Tumor agnostic drug delivery with self-agglomerating nanohydrogels

Abstract

Delivery of oligonucleotides and other agents to tumor microenvironments is challenging due to their rapid clearance, nuclease susceptibility, and inability to traverse plasma membranes, impeding their effective use for the treatment of a variety of disorders. Disclosed herein is a self-agglomerating nanohydrogel and methods of delivering an agent as well as methods of treating cancer with the nanohydrogel.

Description

TUMOR AGNOSTIC DRUG DELIVERY WITH SELFAGGLOMERATING NANOHYDROGELSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 517,215, filed August 2, 2023, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] RNA interference (RNAi) is a powerful tool, which uses small interfering RNA (siRNA) and microRNA (miRNA) to silence expression of target genes in a sequence- specific manner. RNAi molecules hold unique potential as clinically viable modalities for manifold diseases. Oncological indications are of particular interest because the vast majority cancerpromoting genes implicated in growth, apoptosis, migration, and invasion are “undruggable” by conventional and targeted therapies. By contrast, RNAi does not depend on the presence of unique protein conformations and intricate binding pockets to be effective.

[0003] Despite its potential, systemic delivery of RNAi to tumors has been challenging. Rapid clearance, susceptibility to serum nucleases, insufficient delivery to tumor sites, inadequate tumor penetration and inability to traverse plasma membranes all attenuate its intended biological activity. Thus, translation of many RNAi-based therapeutics to the clinic has been limited.

[0004] Numerous strategies have been developed to overcome these barriers including complexation with cationic polymers, or inclusion within nanoliposomes to form lipid nanoparticles (LNPs). While these strategies have improved RNAi delivery, they have primarily consisted of academic demonstrations, with few formulations reaching patients. Moreover, these approaches are not without drawbacks. The vast majority of an injected LNP dose becomes trapped in the liver and is not efficiently taken up by cancerous cells after systemic injection. As a result, indications for LNP formulations have been largely limited to hepatic cancers or those receptive to intra-tumoral injection. This leaves the majority of cancers, especially metastatic lesions, untreated because they require systemic rather than local administration.

[0005] Cationic polymers on the other hand show strong nonspecific binding to many cells types and interact with serum components. This results in short circulation time and toxiceffects. Surface shielding by PEGylation can improve stability, reduce nonspecific binding and clearance, and prolong circulation, but it also impairs cellular uptake by target cells. Targeting specificity can be improved through functionalization with ligand conjugation which facilitates ligand-receptor interactions on target cells. While this does improve specificity, it exposes a plethora of additional drawbacks including increased manufacturing complexity and costs and reduction in stability, the latter of which was the principal reason for a recent clinical trial failure. Moreover, ligand-based targeting requires a prior knowledge of the cell type of interest and necessitates vehicle redesign for each new target, further increasing development time and costs. Collectively, the development of a systemic, tumor agnostic delivery systems that is effective, safe, and simple to manufacture remains an important missing link for therapeutic translation of RNAi.

[0006] These needs and others are at least partially satisfied by the present disclosure.SUMMARY

[0007] Disclosed herein is a self-agglomerating nanohydrogel (also referred to as “SANG”) and methods of delivering a therapeutic agent and treating cancer with the nanohydrogel. Aberrant vasculature in the tumor microenvironment (TME) was demonstrated to cause SANG particles to persist longer and experience more collisions with each other in the tortuous bloodstream. Remarkably, this causes individual SANG particles to aggregate into masses that are too large to escape the tumor. This so called “self-agglomerating” characteristic emerges from the unique topology and physiochemical properties of the SANG platform and ultimately results in much higher concentrations of SANGS in tumors with aberrant vasculature. This unexpected emergent property of the SANGS platform is distinct from the classical EPR (enhanced permeability and retention) effect and results in a large concentration gradient in the TME, promoting the penetration of blood vessels by SANG particles to reach cancerous cells. Because aberrant vasculature is a highly conserved feature among solid tumors cancers, SANGS offers a realistic means to target cancers of multiple tissue origins and molecular profiles with a single platform. This innovation allows targeting of both primary and metastatic loci and will be a potential paradigm shift in cancer treatment.

[0008] In various aspects, disclosed herein is a nanohydrogel comprising a crosslinkable core / shell polymer; wherein the nanohydrogel is configured to self-agglomerate in aberrant vasculature environments.

[0009] In some aspects, the crosslinkable core / shell polymer comprise a gel-forming polymer. In some aspects, the gel-forming polymer comprises a biocompatible polymer. In some aspects, the crosslinkable core / shell polymer comprise polyacrylamide. In some aspects, the crosslinkable core / shell polymer comprises a copolymer of N-isopropylmethacrylamide and N,N'-methylenebis(acrylamide). In some aspects, a shell of the nanohydrogel comprises a cationic polymer ligand (e.g., poly(aminopropyl methacrylate)).

[0010] In some aspects, the crosslinkable core / shell polymer is unfunctionalized. In some aspects, the nanohydrogel has a hydrodynamic size of from about 75 nm to about 200 nm prior to self-agglomeration.

[0011] In some aspects, the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to self- agglomerate to form an agglomerated cluster, wherein the agglomerated cluster has an average hydrodynamic size larger than an average hydrodynamic size prior to self-agglomeration.

