physically entangle in SDS-PAGE.

[0038] ,

[0039] To further ensure that the higher-molecular-weight bands are a result of covalent linking and not artifacts from CP–polymer physical entanglement, Applicant reacted VLPs with mono-valent mPEG17–mal (methoxy-PEG17–maleimide, molecular weight 1k Dalton) (FIGS. 16I-16J). The conjugation resulted in an expected size increase with a CP–PEG at ~21 kDa, compared with native CP at ~20 kDa, but higher-order CP adducts are observed. It is interesting to note that the conjugation with mono-valent mPEG17–mal did not result in complete reaction. This contrasts with results of bi-valent mal– PEG15–mal. Without wishing to be bound by any particular theory, Applicant hypothesizes that although both linkers undergo steric hinderance within the interior, the improved reactivity of bi-valent crosslinkers could be explained by nanoconfinement effect. That is, once one end is conjugated, the rate of reactivity of the other end increases relatively.[6],

[0040] Further, to generalize the scope of this EE strategy, Applicant validated this concept in the Qβ VLP targeting cysteines at the interface, namely C74 and C80. In the native VLP, these residues are covalently linked through disulfide bonds, and Applicant first reduced them to free thiols to render them chemically addressable (FIG. 17A). The mal–PEG15–mal was then conjugated following the methods applied to PhMV. EMSA and SDS-PAGE support chemical linking as evident by mobility changes and laddering effects as a function of reactant ratio (eq. / Cys) (FIG. 17B, FIG. 27). DLS and TEM confirmed that monodisperse and structurally sound EE-Qβ100were obtained (FIGS. 17C-17D). These data validate the proposed EE strategy could be applied to various VLPs, even to those with cysteines at their coat protein interfaces. Thermal Stability of EE-PhMV1000The stability of VLPs, which are macromolecular assemblies of their coat proteins, relies on a delicate balance between short-range, predominantly hydrophobic attraction and long-range electrostatic repulsion,

[0042] ,

[0043] which are considered ‘weak links’ due to the nature of non-covalent interactions.

[0015] Different denaturing conditions, such as extreme temperature,

[0015] ionic strength, acid, base,

[0044] surfactant,

[0045] or chaotropic agents

[0014] ,

[0046] reduce or increase the strength of non-covalent bonds – therefore weaken or strengthen the coat 69 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 protein interactions within the VLPs thus altering stability. The thermal stability of non- modified PhMV and EE-PhMV was probed in buffered media testing a temperature range of 25–90⁰C for 15 min. Visual inspection of the test tubes showed clouding of the non-modified PhMV, and this phenomenon – consistent with protein aggregation and precipitation – was enhanced the higher the temperature (FIG. 18A, inset). In stark contrast, the temperature treated EE-PhMV nanohybrids solutions remained clear. The turbidity was further analyzed by measuring the Abs600(FIG. 18A);

[0047] ,

[0048] the quantitative read-out demonstrates that the polymer endoskeleton improves the thermal stability of PhMV VLPs from 50⁰C for the non- modified VLPs to 80⁰C for the EE-PhMV. Further, TEM images show both nanoparticles remain structurally intact at 65⁰C. At 80⁰C and 95⁰C, the non-modified PhMV VLPs appear in heterogenous shapes and irregular sizes due to CP disassembly and reassembly, indicating the loss of structural integrity (FIG. 18B). In stark contrast, the EE-PhMV nanohybrids appear to maintain structural integrity – this data indicates that the EE-armor increased the thermal stability of the quaternary structure of EE-PhMV relative to PhMV VLPs. Next, to assess the thermal effects on the secondary structure, PhMV and EE- PhMV1000(FIGS. 18C-18D) were analyzed by far-UV circular dichroism (CD) with thermal stressing (95⁰C for 15 min) or without (4⁰C). The CD spectra of PhMV and EE-PhMV show a maximal ellipticity at 218 nm, characteristic of proteins rich in β-sheet structures,

[0030] ,

[0049] which indicates that the introduction of mal–PEG15–mal does not affect the content of β- sheet.

[0015] After 95⁰C exposure, the non-modified PhMV loses signal at 218 nm, while the EE- PhMV1000maintains strong signal. With a fixed-wavelength detection of 218 nm, the β-sheet signals of PhMV and EE-PhMV are monitored within the 25–100⁰C temperature range (FIGS. 18E-18F, FIG. 20).

[0050] The melting temperature (Tm) of PhMV and EE-PhMV1000is 89.2 and >100 °C. Denaturation of the intact PhMV is irreversible, as the cooling of denatured PhMV does not result in the recovery of secondary structures. These results support the conclusion that EE-PhMV is highly thermally stable. A potential mechanism is summarized herein. In summary, this endoskeleton-armored strategy might lead to steric compaction and enhanced attractions between CPs, making EE-VLPs more resistant to the disassociation of both secondary and quaternary structures under various stressors. Potential mechanism for why endoskeleton-armored strategy stabilizes both secondary and quaternary structures 70 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Qβ VLP has either five or six disulfides to form pentamers and hexamers that are covalently linked, providing protection at the level of quaternary structure. The melting temperature (Tm) of Qβ VLP, as detected by CD spectra, is significantly higher in the presence of disulfide bonds than in their absence (85.1⁰C vs 60.5⁰C). This example is similar to the findings presented herein, which suggest that protecting quaternary structures can help preserve the secondary structures. In the work presented herein, compared with the native icosahedral VLP where the short-range hydrophobic attraction dominated,