[0012] In some aspects, self-agglomeration of the population of nanohydrogels is concentration-dependent. In some aspects, a concentration of nanohydrogels causing selfagglomeration is 10 nM or greater (e.g., 20 nM or greater, 30 nM or greater, 40 nM or greater, 50 nM or greater, 60 nM or greater, 70 nM or greater, 80 nM or greater, 90 nM or greater, 100 nM or greater, 110 nM or greater, 120 nM or greater, 130 nM or greater, 140 nM or greater, 150 nM or greater, 156 nM or greater, 200 nM or greater, 250 nM or greater, 500 nM or greater, 1 pM or greater). In some aspects, a concentration of nanohydrogels causing selfagglomeration is from about 10 nM to about 1 pM (e.g., from about 10 nM to about 500 nM, from about 30 nM to about 500 nM, from about 60 nM to about 500 nM, from about 60 nM to about 250 nM, from about 100 nM to about 250 nM, from about 100 nM to about 200 nM, or about 156 nM).

[0013] Also described herein are compositions comprising: any of the populations of nanohydrogel described herein; and a therapeutic agent. In some aspects, the therapeutic agent is encapsulated within a core of the nanohydrogel.

[0014] In some aspects, the therapeutic agent comprises an anti-cancer agent. In some aspects, the therapeutic agent comprises a nucleic acid. In some aspects, the therapeutic agent comprises small interacting RNA (siRNA) and / or microRNA (miRNA). In some aspects, the therapeutic agent comprises an inhibitor of epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus oncogene homolog (KRAS), Glul, and / or Zinc Finger E-Box Binding Homeobox 1 (ZEB1). In some aspects, the therapeutic agent comprises an inhibitor of EGFR. In some aspects, the therapeutic agent comprises an inhibitor of Glul. In some aspects, thetherapeutic agent comprises an inhibitor of ZEB1. In some aspects, the therapeutic agent comprises an inhibitor of KRAS.

[0015] Also described herein is a method of delivering a therapeutic agent to a tumor of a subject. For example, the method can include: administering any of the compositions described herein to the subject; wherein the population of nanohydrogels is inducible to self- agglomerate to form an aggregated cluster of nanohydrogels via the aberrant microvasculature of a tumor microenvironment.

[0016] In some aspects, the method further includes: allowing the therapeutic agent to be released from the aggregated cluster of nanohydrogels.

[0017] Further described herein are methods of treating cancer in a subject, comprising: administering any of the compositions described herein to the subject; wherein the population of nanohydrogels is inducible to self-agglomerate to form an aggregated cluster of nanohydrogels via the aberrant microvasculature of a tumor microenvironment; and allowing the therapeutic agent to be released from the aggregated cluster of nanohydrogels.

[0018] In some aspects, the cancer is ovarian cancer, colorectal cancer, lung cancer or breast cancer. In some aspects, the cancer is ovarian cancer. In some aspects, the cancer is colorectal cancer. In some aspects, the cancer is breast cancer. In some aspects, the cancer is lung cancer.

[0019] In some aspects, the cancer is breast cancer. In some aspects, the cancer is a cancer having an undruggable cancer-promoting gene. In some aspects, the undruggable cancerpromoting gene comprises one or more of c-Myc. APC, BRAF, and / or KRAS.

[0020] In some aspects, a selectivity of delivery of the therapeutic agent to the tumor microenvironment is 50% or greater (e.g., 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 99% or greater, 99.5% or greater). In some aspects, 2.5% or more of the population of nanohydrogels is retained after 24 hours from an initial dose (e.g., 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 10% or more).

[0021] In some aspects, the administration of the composition comprises administering a first composition comprising nanohydrogels having a first therapeutic agent and a second composition comprising nanohydrogels having a second therapeutic agent. In some aspects, the first composition and second composition are administered concurrently. In some aspects, the first therapeutic agent and the second therapeutic agent are the same. In some aspects, the first therapeutic agent and the second therapeutic agent are different. In some aspects, the firsttherapeutic agent comprises an RNAi agent and the second therapeutic agent comprises a chemotherapeutic agent.

[0022] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 depicts physicochemical property characterization of SANG and its cellular uptake, (a) A schematic diagram of the protocol for the synthesis and preparation of fluorescently labeled siRNA-loaded SANGs. (b) Physicochemical property characterization of SANGs, which includes the hydrodynamic size, polydispersion index (PDI), zeta potential, molecular weight (MW in kDa), N:P ratio, and encapsulation efficiency (E.E.). (c) Negativestain TEM image of SANGs. (d) reproducibility of SANG production across 3 independent batches (n = 30 independent measures / batch for each core and shell) showing the complete size distribution of the core and shell components prior to final cleaning and purification, (e) Chemical stability of siRNA loaded and unloaded SANGs at -25 °C, 4°C and 25 °C (mean of n = 3 independent batches per condition, (f) Cell viabilities (Hey-A8-F8) of SANGs (75nM) differentially loaded with negative control (NC) siRNA-EGFR (siRNA) and mir-429 (miRNA), normalized to saline treated cells (n = 4 independent experiments), (g) Cellular uptake in Hey-A8-F8 ovarian cancer cells following overnight incubation (18 h) with Texas- Red labeled (top) and unlabeled (bottom) SANGs for both low (left, 20x) and high (right, 63 x) magnification views. Cells (purple) visualized with Lectin DyLight™ 649 (Vector Laboratories), (h) Semi-quantitative analysis of the dose-dependent mean fluorescent intensity (MFI) measured in Hey-A8-F8 ovarian cancer cells 18 h following incubation Texas-Red labeled SANGs derived from confocal images acquired under identical conditions. Data are presented as mean ± sd. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model.

[0024] Figure 2 shows an example mechanism of internalization, endosomal escape, and delivery of RNAi payloads, (a) Sodium azide (NaN3) dependent reduction of cellular uptake as measured by mean fluorescence intensity, (b) Representative confocal fluorescence images (maximum intensity projections from z-stack) showing suppressed SANG internalization by clathrin-mediated endocytosis inhibitor (chlorpromazine [Cpz]) and quantification ofintracellular mean fluorescent intensity of SANGs following exposure to different doses of Cpz. (c) Representative confocal fluorescence images (maximum intensity projections from z- stack) showing suppressed SANG internalization by macropinocytosis inhibitor (cytochalasin D [CytoD]) and quantification of intracellular mean fluorescent intensity of SANGs following exposure to different doses of CytoD. SANGs were transfected at a final concentration of 75nM. (d) Maximum intensity projections from z-stack (10 pm) confocal images of Hey-A8- F8 cells 18 h following incubation with Cy3-siRNA-loaded SANGs (Alexa Fluor 488) at both low (right, 20x) and high (left, 63x) magnifications showing intracellular distribution, (e) Three-dimensional rendering of confocal images dual-labeling endosomes (CellLight™ Endosomes-GFP) and siRNA (Cy3) 18 h following Hey-A8-F8 incubation with siRNA-loaded SANGs. (f) Individual fluorescent channels shown for clarity, (f, lower left) Results of colocalization analysis performed on a pixel-by-pixel basis between endosomes and siRNA channels, where every pixel is plotted based on its intensity level. Scatterplot colors represent pixel density, (g) Quantitative results of colocalization analysis summarized by the Pearson’s correlation coefficients (R) between endosomes and siRNA, n = 24 cellular replicates, (h) siRNA release from SANGs quantified by colocalization coefficients (mean ± sd) between SANGs loaded with Cy3-labeled siRNA at 6 and 18 hours after incubation with Hey-A8-F8 cells, n = 24 cellular replicates. SANGs were transfected at a final particle concentration of 75 nM particle concentration. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model (stan_glm): 95% highest density intervals do not overlap between groupwise contrasts.

[0025] Figure 3 illustrates tumor agnostic, cross species in vivo and ex vivo biodistribution, (a) Whole-body imaging of the mice after intravenous injection of free siRNA (Cy3-labeled) or SANGs (1 mg»kg-l). Tumor bioluminescence and SANGs fluorescence were imaged sequentially in ovarian cancer bearing and control mice showing distribution at 30 min and 24 hours after i.v. injection, (b) Whole-body imaging of ovarian cancer-bearing mice 72 hours after intravenous injection of SANGs (1 mg»kg-l) or saline control (top row). Ex vivo imaging of tumors (Hey-A8-F8) and major organs immediately imaged after in vivo imaging was completed, (c) Quantification of SANG delivery and retention to tumors, normalized against SANG fluorescence signal in heart. Intravenous delivery of free siRNA (Cy3- labeled) used as quantitative control, (d) Representative ex vivo images at indicated time points after intravenous injection of SANGs (1 mg»kg-l) to mice following metastatic tumor induction (typically 18-21days). (e) Quantification of SANG delivery and retention to metastatic ovarian tumors, normalized against SANG fluorescence signal in heart. Intravenous delivery of freesiRNA (Cy3-labeled) used as quantitative control, (f) Colocalization analysis of ex vivo SANG fluorescence and tumor bioluminescence. Dotted line of identity indicates perfect colocalization with ±15% bounding conditions (light blue). Upper quadrant shows low (dark blue) and high-intensity (red) off-target SANGs. Lower quadrant shows low (dark blue) and high-intensity (orange) untargeted cancer, (g) Quantification of SANG delivery and retention across specified time points across tumors, liver, kidney, spleen, heart, lungs, visceral fat, skeletal muscle and GI tract all normalized against SANG fluorescence signal in heart. Intravenous delivery of free siRNA used as quantitative control, (h) Flow cytometry uptake studies of LNP-LP01 formulated to carry AlexaFluor-647 labeled siRNA against EGFR (n=3 biologically independent experiments) compared to (i) SANGs formulated to carry AlexaFluor-647 labeled siRNA against EGFR (n=4 biologically independent experiments) and PBS control (n=3) in CD31-CD45-, CD31+CD45-, and CD31-CD45+ cell populations isolated from liver and kidneys, (j) Whole-body imaging of Pirc rats after intravenous injection of free siRNA (Cy3-labeled) or SANGs (1 mg*kg-l ). SANGs fluorescence were imaged sequentially in cancer bearing showing the immediate (90 min), early distribution (24 hours), and retention (1 week), (k) Ex vivo imaging of tumors and major organs and (1) quantification of the specificity of SANG delivery and retention to tumors and major organs (liver, kidney, spleen, heart, lungs), normalized against SANG fluorescence signal in heart. Top inset shows enlarged colorectal tumor biodistribution of SANGs across time. Representative color photograph of a section the internal lumen of the descending colon of a Pirc rat indicating diffuse adenocarcinomas. Data are presented as mean ± sd. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model (stan_glm): 95% highest density intervals do not overlap between groupwise contrasts.