[0042] it is a hybrid mechanism for EE-VLPs: (1) Steric compaction. The PEG linkers help CPs to keep a compact arrangement under thermal stressor (FIG. 18B, TEM of PhMV and EE-PhMV1000under 95⁰C), thereby sterically compact the surrounding CPs, and preventing the CPs from further unfolding, i.e., secondary structures. (2) Enhanced attractions. Compared with a monomer, the PEG-linked dimers increase the area of the hydrophobic site on one dimer’s sides where it interacts with the surrounding dimes, which enhances the already-existed hydrophobic attraction and van der Waals forces. In summary, the endoskeleton strategy might lead to steric compaction and enhanced attractions between CPs, making EE-VLPs more resistant to the disassociation of secondary and quaternary structures under various stressors. Comprehensive Stability of EE-PhMV1000under Various Denaturants Applicant tested whether the EE-VLP1000would also be more resistant to various chemical denaturants. Non-modified PhMV and EE-PhMV1000were exposed to SDS (1% w / v, 24 h, RT), NaCl (1–4 M, 6 h), pH (1.2–12, 24 h), urea (1–6 M, 24 h), guanidine hydrochloride (1–6 M, 24 h), and methanol (25–90%, 2 h); pre- and post-treatment samples were analyzed by DLS (FIG. 18G, Table 6). Under SDS and NaCl treatment, the size distribution of PhMV and EE-PhMV1000remains roughly unchanged. Stressed by different pHs, urea, and GdnHCl concentrations, the size distributions of PhMV changed dramatically, whereas EE-PhMV remained comparatively stable with minor changes in sizing. Notably, the size distribution of EE-PhMV1000is only slightly affected at 50% methanol, indicating that stability was enhanced and suggesting that a higher degree of stability may be conferred through a more robust EE strategy to stabilize the VLPs in organic solvents, which potentially lead to the use of endohedrally catalyst-functionalized cages for catalysis.

[0051] It is 71 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 interesting that the stability profile could be enhanced so significantly through a simple bi- valent linking strategy – in prior research enhanced stability of VLPs was only reported through external multi-point-attachment crosslinkers.

[0015] Single-Molecule Force Spectroscopy of PhMV and EE-PhMV1000To determine the mechanical performance of the intact PhMV and EE-PhMV1000, a single-molecule force spectroscopy (SMFS) study was performed by using AFM at the force- extension operational mode, with a pulling velocity of 1000 nm per second.

[0053] ,

[0054] PhMV and EE-PhMV1000each exhibits five exterior-exposed carboxylates (namely, side chains of D58, D83, E39, and E47, and C-terminus). The PhMV or EE-PhMV1000was immobilized on an amine-functional substrate, followed by conjugating to an amine-functionalized AFM cantilever by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) crosslinker (FIGS. 29- 30). The external surface and size of the VLPs determine the coupling of the VLPs to the surface and tip and the starting point of force-extension curves, and these characteristics are effectively identical for PhMV and EE-PhMV1000. Thus, Applicant attributes any differences in the SMFS force-extension curves to the endoskeleton. Two key parameters, rupture force (Frup) and rupture distance (Drup), are extracted from the force-extension curves. The Drupis the change in tip-substrate separation at which the force returns to zero following either rupture of the particle, detachment of the particle, or other scission event along the analyte.

[0055] ,

[0056] According to FIG. 19A, (1) the NP (with externally exposed carboxylate groups) is immobilized onto the amine-functionalized station by EDC crosslinker; (2) the NP is conjugated onto an amine-functionalized AFM cantilever by second round of EDC chemistry; (3) the force and distance are monitors between NP and the cantilever. It therefore captures any difference in mechanical extension of the VLP that might be facilitated by unfolding of the inter-connected CPs (FIG. 19A).

[0057] The key difference is shown in selected SMFS curves in FIG. 19B. The SMFS curve of PhMV reflects a single, smooth extension and rupture event with no evidence of multi-domain protein unfolding. The curve is instead consistent with a single distortion / stretching event that culminates in an abrupt dissociation (e.g., that of a tip- or surface- tethered CP from the VLP). In stark contrast, SMFS of EE-PhMV1000shows a sawtooth-like curve that is consistent with a series of multiple mechanical rupture events.

[0058] ,

[0059] These characteristics are general for the two analytes; additional examples of pulling events of PhMV and EE- 72 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 PhMV1000are provided (FIG. 31). The data show that the chemically connections between the CPs connects them mechanically as well, as characterized by net rupture force and distance that increase due to the sawtooth-like extension brought about by mal–PEG15–mal linking (FIGS. 32-33).

[0057] The intact PhMV NPs show a relatively narrow distribution in rupture distance, while that of IC-PhMVeq.1000 is shifted toward a longer rupture distance. The statistics of Frupand Drup(FIGS. 19C-19D) show increases in both upon endoskeleton formation; Drupincreased from 22 ± 16 to 136 ± 107 nm, and the Frupincreased from 422 ± 466 to 814 ± 677 pN. Notably, the multiple-pulling artifacts that multiple viruses involved in a single-pulling event were manually ruled out (FIG. 34). Because the total energy absorbed before dissociation is a function of both force and distance achieved by the extended analyte, the endoskeleton formation results in a substantial mechanical toughening of the VLPs. The average rupture distance of PhMV (i.e., 24 nm) is considerably shorter than the stored length of one CP (estimated to be approximately 73 nm; see equation I),