[0026] Figure 4 demonstrates In vivo tumor penetration and efficacy, (a) representative 20x LSCM images of tumors sections (orthotopic Hey-A8-F8) from mice 48 hours after intravenous administration of either negative control siRNA loaded SANGs (top row) or siRNA against EGFR loaded SANG (bottom row). Three-dimensional (z-stack) confocal microscopic images show expanded view (63x) of white dotted sections (a). Intracellular distribution of endosomes and siRNA 18 hours following Hey-A8-F8 incubation with SANGs loaded with Cy3-labeled siRNA. Merged images of cells nuclei (DAPI), EGFR (Alexa Fluor 488) and SANGs (Texas Red), (d) Down-regulation of EGFR, KRAS, and Glutl mRNA levels as quantified by RT-qPCR (n - 3 independent experiments) in Pirc rats, (e) Dose-dependent down-regulation of EGFR and KRAS mRNA levels as quantified by RT-qPCR (n = 3 independent experiments) in tumor bearing mice. The relative level of mRNA expression wascalculated over the NC siRNA control each of which was normalized to GAPDH (mice) or RPS18 (rats), (f) Whole-body in vivo imaging of representative Pirc rat after 1 week after intravenous injection of SANGs (1 mg*kg-l) with corresponding confocal microscopic images of EGFR overexpressing colorectal cancer cells and SANGs near-infrared fluorescence demonstrating tumor penetration and retention behavior, (g) Whole-body in vivo bioluminescence imaging OC tumor-bearing mice assessing tumor load prior to and 6 days after cohorts of animals were treated with saline (n=5), cisplatin (n=4), or combined miRNA- 429 and siRNA against EGFR loaded SANGs (n=5). Quantification of wet tumor weights following last in vivo imaging. Data are presented as mean ± sd. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model (stan_glm): 95% highest density intervals do not overlap between groupwise contrasts.

[0027] Figure 5 shows tests of tumor-agnostic targeting mechanism, (a) Representative phase contrast transmission electron microscopy (TEM) images showing reversible SANG agglomeration when going from 52 nM to 156 nM, and back again (re-diluted), (b) Observed (solid lines, data points = mean + sd) and expected (dotted lines) size and number of SANGs particles over the entire concentration range studied by TEM within a standard field of view (area), (c) Hydrodynamic size distributions of SANGs at 52 nM and 156 nM concentrations (n = 3 independent experiments), (d) Diffusion coefficients derived from diffusion-ordered NMR (DOSY) for 20 ms and 100 ms pulse sequences and T2 relaxation times across a 10-fold concentration range, (e) Representative 20x LSCM tiled image of colon with diffuse adenocarcinomas characterized by marked dysplasia in crypts and villus (box 1) and that penetrate the smooth muscle layers (box 2) showing merged view of both fluorescently labeled SANG populations and DAPI. (el-2) High resolution (63x) maximal projection confocal microscopic images show expanded view of white dotted sections (e). Scatter plot of pixelbased colocalization analysis of two representative areas (Costes randomization based colocalization R=0.85±0.02, / ?<2.2-16, n=3 animals, 3 sections each), (f) Size distribution of colocalized or not-colocalized SANGs derived from object-based analysis of 63x views (n=3 animals, 3 sections), (g) Graphical depiction of provisional mechanism by which SANGs achieve preferential tumor by exploiting maladaptive vasculature observed uniquely in tumor microenvironments (bottom) compared to healthy vasculature (top). (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model (stan_glm): 95% highest density intervals do not overlap between groupwise contrasts.

[0028] Figure 6 depicts a schematic diagram of the protocol for the synthesis and preparation of fluorescently labeled siRNA-loaded SANGs. PDI, poly dispersity index; DLS, dynamic light scattering.

[0029] Figure 7 depicts in vitro functionality, (a) representative confocal fluorescence microscopy images (20x) show suppression of EGFR protein expression levels in Hey-A8-F8 cells 48 hours after siRNA against EGFR loaded SANG (top row) as compared to negative control siRNA loaded SANGs (bottom row), n = 3 replicates. In both instances, cells were incubated with SANGs for 12 hours after which media was replaced for remainder of the study, (b) Down-regulation of EGFR, KRAS, ZEB1, and Glutl mRNA levels as quantified by RT- qPCR. The relative level of mRNA expression was calculated over the NC siRNA control, n = 3 replicates, (c) phenotypic shift of canonically mesenchymal Hey-A8-F8 ovarian cancer cells to epithelia phenotype following 12 hours incubation with SANGs loaded with mir-429. Scalebar, I OLI M. Data are presented as mean ± sd. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model (stan_glm): 95% highest density intervals do not overlap between groupwise contrasts.

[0030] Figure 8 depicts dose- and time-dependent internalization, (top row) Representative merged confocal fluorescence microscopy images (63x) of Hey-A8-F8 ovarian cancer cells after 18 hours of incubation as function of SANG concentration (red), (middle row) Individual SANG fluorescence channel, (bottom row) Representative merged confocal fluorescence microscopy images (20x) of Hey-A8-F8 ovarian cancer cells as a function of SANG incubation time.

[0031] Figure 9 depicts SANG internalization in a variety of cell types, representative confocal fluorescence microscopy images (63x) of MCF7 breast cancer, OVCAR3 ovarian cancer, and IOSE epithelial ovarian cells following overnight incubation (18 h) with Texas- Red labeled (top) SANGs.

[0032] Figure 10 depicts Dose-dependent internalization inhibitors. Quantification of intracellular mean fluorescent intensity of SANGs following exposure to different doses of clathrin-mediated endocytosis inhibitor (chlorpromazine [Cpz]) macropinocytosis inhibitors (latrunculin A [Lat]; cytochalasin D [CytD]), and a caveolae-mediated inhibitor (methyl-P- cyclodextrin [Mpcd]). Maximum intensity projections from z-stack (63x) confocal images of Hey-A8-F8 cells 18 h following incubation with SANGs. SANGs were transfected at a final concentration of 75nM. Data are presented as mean ± sd. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model.

[0033] Figure 11 depicts SANGs escape endosomes. Three-dimensional rendering of confocal images duallabeling endosomes (CellLight™ Endosomes-GFP) and SANGs (Texas- red) 18 h following Hey-A8-F8 incubation, (f) Individual endosome and SANG channels shown for clarity. siRNA release from SANGs quantified by colocalization coefficients (mean + sd) between SANGs loaded with Cy3-labeled siRNA at 6 and 18 hours after incubation with Hey-A8-F8 cells, n = 24 cellular replicates. SANGs were transfected at a final particle concentration of 75 nM particle concentration. Surface renderings of SANGs and Cy3-siRNA channels in two representative cells studied at 6 and 18 hours after incubation. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model (stan_glm): 95% highest density intervals do not overlap between group wise contrasts.

[0034] Figure 12 depicts biodistribution comparison across models, (a) Whole-body imaging of the mice 24 h after intravenous injection of SANGs (1 mg«kg- l ). Tumor bioluminescence and SANGs fluorescence were imaged sequentially in breast cancer bearing mice (orthotopically implanted MDA-MD-231). (b) Whole-body imaging of cancer-bearing mice 72 hours after intravenous injection of SANGs (1 mg‘kg-1 ). (c) Ex vivo imaging of tumors (MDA-MB-231) and major organs immediately imaged after in vivo imaging was completed.

[0035] Figure 13 depicts Epidural metastasis spinal cord (a) Ex vivo imaging of the brain and spinal cord from an ovarian cancer (Hey-A8-F8) bearing mouse 72 h after intravenous injection of SANGs (1 mg‘kg-1). Tumor bioluminescence and SANGs fluorescence were imaged sequentially.

[0036] Figure 14 depicts met targeting quantification, (a) Representative ex vivo images of Tumor bioluminescence and SANGs fluorescence from ovarian cancer (Hey-A8-F8) bearing mouse following metastatic tumor induction (typically 18-21 days) 4 h after intravenous injection of SANGs (1 mg«kg- l ) as shown in Figure 3. (b) Image masks utilized for colocalization analysis, (c) Computed distributions of colocalization among BLI and SANG channels following 1000 iterations of Cosies randomization and observed colocalization with one representative shuffled image shown (d). Scatter plot of pixel-based colocalization analysis (Costes randomization based colocalization R=0.85+0.02, p

[0037] Figure 15 depicts the time-dependent internalization in rats with advanced colorectal cancer. Whole-body imaging of Pirc rats after intravenous injection of free siRNA or SANGs (1 mg»kg-l).

[0038] Figure 16 depicts empirical quantification of SANG concentration in blood along with bi-exponential model fit (red) after single bolus injections in SD rats. Dotted and solid lines shows siRNA concentration and background fluorescence over time. Inset shows urine and feces elimination of naked siRNA and SANG dose during the first 24 h. Data are presented as mean ± sd (n= 3 rats per group).

[0039] Figure 17 depicts tumor Penetration, (left) Representative merged confocal fluorescence microscopy image (20x) and expanded (63x of dotted white box) of advanced colorectal tumors, 72 hours after i.v. injection of SANGs.

[0040] Figure 18 shows that SANGs are minimally toxic, (a) Percentage change of starting weight in response to two i.v. 7mg»kg-l doses of either (1) empty NG or (2) NG loaded with siRNA or miRNA administered to female CD-I mice (5 animals / group) separated by 24hours. The error bands represent ± 95% CI. (b) Complete blood chemistry metrics 14 days after the final dose in CD-I mice, (c) Complete blood chemistry metrics at 6 and 24 hours after i.v. administered of SANGs loaded with siRNA against EGFR at 0, 7, 12, 17 mg*kg-l (3 animals / sex / dose) in Sprague-Dawley rats. Serial blood chemistry metrics (0, 5min, 15min, 30min, Ihr, 2hr, 3hr, 4hr 5hr, and 6hr) after a single bolus i.v. injection of SANGs loaded with siRNA against EGFR (7mg»kg-l) in female Domestic Yorkshire Crossbred Swine (6 month, 73kg). Data are presented as mean ± sd unless otherwise noted. (*) indicates statistically significant differences between experimental groups as empirically derived from hierarchical Bayesian model (stan_glm): 95% highest density intervals do not overlap between groupwise contrasts.

[0041] Figure 19 depicts SANG toxicity in NOD scid. Hey-A8-F8 ovarian tumor-bearing mice received the treatments as described in Figure 18. Serum levels of ALP, BUN and IL6 at the completion of the therapy study. Mouse weights were measured once every 2 days. Data are presented as mean + sd (n= 5 mice per group).