[0054] which is consistent with an absence of CP unfolding in the dissociation process. A precise physical picture underlying the sawtooth pattern observed in EE-PhMV1000cannot be inferred from the phenomenological data available, but a possible mechanism involves a combination of multiple CP dissociations, where entanglements between the congested and overlapping PEG15linkers prevent simple dissociation of one or two CPs from breaking the mechanical connection between tip and substrate, which is also observed in SDS-PAGE (FIG. 16C). The physical entangling between linked CPs is typically invoked for the aggregation observed in gel electrophoresis, as noted above, and so it is reasonable to expect that similar effects are present within the intact, modified VLP. In addition to the extension enabled by multiple CP dissociation events, unfolding of dissociated CPs might allow further extension. Conclusion To summarize, Applicants have outlined a method for improving the stability and mechanical properties of VLPs; PhMV was used as a testbed, and Applicant also demonstrated feasibility of the endoskeleton-enhancement strategy for Qβ VLPs. The endoskeleton armor is synthesized through covalently linking adjacent CPs using bi-valent crosslinkers. Superior stability is conferred through introduction of covalent bonding of the otherwise non-covalently bonded macromolecular protein assembly. The crosslinkers act as a “backbone” to enhance the VLPs' overall performance. Applicants highlight several points: 73 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 (1) The endoskeleton-armored strategy does not occupy the external surface or derivatize motifs, allowing for future modular ‘add-on’ functionalities. (2) The endoskeleton-armored VLPs were significantly more stable to heat, bathing conditions with various denaturants, and mechanical stressors; this provides opportunity for synthesis and modification under harsh conditions, or integration in devices that may require extreme conditions. (3) The polymer-based endoskeleton allows for the exploration of the quaternary structure's hidden length through single-molecule force spectroscopy. Overall, this endoskeleton strategy provides an armored VLP with increased stability profile that may find utility in physical virology, biomedicine or advanced materials. Materials and Methods Materials Maleimide–PEG crosslinkers were purchased from Creative PEGWorks. TEM grids (200 mesh) were obtained from Ted Pella, Inc. High-purity water with a resistivity of 18.2 MΩ cm from an inline Millipore RiOs / Origin water purification system. Unless otherwise specified, other materials were purchased from Millipore Sigma and supplies were purchased from ThermoFisher Scientific (Waltham, MA, USA). General Characterization Dynamic light scattering (DLS) measurements were performed on a Malvern NANO- ZS90 Zetasizer to determine hydrodynamic sizes. Final concentrations of 0.05–0.10 mg mL-1were used for measurements. All data were presented by intensity (%). Measurements were triplicated and averaged. TEM images were acquired using a JEOL JEM 1400 Plus operating at 80 kV with a Gatan 4k digital camera. Alternatively, ThermoFisher Talos 200X was performed at 200 kV. Fast protein liquid chromatography was performed to analyze the integrity of samples using a Superose-6 increase 10 / 300 GL column (Pi= 5 MPa, ΔP = 5 MPa, column volume = 25 mL) on the AkTA Pure system (GE Healthcare). The column was loaded with a 100 μL sample (1.0 mg mL-1), and the sample was run for 50 min at a flow rate of 0.5 mL min−1in PBS buffer. Dual-wavelength detection channels of 260 and 280 nm were selected. Circular dichroism (CD) spectra were acquired using an Aviv model 215 CD spectrometer with a 2 mm pathlength quartz cuvette. Samples were performed at a 74 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 concentration of 0.5 mg mL-1in PBS. Temperature-dependent experiments were conducted with a detector fixed at 218 nm. Settings of temperature ramp were listed here: temperature increase rate was 20 °C min-1, temperature interval was 2.5 °C, and equilibration time at each point was 2.5 min. UV-visible absorption measurements were performed on a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Bicinchoninic acid (BCA) assay was performed to quantify the protein concentrations using established methods.

[60] Expression of the PhMV and Qβ VLPs The 564-bp PhMV coat protein gene (NCBI Gene ID 940246) was inserted into pET and pRSETa bacterial expression vectors using established methods.

[0061] The resulting pET- PhMV CP and pRSETa-PhMV CP vectors were used to double transform BL21(DE3) cells (NEB). A single colony was isolated and used to inoculate Lysogeny Broth (LB) supplemented with 50 μg mL-1carbenicillin and 50 μg mL-1kanamycin, which was incubated for 18 hours at 37 °C with shaking (225 rpm). Glycerol (Sigma-Aldrich) was added to a final concentration of approximately 25% (v / v) and the resulting solution was aliquoted into small vials and stored at -80 °C. The expression and purification of PhMV VLPs were then carried out as previously reported.

[0020] ,

[0031] A small amount of BL21 cells in glycerol stock containing pRSETa-PhMV CP and pET-PhMV CP were inoculated into 50 mL of LB containing 50 μg ml-1of carbenicillin and 50 μg ml-1kanamycin and the medium was incubated for 18 hours (37 °C, 225 rpm). Ten ml of the preculture were then inoculated into 500 mL of Terrific Broth (TB) also containing carbenicillin and kanamycin 50 μg mL-1and left to incubate (37 °C, 250 rpm). When culture density reaches OD600= ~1.0 (approximately 8 hours), protein expression was induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG; Gold Biotechnology), and the temperature was reduced to 30 °C overnight. The culture was then centrifuged (7500 × g, 10 min, 4 °C), the pellet was resuspended in 50 mM sodium citrate buffer (SCB), and the resulting suspension was lysed by sonication (10 min, 30% amplitude). The resulting solution was cleared by centrifugation (10000 × g, 10 min, 4 °C, then 27,000 × g, 30 min, 4 °C), and the supernatant was subjected to ultracentrifugation (111,000 × g, 50.2 Ti rotor, Beckman Coulter Inc., 3 hours, 4 °C). The resulting pellets were resuspended in SCB overnight. Insoluble material was removed by centrifugation (20,500 × g, 10 min, 4 °C). 75 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Supernatant was extracted with 1:1 CHCl3 / BuOH and the aqueous layer was isolated by centrifugation (5000 × g, 10 min, 4 °C). The resulting solution was centrifuged (15,000 × 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 separated by centrifugation (93,900 × g, SW32 rotor, Beckman Coulter Inc., 3 hours, 4 °C). The light scattering fractions were collected and pooled with syringe metal needle, diluted in SCB 1:1, and concentrated in ultra-centrifuge (165,000 × g, 50.2 Ti rotor, Beckman Coulter Inc., 3 hours, 4 °C). The clear colorless pellet was finally resuspended in PBS and stored at 4 °C. The VLP concentration was measured using the bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as a standard. The Bacteriophage Qβ VLPs (NCBI accession: P03615) were expressed and purified from BL21(DE3) as previously described.