[0042] Figure 20 depicts SANG toxicity in Pirc rats. Rats with advanced colorectal cancer (Pirc) received a modeled anticipated clinical treatment schedule. SANG-siEGFR (7mg»kg-l) was infused twice weekly for 5 weeks along with a co-infusion of oxaliplatin (5mg*kg-l). Weights were measured twice weekly as a surrogate measure for tolerability. Data are presented as mean + sd (n= 5 mice per group).

[0043] Figure 21 depicts Histology of spleen, kidney, and liver in SD rats 24 hours after randomly assigned to receive saline control, or escalating SANG-siEGFR doses 7, 13, or 17mg»kg-l as described in Figure 18. Shown are representative images from 4 independent experiments.

[0044] Figure 22 depicts (a) Fixed view negative-stain TEM images as a function of the SANG concentration and corresponding particle analysis masks (b). high magnification SEM of gold coated SANGs.

[0045] Figure 23 depicts SANG concentration dependent size change. Hydrodynamic sizes of SANGs at sequentially escalating sample concentrations followed by re-dilution. First and second dynamic light scattering peak data are presented as mean ± sd (n=3).DETAILED DESCRIPTION

[0046] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0048] The following definitions are provided for the full understanding of terms used in this specification.Terminology

[0049] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0050] Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and are not intended to exclude, for example, other additives, segments, integers, or steps. Furthermore, it is to be understood that the terms comprise, comprising, and comprises as they relate to various aspects, elements, and features of the disclosed invention also include the more limited aspects of “consisting essentially of” and “consisting of.”

[0051] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “polymer” includes aspects having two or more such polymers unless the context clearly indicates otherwise.

[0052] Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It should be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0053] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0054] For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise.

[0055] The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0056] “Administration” to a subject or “administering” includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, intravenous, intraperitoneal, intranasal, inhalation and the like. Administration includes self-administration and the administration by another.

[0057] The term “biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

[0058] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is tobe understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0059] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0060] As used herein, the term “crosslinkable” in the context of crosslinkable macromolecules or polymers refers to the ability of macromolecules or polymers to form at least one or more covalent bonds (crosslinks) with one another to form a polymeric network (nanohydrogel) under a conditions effective.

[0061] By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

[0062] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0063] The “fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impairedcompared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as regulating the transcription of the target gene.

[0064] The term "gene" or "gene sequence" refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a "gene" as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term "gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, control sequences upstream of the coding sequence.

[0065] The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for example, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0066] The term “reduced”, “reduce”, “reduction”, or “decrease” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0067] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0068] The term "nucleic acid" as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides (DNA) or ribonucleotides (RNA). The terms "ribonucleic acid" and"RNA" as used herein mean a polymer composed of ribonucleotides. The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxyribonucleotides. (Used together with “polynucleotide” and “polypeptide”.)

[0069] "Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0070] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents.

[0071] As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New lersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).

[0072] As used herein, the term “subject” or “host” can refer to living organisms such as mammals, including, but not limited to humans, livestock, dogs, cats, and other mammals. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a human.

[0073] The term “selectivity” in the context of therapeutic delivery refers to the relative portion of an administered dose that reaches the targeted location. Thus, the term “tumor selectively” or the like refers to the amount of the administered dose of agent localized within a particular tumor microenvironment.

[0074] The term "polynucleotide" refers to a single or double stranded polymer composed of nucleotide monomers.

[0075] The term "polypeptide" refers to a compound made up of a single chain of D- or L- amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

[0076] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0077] The term “target” refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species.

[0078] The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of cancer or condition and / or alleviating, mitigating or impeding one or more symptoms of cancer. Treatments according to the invention may be applied preventively, prophylactically, palliatively or remedially. Prophylactic treatments are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), after an established development of cancer, or during prevention or mitigation of cancer relapse. Prophylactic administration can occur for several minutes to months prior to the manifestation of a disease.

[0079] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeuticagent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0080] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0081] The term “tumor” refers to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign tumor” is generally well differentiated, has characteristically slower growth than a malignant tumor, and remains localized to the site of origin. In addition, a benign tumor does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign tumors include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant tumor, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as “pre-malignant tumor.” An exemplary pre-malignant tumor is a teratoma. In contrast, a “malignant tumor” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant tumor generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to anotherorgan or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue. Reference is also made to the term “tumor microenvironment,” which refers to any and all elements of the tumor milieu including elements that create a structural and or functional environment for the malignant process to survive and / or expand and / or spread.Nanohydrogels and Compositions

[0082] In various aspects, disclosed herein is a nanohydrogel comprising a crosslinkable core / shell polymer; wherein the nanohydrogel is configured to self-agglomerate in aberrant vasculature environments.

[0083] In some aspects, the crosslinkable core / shell polymer comprises a gel-forming polymer. In some aspects, the gel-forming polymer comprises a biocompatible polymer.

[0084] Various gel-forming polymers are known in the art and include both natural and synthetic materials or combinations and copolymers thereof. In some aspects, the gel-forming polymer comprises a synthetic material. Examples of synthetic gel-forming polymers include, but are not limited to, polyacrylamide, polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), polyethylene glycol (PEG), polyethylene glycol-co-propylene glycol (PEO-PPO), and poly acrylates. In some aspects, the gel-forming polymer comprises a natural gel-forming material. Examples of natural gel-forming polymers include but are not limited to polysaccharides, such as starch, alginate, agarose, cellulosic derivatives, such as, for example, but not limited to, hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) and n-ethyl cellulose, hyaluronic acid, chitosan, xanthan gum, dextran, alginate, and agar.