[0062] Final pure VLPs were resuspended in PBS and stored at 4 ºC. Final concentration was determined by BCA assay using BSA as a standard. Synthesis of Endoskeleton-Enhanced PhMV Nanoparticles The standard preparation process was described as follows: in a 2-mL tube, 100 μL of PhMV stock (10 mg mL-1, in PBS buffer) was first added dropwise to 770 μL of PBS buffer with a gentle vortex. Next, 130 μL of maleimide–PEG15–maleimide (Creative PEGWorks) stock solution (400 mg mL-1, dissolved in DMSO) was freshly made and added dropwise while gently vortexing. The aqueous mixture was fully vortexed to yield the final concentrations (PhMV: 2.78 × 10-7M; mal–PEG15–mal: 5.00 × 10-2M, DMSO: 12.95% in v / v), then was incubated at 4 °C for 16 hours while avoiding the exposure of light to yield the EE-PhMV nanoparticles. For purification, to a 1.5 mL ultra-clear centrifuge tube (Beckman Coulter Inc.), 1 mL of the resulting EE-PhMV nanoparticle mixture was then layered onto 100 μL of 20% sucrose cushion (w / v, in PBS buffer), and were pelleted by ultra- centrifugation (124,000 × g, 90 min, TLA-55 rotor). Finally, the pellets were resuspended overnight by PBS at 4 °C. The final concentration was determined by bicinchoninic acid (BCA) assay for further characterization. Synthesis of Endoskeleton-Enhanced Qβ Nanoparticles In a tube, 12 μL of tris(2-carboxyethyl)phosphine (TCEP) solution (2 mg mL-1) was added into 600 μL of Qβ VLPs (1 mg mL-1in PBS) to yield the final concentrations (0.070 μmol CP, 0.070 μmol disulfides; 0.70 μmol TCEP), followed by incubation at room temperature for 1 h. Next, maleimide–PEG crosslinkers (400 mg mL-1) were added to yield 76 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 different eq. / Cys, followed by incubation at 4 °C for 16 h. Electrophoretic Mobility Shift Assay and Denaturing SDS-PAGE Analysis Denatured protein CPs (2.5–3.75 μg per lane) were analyzed by polyacrylamide gel electrophoresis using 12% NuPAGE gels and 1× MOPS buffer (Invitrogen). For denaturing gel electrophoresis, all samples were denatured by boiling in 1:10 reducing agent and 1:4 SDS blue loading dye for 5 min, followed by running under 180 V for 60 min. For native gel electrophoresis, PhMV VLPs (5 μg per lane) were characterized using 0.8% (w / v) agarose in 0.1 M Tris-borate-EDTA (TBE) running buffer (pH 8.3); Qβ VLPs were characterized using 1.2% (w / v) agarose in 0.1 M Tris-acetate-EDTA (TAE) running buffer (pH 8.3). The gels were then stained in Coomassie blue staining solution, followed by photography under UV light in a FluorChem R system (Bio-techne). The fluorescently labeled VLPs were analyzed under UV light with a red-light filter before Coomassie Blue staining. Comprehensive Stability Assay For the study of thermal stability, PhMV and EE-PhMV were incubated in PBS buffer, followed by putting the tube in a heat block with desired temperatures for 15 min. The resulting samples were taken to DLS or microplate reader (Tecan Infinite M200) for further analysis. For other stressors, the PhMV and EE-PhMV were incubated in diverse stressors at desired final concentrations at room temperature, followed by DLS measurement. Specifically, the conditions used included SDS (1% w / v, 24 h, RT), NaCl (1–4 M, 6 h), pH (1.2–12, 24 h), urea (1–6 M, 24 h), guanidine hydrochloride (1–6 M, 24 h), and methanol (25–90%, 2 h). The DLS was taken in triplicated, and the averages were presented by intensity (%). The final concentrations of all samples were 1 mg mL-1. Single-Molecule Force Spectroscopy For SMFS experiments Applicants have employed a ‘homebuilt’ instrument, constructed using Multimode AFM (Bruker, Inc) head and Physick Instumente piezo stages similar to the one previously described in detail.59Sharp Microlever Silicon probes (MSNL) were purchased from Bruker (Camarillo, CA) and calibrated using thermal method with typical spring constant being ~20 pN nm-1. Silicon wafers were cut into ~1×1 cm pieces and immersed in a hot piranha solution (H2SO4: H2O2= 3:1) for 30 min (Extreme caution should be taken when working with piranha), rinsed with deionized water and dried under nitrogen stream. AFM probes were 77 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 rinsed with dichloromethane. To achieve amine-functionalized surface silicon substrates were subsequently incubated in a 2% v / v solution of N-[3-(trimethoxysilyl)propyl]ethylenediamine in methanol for 30 min, and AFM probes were incubated in the same solution for 10 min. After that both were extensively rinsed with methanol and annealed at 110 °C for 30 min. Adding 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, dissolved in DMSO) to 1 mg mL-1PhMV or EE-PhMV nanoparticles to yield a final concentration of EDC of 0.03 followed by incubation for 1 h at room temperature to allow the EDC reaction with carboxylates. 100 uL of EDC-coupled VLP solution was placed on the amine-functionalized substrate and incubated for 2 h to allow immobilization, followed by gently rinsing with PBS to remove free VLPs and EDC. SMFS experiments were conducted at room temperature (~23 °C) in the PBS solution. The amine-functionalized cantilever tip was repeatedly brought in contact with surface up to the force of 50 pN and retracted at the different spots on the substrate at the raster pattern with pulling velocity of 1000 nm sec-1. Data was collected and processed using Matlab (MathWorks, Natick, MA). Estimation of Stored Length within One CP In theory, the stored length of one CP can be estimated by Formula I

[0054] ^^^ൌ ^^^ ൈ ^^Ȁ^^^ െ ^ி(1), where ^^^is the contour length increment, ^^^ is the number of the amino acids of the protein (taken as 180), ^Ȁ^^ is the average length per amino acid (taken as 0.39 nm), and ^ிis the folded length of the protein (taken as 35.6 Å), measured by the average end-to-end distance between N and C terminals from the crystal structure by UCSF Chimera. It is thus one CP’s stored length can be taken as 73.32 nm. Table 3. The calculation of Flory radius and contour length of maleimide–PEG crosslinkers.aFor linear polymer:^^ൌ ^^^Ǥ^; for branched polymers: where ^ = length of one ethylene glycol (EG) repeats (taken as 3.50 Å), ^ = number of EG repeat per branch, and ^ = number of branches.

[0065] bEthylene glycol repeat is taken as 2.78 Å in water where PEG chain is in helical conformation.

[0066] , [1]Contour length ൌ ^ ൈ ʹǤ^ͺ 78 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Table 4. Size comparison of PhMV and EE-PhMV, Qβ and EE-Qβ with different degrees of linking. The data is acquired in PBS buffer by DLS, plotted by intensity (%). All measurements were taken in triplicate. 79 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Table 5. Band analysis of PhMV and EE-PhMV1000. *MW of different components: CP = 19,974 Da; mal–Cy5 = 606 Da; mal–PEG15–mal = 1036 Da. *In SDS- PAGE, the dimers formed by physical entanglement of two individual CPs are denoted by a symbol This analysis is based on gel in FIGS. 16C and 16H. 80 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Table 6. Summary of the polydispersity index (PDI) values of samples used in comprehensive stability assay. The data is acquired by DLS and plotted by intensity (%). All measurements were taken in triplicate. 81 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 82 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 Embodiments 1. A method to prepare an engineered virus-like particle capsid protein (E-VLP- CP) conjugated to a thiol-reactive small molecule: preparing a cysteine-mutated VLP CP by mutating one or more internal amino acids of a wild-type VLP-CP; contacting the cysteine-mutated VLP CP with one or more thiol-reactive small molecules to prepare a thiol-containing VLP CP; and contacting one or more maleimide-containing small molecules with the thiol-containing VLP CP, thereby preparing the engineered VLP-CP conjugated to the thiol-reactive small molecules. 2. The method of embodiment 1, wherein the wildtype VLP-CP is icosahedral. 3. The method of embodiment 1, wherein the wild-type VLP-CP is selected from a CP from a plant virus selected from the Tymoviridae, Secoviridae, Bromoviridae, Virgaviridae, Kitaviridae, or Alphaflexiviridae families. 83 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 4. The method of embodiment 1, wherein the wild-type VLP-CP is selected from a CP from a virus selected from a cytoplasmic type citrus leprosis virus, bacteriophages Qbeta, P22, and MS2, the mammalian hepatitis B virus (HBV), and plant viruses Tobacco mosaic virus (TMV), Cowpea chlorotic mottle virus (CCMV), Potato virus X (PVX) or Physalis mottle virus (PhMV). 5. The method of any of embodiments 1-4, wherein the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. 6. The method of embodiment 5, wherein the one or more internal amino acids are selected from A31C and S137C. 7. The method of any of embodiments 1-6, wherein the one or more thiol reactive small molecules are anti-cancer agents. 8. The method of any one of embodiments 1-7, wherein the one or more thiol reactive small molecules are selected from aldoxorubicin, monomethyl auristatin E (MMAE), valine-citrulline MMAE, gadolinium (Gd)- 2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane- 1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), and STG982-maleimide. 9. The method of any of embodiments 1-6, wherein the one or more thiol- reactive small molecules are toll-like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists), wherein the thiol-reactive small molecules comprise a maleimide moiety. 10. The method of embodiment 8 or 9, wherein the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. 11. The method of embodiment 10, wherein the one or more internal amino acids are A31C or S137C. 84 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 12. An engineered virus like particle capsid protein (E-VLP-CP) conjugated to one or more thiol-reactive small molecules, the E-VLP-CP comprising one or more internally cysteine-mutated amino acids in a wild-type VLP-CP. 13. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of embodiment 12, wherein the wild-type VLP-CP is icosahedral. 14. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of embodiment 12, wherein the wild-type VLP-CP is selected from a CP from a plant virus selected from the Tymoviridae, Secoviridae, Bromoviridae, Virgaviridae, Kitaviridae, or Alphaflexiviridae families. 15. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of embodiment 12, wherein the wild-type VLP-CP is selected from a CP from a virus selected from a bacteriophages Qbeta, P22, and MS2, the mammalian hepatitis B virus (HBV), and plant viruses Tobacco mosaic virus (TMV), cytoplasmic type citrus leprosis virus, Cowpea chlorotic mottle virus (CCMV), Potato virus X (PVX) or Physalis mottle virus (PhMV). 16. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-15, wherein the diameter of the wild-type VLP-CP is selected from: about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm. 17. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-16, wherein the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. 18. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-16, wherein the one or more internal amino acids are A31C or S137C. 85 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 19. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of embodiment 17, wherein the one or more internal amino acids are selected from A31C or S137C. 20. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-19, wherein the one or more thiol-reactive small molecules are selected from aldoxorubicin, monomethyl auristatin E (MMAE), valine-citrulline MMAE, gadolinium (Gd)-2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982-maleimide. 21. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-19, wherein the one or more thiol-reactive small molecules are toll- like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists). 22. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of embodiment 20 or 21, wherein the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C, and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. 23. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of embodiment 22, wherein the one or more internal amino acids are selected from A31C or S137C. 24. A composition comprising a carrier and the E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-23. 25. The composition of embodiment 24, wherein the carrier is a pharmaceutically acceptable carrier. 26. A method to deliver a thiol-reactive small molecule to a cell, comprising contacting the cells with the E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-23, or the composition of embodiment 24 or 25. 27. The method of embodiment 26, wherein the contacting is in vitro or in vivo. 86 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 28. A method to deliver a thiol-reactive small molecule to a subject, comprising administering to the subject the E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-23, or the composition of embodiment 24 or 25. 29. The method of embodiment 28, wherein the administration is local or systemic. 30. A method for treating cancer in a subject in need thereof, comprising administering to the subject the E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-23, or the composition of embodiment 24 or 25. 31. The method of embodiment 30, wherein the cancer is selected from a blood cancer, a carcinoma, a sarcoma, a lung cancer, an intraperitoneal cancer, a colorectal cancer, a colon cancer, an ovarian cancer, a melanoma, or a breast cancer. 32. The method of embodiment 30 or 31, wherein the E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of embodiments 12-23 or the composition of embodiment 24 or 25 is administered to the patient by oral, intratumoral, nasal, topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, epidural or intrathecal administration 33. The method of embodiment 32, wherein the cancer is a melanoma. 