[0085] In some aspects, the crosslinkable core / shell polymer comprises polyacrylamide. In some aspects, the crosslinkable core / shell polymer comprises a copolymer of N- isopropylmethacrylamide and N,N'-methylenebis(acrylamide). In some aspects, a shell of the nanohydrogel comprises a cationic polymer ligand (e.g., poly(aminopropyl methacrylate)). In some aspects, the shell of the nanohydrogel comprises an amine-doped polymer. In some aspects, the shell of the nanohydrogel comprises aminomethyl propionic acid (AMPA).

[0086] Methods of synthesizing nanohydrogels are generally known in the art. As an example, core / shell nanohydrogels can be synthesized via precipitation polymerization, such as that described by Lyon et al. (2009), which is incorporated by reference in its entirety.Briefly, precipitation polymerization involves the use of solvents or solvent mixtures in which the monomers to be polymerized are soluble, but not the polymer which is formed. In some aspects, the nanohydrogels are formed using free-radical precipitation in which monomers are dissolved / dispersed in a polar solvent or solvent mixtures before initiating the polymerization using a free radical-forming compound.

[0087] In some aspects, the crosslinkable core / shell polymer is unfunctionalized. Prior published uses of hydrogels invariably included the attachment of molecules (termed “targeting ligands” here) known to bind to cell surface markers characteristic of cancer cells or other target cells. It was shown that SANGs lacking any targeting ligand (designated as “unfunctionalized SANGs”) were able to effectively and selectively distribute to tumor tissue upon intravenous administration and to operate as a therapeutic delivery agent. This is entirely unexpected, as evidenced by the lack of testing of such unfunctionalized SANGs in any reported account of related materials: those skilled in the art would assume that functionalization would be necessary for selective delivery to tumor tissue. Remarkably, unfunctionalized SANGs can thereby function as a drug delivery platform that exhibits comparable performance among a variety of different cancer models, all of which share common properties of tortuous tumor vasculature but do not necessarily share common cellsurface markers. The term “unfunctionalized” generally refers to a material that is not augmented with a functional group to impart a specific property (e.g., tumor targeting) to the nanohydrogel. For example, the polymer shell can be prepared such that it does not have a targeting moiety selective for a specific tumor. In this regard, the resulting nanohydrogel can respond to a variety of different disorders. In some aspects, the nanohydrogel has a hydrodynamic size of from about 75 nm to about 200 nm prior to self- agglomeration.

[0088] In some aspects, the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to self- agglomerate to form an agglomerated cluster, wherein the agglomerated cluster has an average hydrodynamic size larger than an average hydrodynamic size prior to self-agglomeration.

[0089] In some aspects, self-agglomeration of the population of nanohydrogels is concentration-dependent. In some aspects, a concentration of nanohydrogels causing selfagglomeration is 10 nM or greater (e.g., 20 nM or greater, 30 nM or greater, 40 nM or greater, 50 nM or greater, 60 nM or greater, 70 nM or greater, 80 nM or greater, 90 nM or greater, 100 nM or greater, 110 nM or greater, 120 nM or greater, 130 nM or greater, 140 nM or greater, 150 nM or greater, 156 nM or greater, 200 nM or greater, 250 nM or greater, 500 nM or greater, 1 pM or greater). In some aspects, a concentration of nanohydrogels causing self-agglomeration is from about 10 nM to about 1 gM (e.g., from about 10 nM to about 500 nM, from about 30 nM to about 500 nM, from about 60 nM to about 500 nM, from about 60 nM to about 250 nM, from about 100 nM to about 250 nM, from about 100 nM to about 200 nM, or about 156 nM). It was unexpectedly determined in the exemplified cases that a concentration of the nanohydrogels at or above 156 nM promoted the agglomeration characteristic, thereby increasing retention and delivery of a therapeutic agent.

[0090] Also described herein are compositions comprising: any of the populations of nanohydrogel described herein; and an agent (e.g., a therapeutic agent). In some aspects, the therapeutic agent is encapsulated within a core of the nanohydrogel

[0091] In some aspects, the therapeutic agent comprises an anti-cancer agent or a chemotherapeutic agent. The term “anti-cancer agent” as used herein refers to any therapeutic agent that directly or indirectly kills cancer cells or directly or indirectly prohibits stops or reduces the proliferation of cancer cells. It should also be noted that the phrase “anti-cancer agent” should not be interpreted as being limited to a single anti-cancer agent and may further include combinations of agents to achieve the intended effect. In some aspects, the anti-cancer or chemotherapeutic agent comprises mRNA encoding said agent, wherein said agent is a protein, polypeptide, or a peptide. By way of example, various aspects include mRNA encoding a toxic proteins or anticancer immunogens configured to produce a therapeutic immune response.

[0092] Examples of suitable chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines (e.g., altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine); acetogenins; delta-9-tetrahydrocannabinol (e.g., dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fhidarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifhiridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine1(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

[0093] In some aspects, the therapeutic agent comprises a nucleic acid. In some aspects, the therapeutic agent comprises small interacting RNA (siRNA) and / or microRNA (miRNA). In some aspects, the therapeutic agent comprises an inhibitor of epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus oncogene homolog (KRAS), Glul, and / or Zinc Finger E-Box Binding Homeobox 1 (ZEB1). In some aspects, the therapeutic agent comprises an inhibitor of EGFR. In some aspects, the therapeutic agent comprises an inhibitor of Glul. In some aspects, the therapeutic agent comprises an inhibitor of ZEB1. In some aspects, the therapeutic agent comprises an inhibitor of KRAS.