34. A method for treating cancer in a subject in need thereof, comprising administering to the subject an engineered virus like particle capsid protein (E-VLP-CP) comprising one or more internally cysteine-mutated amino acids conjugated to one or more thiol-reactive small molecules, wherein one of the thiol-reactive small molecules is selected from aldoxorubicin, valine-citrulline MMAE, and STG982-maleimide, wherein the VLP-CP is from PhMV. 35. The method of embodiment 34, wherein the E-VLP-CP conjugated to one or more thiol-reactive small molecules is administered intratumorally. 36. An engineered virus-like particle (E-VLP) comprising: a first capsid protein; a second capsid protein; and a maleimide-PEGN-maleimide linker internally cross-linking the 87 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 first and the second capsid proteins to each other through internally surface exposed cysteines. 37. The E-VLP of embodiment 36, wherein the N in the PEGNrepresents an integer in a range selected from: 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15. 38. The E-VLP of embodiment 36 or 37, wherein the maleimide-PEGN-maleimide linker has a molecular weight in a range selected from: about 0.5k Da to about 15k Da, from about 0.5k Da to about 10k Da, from about 0.5k Da to about 5k Da, from about 0.5k Da to about 2.5k Da, from about 0.5k Da to about 1k Da, or about 1k Da. 39. The E-VLP of any of embodiments 36-38, wherein the maleimide-PEGN- maleimide linker has 1, 4, or 8 branches. 40. The E-VLP of any of embodiments 36-39, wherein the first and second capsid proteins are the same or different from each other. 41. The E-VLP of any of embodiments 36-40, wherein the capsid proteins are from the group of VLP selected from a plant Picornavirus nanoparticle, a Comovirus nanoparticle, a Tymovirus nanoparticle, a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic virus nanoparticle, a bean pod mottle viral nanoparticle, a rice tungro spherical viral nanoparticle, a Physalis mottle virus (PhMV), or a Qbeta bacteriophage. 42. The E-VLP of embodiment 41, wherein the VLP is a PhMV. 43. The E-VLP of any of embodiments 36-42, wherein the N in the PEGNrepresents an integer in a range selected from: 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15, and the first and / or the second capsid proteins are from PhMV. 44. The E-VLP of embodiment 43, wherein the N in the PEGNrepresents an integer in a range from about 10 to about 20, or about 15. 45. The E-VLP of any of embodiments 36-44, wherein the ratio of maleimide- PEGN-maleimide linkers to internally surface exposed cysteines is selected from: about 1 to 88 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 about 2000, about 10 to about 2000, about 10 to about 1500, about 10 to about 1000, about 10 to about 500, about 10 to about 100, or about 100, or about 1000. 46. The E-VLP of any of embodiments 35-45, wherein the size of the E-VLP is selected from: about 1 nm to about 100 nm, or about 1 nm to about 90 nm, or about 1 nm to about 80 nm, or about 1 nm to about 70 nm, or about 1 nm to about 60 nm, or about 1 nm to about 50 nm, or about 1 nm to about 40 nm, or about 1 nm to about 30 nm, or about 40 nm, or about 30 nm. 47. The E-VLP of any of embodiments 36-46, wherein the change in tip-substrate separation at which the force returns to zero following an event selected from rupture of the E-VLP, detachment of the E-VLP, or other scission event of the E-VLP (DRUP) is selected from: about 1 nm to 500 nm, about 10 nm to about 400 nm, about 30 nm to about 300 nm, about 40 nm to about 200 nm, about 50 to about 100 nm, about 10 to about 100 nm, about 50 nm, or about 100 nm. 48. The E-VLP of any of embodiments 36-46, wherein the DRUPof the E-VLP is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10- fold, or about 100-fold higher than the DRUPof a non-engineered control VLP. 49. The E-VLP of any of embodiments 36-46, wherein the force required to rupture (FRUP) the E-VLP is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5- fold, or about 10-fold higher than the FRUPrequired to rupture a non-engineered control VLP. 50. A plurality of E-VLPs of any of embodiments 36-49, wherein the E-VLP of the plurality are the same or different from each other. 51. A substantially homogenous plurality of E-VLPs of any of embodiments 36- 49. 52. A composition comprising the E-VLP of any of embodiments 36-49 or the plurality of claim 50 or 51, and a carrier. 53. The composition of embodiment 52, wherein the carrier is a pharmaceutically acceptable carrier. 54. A method to deliver a E-VLP to a cell or tissue, comprising contacting the cell or tissue with the E-VLP with one or more of: the E-VLP of any of embodiments 36-49 or the plurality of E-VLP of embodiments 50 or 51, or the composition of embodiment 52 or 53, thereby delivering the E-VLP to the cell or tissue. 89 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 55. The method of embodiment 54, wherein the contacting is in vitro, ex vivo, or in vivo. 56. The method of embodiment 54 or 55, wherein the cell or tissue is animal or plant cell or tissue. 57. The method of embodiment 54, wherein the contacting is in vitro or in vivo, and the cell or tissue is an animal cell or tissue. 58. The method of embodiment 54, wherein the cell or tissue is a plant cell or tissue and the contacting comprising depositing the E-VLP on one or more of the stem, roots or leaves of the plant. 59. The method of embodiment 54, wherein the animal cell or tissue is selected from canine, feline, equine, bovine, rat, simian or human. 60. A kit comprising one or more of the E-VLP of any of embodiments 36-49, the plurality of embodiment 50 or 51, or the composition of embodiment 52 or 53, and instructions for use. 61. A method of forming an engineered virus-like particle (E-VLP), the method comprising: combining a plurality of capsid proteins, and a multiarm maleimide-PEGN- maleimide polymer to form the E-VLP, wherein each of the plurality of capsid proteins comprises internally surface exposed cysteine groups that react with a maleimide group of the multiarm maleimide-PEGN-maleimide polymer, and wherein the E-VLP comprises capsid proteins linked to one another by the multiarm maleimide-PEGN-maleimide polymer. 62. The method of embodiment 61, wherein the multiarm maleimide-PEGN- maleimide polymer comprises two or more maleimide arms on the PEGNgroup. 63. The method of embodiment 62, wherein the multiarm maleimide-PEGN- maleimide polymer comprises 2, 4, or 8 maleimide arms on the PEGNgroup. 64. The method of any one of embodiments 61-63, wherein the combining is conducted in a buffer solution. 65. The method of any one of embodiments 61-64, wherein the combining is conducted from greater than 0⁰C to about 20⁰C. 66. The method of any one of embodiments 61-65, wherein the combining is conducted by incubating the plurality of capsid proteins, and the multiarm maleimide-PEGN- maleimide polymer at greater than 0⁰C to about 10⁰C for about 1 hour to about 72 hours. 90 4866-2001-7873.2 Atty. Dkt. No. 114198-4960 67. The method of any one of embodiments 61-66, further comprising collecting the E-VLP by centrifugation. 68. The method of embodiment 67, wherein the E-VLP is collected in pellet form. Equivalents 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 technology belongs. The present technology 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 described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed. Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology. It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can 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 present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology 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. 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Claims

Atty. Dkt. No. 114198-4960 WHAT IS CLAIMED IS:

1. A method to prepare an engineered virus-like particle capsid protein (E-VLP-CP) conjugated to a thiol-reactive small molecule, the method comprising: a. preparing a cysteine-mutated VLP CP by mutating one or more internal amino acids of a wild-type VLP-CP; b. contacting the cysteine-mutated VLP CP with one or more thiol-reactive small molecules to prepare a thiol-containing VLP CP; and c. contacting one or more maleimide-containing small molecules with the thiol- containing VLP CP, thereby preparing the engineered VLP-CP conjugated to the thiol-reactive small molecule, optionally wherein the wild-type VLP-CP is icosahedral, optionally wherein the wild-type VLP-CP is selected from a CP from a plant virus selected from the Tymoviridae, Secoviridae, Bromoviridae, Virgaviridae, Kitaviridae, and Alphaflexiviridae families, and optionally wherein the one or more thiol reactive small molecules are anti-cancer agents, toll-like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists), wherein the thiol-reactive small molecules comprise a maleimide moiety.

2. The method of claim 1, wherein the wild-type VLP-CP is selected from a cytoplasmic type citrus leprosis virus, bacteriophages Qbeta, P22, and MS2, the mammalian hepatitis B virus (HBV), and plant viruses Tobacco mosaic virus (TMV), Cowpea chlorotic mottle virus (CCMV), Potato virus X (PVX) or Physalis mottle virus (PhMV).

3. The method of claim 1 or 2, wherein the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof. 104 4866-2001-7873.2Atty. Dkt. No. 114198-4960 4. The method of claim 3, wherein the one or more internal amino acids are A31C or S137C.

5. The method of any one of claims 1-4, wherein the one or more thiol reactive small molecules are selected from aldoxorubicin, monomethyl auristatin E (MMAE), valine- citrulline MMAE, gadolinium (Gd)- 2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane-1,4,7,10- tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982-maleimide.

6. The method of claim 5, wherein the wild-type VLP of the VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof.

7. An engineered virus like particle capsid protein (E-VLP-CP) conjugated to one or more thiol-reactive small molecules, the E-VLP-CP comprising one or more internally cysteine-mutated amino acids in a wild-type VLP-CP, optionally wherein the wild-type VLP-CP is icosahedral, optionally wherein the wild-type VLP-CP is selected from a CP from a plant virus selected from the Tymoviridae, Secoviridae, Bromoviridae, Virgaviridae, Kitaviridae, or Alphaflexiviridae families, and optionally wherein the one or more thiol reactive small molecules are anti-cancer agents, toll-like receptor (TLR) agonists, retinoic acid-inducible gene 1 (RIG-1) agonists, immune checkpoint inhibitors (i.e. anti-PD-1 / PD-L1), or chemokine receptor drugs (i.e. CXCR4 antagonists).

8. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of claim 7, wherein the wild-type VLP-CP is selected from a CP from a virus selected from a bacteriophages Qbeta, P22, and MS2, the mammalian hepatitis B virus (HBV), and plant viruses Tobacco mosaic virus (TMV), cytoplasmic type citrus leprosis virus, Cowpea chlorotic mottle virus (CCMV), Potato virus X (PVX) or Physalis mottle virus (PhMV).

9. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of claim 7 or 8, wherein the diameter of the wild-type VLP-CP is selected from: about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from 105 4866-2001-7873.2Atty. Dkt. No. 114198-4960 about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm.

10. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of claims 7-9, wherein the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof.

11. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of claim 10, wherein the one or more internal amino acids are selected from A31C and S137C.

12. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of claims 7-10, wherein the one or more thiol-reactive small molecules are selected from aldoxorubicin, monomethyl auristatin E (MMAE), valine-citrulline MMAE, gadolinium (Gd)-2,2’,2’’,2’’’-(1,4,7,10-tetrazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-gadolinium (DOTA(Gd)), or STG982-maleimide.

13. The E-VLP-CP conjugated to one or more thiol-reactive small molecules of claim 12, wherein the wild-type VLP-CP is a PhMV CP and the one or more internal amino acids are selected from PhMV A31C, PhMV S137C, and PhMV A31C, and PhMV S137C, wherein the wild-type VLP-CP comprises the sequence according to SEQ ID NO: 4, or an equivalent thereof.

14. A composition comprising a carrier and the E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of claims 7-13, optionally wherein the carrier is a pharmaceutically acceptable carrier.

15. A method to deliver a thiol-reactive small molecule to a cell, comprising contacting the cells with the E-VLP conjugated to one or more thiol-reactive small molecules of any of claims 7-13, or the composition of claim 14, wherein the contacting is in vitro or in vivo.

16. A method to deliver a thiol-reactive small molecule to a subject, comprising administering to the subject the E-VLP-CP conjugated to one or more thiol-reactive small 106 4866-2001-7873.2Atty. Dkt. No. 114198-4960 molecules of any of claims 7-13, or the composition of claim 14, wherein the administration is local or systemic.

17. A method for treating cancer in a subject in need thereof, comprising administering to the subject the E-VLP-CP conjugated to one or more thiol-reactive small molecules of any of claims 7-13, or the composition of claim 14.

18. The method of claim 17, wherein the cancer is selected from a blood cancer, a carcinoma, a sarcoma, a lung cancer, an intraperitoneal cancer, a colorectal cancer, a colon cancer, an ovarian cancer, a melanoma, or a breast cancer.

19. The method of claim 18, wherein the E-VLP-CP conjugated to one or more thiol- reactive small molecules of any of claims 7-13 or the composition of claim 14 is administered to the patient by oral, intratumoral, nasal, topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, epidural or intrathecal administration.

20. A method for treating cancer in a subject in need thereof, comprising administering to the subject an engineered virus like particle capsid protein (E-VLP-CP) comprising one or more internally cysteine-mutated amino acids conjugated to a one or more thiol-reactive small molecules, wherein one of the thiol-reactive small molecules is selected from aldoxorubicin, valine-citrulline MMAE, and STG982-maleimide, wherein the VLP-CP is from PhMV, optionally wherein the E-VLP-CP is administered intratumorally.

21. An engineered virus-like particle (E-VLP) comprising: a first capsid protein; a second capsid protein; and a maleimide-PEGN-maleimide linker internally cross-linking the first and the second capsid proteins to each other through internally surface exposed cysteines, wherein the first and second capsid proteins are the same or different from each other, optionally wherein the maleimide-PEGN-maleimide linker has 1, 2, 4, or 8 branches.

22. The E-VLP of claim 21, wherein the N in the PEGNrepresents an integer in a range selected from: 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15. 107 4866-2001-7873.2Atty. Dkt. No. 114198-4960 23. The E-VLP of claim 21 or 22, wherein the maleimide-PEGN-maleimide linker has a molecular weight in a range selected from: about 0.5k Da to about 15k Da, from about 0.5k Da to about 10k Da, from about 0.5k Da to about 5k Da, from about 0.5k Da to about 2.5k Da, from about 0.5k Da to about 1k Da, or about 1k Da.

24. The E-VLP of any of claims 21-23, wherein the capsid proteins are from the group of VLP selected from a plant Picornavirus nanoparticle, a Comovirus nanoparticle, a Tymovirus nanoparticle, a Cowpea mosaic viral nanoparticle, a Broad bean wilt virus 1 nanoparticle, a Tobacco ringspot viral nanoparticle, a cowpea severe mosaic virus nanoparticle, a bean pod mottle viral nanoparticle, a rice tungro spherical viral nanoparticle, a Physalis mottle virus (PhMV), or a Qbeta bacteriophage.

25. The E-VLP of claim 21, wherein the N in the PEGNrepresents an integer in a range selected from: 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; 1 to about 5; about 5 to about 30; about 5 to about 25; about 5 to about 20; about 5 to about 15; about 5 to about 10; or about 15, and the first and / or the second capsid proteins are from PhMV, optionally wherein the N in the PEGNrepresents an integer in a range from about 10 to about 20, or about 15.

26. The E-VLP of any of claims 21-25, wherein the ratio of maleimide-PEGN-maleimide linkers to internally surface exposed cysteines is selected from: about 1 to about 2000, about 10 to about 2000, about 10 to about 1500, about 10 to about 1000, about 10 to about 500, about 10 to about 100, or about 100, or about 1000.

27. The E-VLP of any of claims 21-26, wherein the size of the E-VLP is selected from: about 1 nm to about 100 nm, or about 1 nm to about 90 nm, or about 1 nm to about 80 nm, or about 1 nm to about 70 nm, or about 1 nm to about 60 nm, or about 1 nm to about 50 nm, or about 1 nm to about 40 nm, or about 1 nm to about 30 nm, or about 40 nm, or about 30 nm.

28. The E-VLP of any of claims 21-27, wherein the change in tip-substrate separation at which the force returns to zero following an event selected from rupture of the E-VLP, detachment of the E-VLP, or other scission event of the E-VLP (DRUP) is selected from: about 1 nm to 500 nm, about 10 nm to about 400 nm, about 30 nm to about 300 nm, about 40 nm to about 200 nm, about 50 to about 100 nm, about 10 to about 100 nm, about 50 nm, or about 100 nm. 108 4866-2001-7873.2Atty. Dkt. No. 114198-4960 29. The E-VLP of any of claims 21-27, wherein the DRUPof the E-VLP is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, or about 100-fold higher than the DRUPof a non-engineered control VLP, optionally wherein the force required to rupture (FRUP) the E-VLP is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold higher than the FRUPrequired to rupture a non-engineered control VLP.

30. A plurality of E-VLPs of any of claims 21-29, wherein that E-VLP of the plurality are the same or different from each other, optionally wherein the plurality of E-VLPs is substantially homogenous.

31. A composition comprising the E-VLP of any of claims 21-29 or the plurality of claim 30, and a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.

32. A method to deliver a E-VLP to a cell or tissue, comprising contacting the cell or tissue with the E-VLP with one or more of: the E-VLP of any of claims 21-29 or the plurality of E-VLP of claim 30, or the composition of claim 31, thereby delivering the E-VLP to the cell or tissue, wherein the contacting is in vitro, ex vivo, or in vivo, optionally wherein the cell or tissue is animal or plant cell or tissue.

33. The method of claim 32, wherein the cell or tissue is a plant cell or tissue and the contacting comprising depositing the E-VLP on one or more of the stem, roots or leaves of the plant.

34. The method of claim 32, wherein the animal cell or tissue is selected from canine, feline, equine, bovine, rat, simian or human.

35. A kit comprising one or more of the E-VLP of any of claims 21-29, the plurality of claim 30, or the composition of claim 31, and instructions for use.

36. A method of forming an engineered virus-like particle (E-VLP), the method comprising: combining a plurality of capsid proteins, and a multiarm maleimide-PEGN- maleimide polymer to form the E-VLP, wherein each of the plurality of capsid proteins comprises internally surface exposed cysteine groups that react with a maleimide group of the multiarm maleimide-PEGN-maleimide polymer, and wherein the E-VLP comprises capsid proteins linked to one another by the multiarm maleimide-PEGN-maleimide polymer, 109 4866-2001-7873.2Atty. Dkt. No. 114198-4960 optionally wherein the combining is conducted in a buffer solution, optionally wherein the combining is conducted from greater than 0⁰C to about 20⁰C.

37. The method of claim 36, wherein the multiarm maleimide-PEGN-maleimide polymer comprises two or more maleimide arms on the PEGNgroup.

38. The method of claim 36, wherein the multiarm maleimide-PEGN-maleimide polymer comprises 2, 4, or 8 maleimide arms on the PEGNgroup.

39. The method of any one of claims 36-38, wherein the combining is conducted by incubating the plurality of capsid proteins, and the multiarm maleimide-PEGN-maleimide polymer at greater than 0⁰C to about 10⁰C for about 1 hour to about 72 hours.

40. The method of any one of claims 36-39, further comprising collecting the E-VLP by centrifugation, optionally wherein the E-VLP is collected in pellet form. 110 4866-2001-7873.2

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