[0094] In some aspects, the therapeutic agent is one or more of monoclonal antibodies, chimeric antibodies, humanized antibodies, nanobodies, antibody fragments, cholesterol, hormones, peptides, proteins, chemotherapeutics, antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, polynucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys, analogs, plasmids, expression vectors, small nucleic acid molecules, mRNA, RNAi agents, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), peptide nucleic acid (PNA), locked nucleic acid ribonucleotides (LNA), morpholino nucleotides, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (assymetrical interfering RNA), and siRNA with 1, 2, or more mismatches between the sense and anti-sense strand to relevant cells or tissues, or a combination thereof.

[0095] For example, the therapeutic agent may comprise a nucleic acid, such as RNAi agents. The term “RNAi agent” may refer to any RNA, RNA derivative, and / or nucleic acid encoding an RNA that induces an RNAi effect (e.g., degradation of target RNA and / or inhibition of translation). Generally, an RNAi agent is an RNA molecule (or combination of molecules) that includes a structure characteristic of molecules that can mediate inhibition ofgene expression through an RNAi mechanism. Examples of RNAi mechanisms include inhibition of gene expression through the degradation of target transcripts or by inhibiting translation of target transcripts. Generally, an RNAi agent includes a portion that is substantially complementary to a target RNA. In some aspects, RNAi agents are at least partly double-stranded. However, in some examples, RNAi agents are single-stranded. Exemplary RNAi agents can include siRNA, shRNA, and / or miRNA. RNAi agents can be composed entirely of natural RNA nucleotides (i.e., adenine, guanine, cytosine, and uracil) or include one or more non-natural RNA nucleotides (e.g., nucleotide analogs, DNA nucleotides, etc. In some examples, RNAi agents can include a combination of siRNA and miRNA constructs to silence expression of target genes in a sequence specific manner, which is particular advantageous for use in a variety of cancer models. As discussed above, previous efforts of systemic delivery of RNAi to tumors has been challenging due to rapid clearance, susceptibility to serum nucleases, insufficient delivery to tumor sites, inadequate tumor penetration and inability to traverse plasma membranes all attenuate its intended biological activity. Remarkably, the described nanohydrogels are shown to interact with aberrant vasculature in the tumor microenvironment (TME), which increases retention in the tortuous supplies (Figure 2). This local increase in SANG concentration causes individual SANG particles to glom onto each other, and, once a threshold is reached, a positive-feedback loop can be initiated. This so called “selfagglomerating” characteristic emerges from the size and surface topology of the SANG platform and ultimately results in much higher concentrations of SANG in tumors with aberrant vasculature - providing an effective solution to the systemic delivery of a variety of therapeutic agents, including RNAi agents.Methods

[0096] Also described herein is a method of delivering an agent to a tumor of a subject. For example, the method can include: administering any of the compositions described herein to the subject; wherein the population of nanohydrogels is inducible to self- agglomerate to form an aggregated cluster of nanohydrogels via the aberrant microvasculature of a tumor microenvironment.

[0097] In some aspects, the method further includes: allowing the agent to be released from the aggregated cluster of nanohydrogels. Advantageous to the disclosed platform, the selfagglomeration property permits consolidation and sustained release of the agent to the tumor microenvironment. After sufficient exposure, the agglomerated nanohydrogels begin todissociate and extravasate to tumor interstitial spaces, leading to the selective delivery of the agent.

[0098] Further described herein are methods of treating cancer in a subject, comprising: administering any of the compositions described herein to the subject; wherein the population of nanohydrogels is passively inducible to self-agglomerate to form an aggregated cluster of nanohydrogels via the aberrant microvasculature of a tumor microenvironment; and allowing the therapeutic agent to be released from the aggregated cluster of nanohydrogels.

[0099] In some aspects, the cancer is ovarian cancer, colorectal cancer, or breast cancer. In some aspects, the cancer is ovarian cancer. In some aspects, the cancer is colorectal cancer.

[0100] In some aspects, the cancer is breast cancer. In some aspects, the cancer is a cancer having an undruggable cancer-promoting gene. In some aspects, the undruggable cancerpromoting gene comprises one or more of c-Myc. APC, BRAF, and / or KRAS.

[0101] One major limitation of existing nanoparticle platforms for therapeutic delivery is the low delivery efficiency to the tumor microenvironment. A review of the biodistribution of these nanostructures reveals a representative example in which a significant portion (~ 11.25%) of the dose is entrained in the liver and only about 0.67 % of the dose is ultimately delivered to the tumor. (Chen, 2023). Comparatively, the present nanohydrogels offer substantially higher tumor delivery efficacy relative to these existing platforms. In some aspects, a tumor selectivity of the nanohydrogel is 1% or greater (e.g., 1.5% or greater, 2% or greater, 2.5% or greater, 3% or greater, 3.5% or greater, 4% or greater, 4.5% or greater, 5% or greater, 5.5% or greater, 6% or greater, 6.5% or greater, 7% or greater, 7.5% or greater, 8% or greater, 9.5% or greater, 10% or greater, 10.5% or greater, 11% or greater, 11.5% or greater, 12% or greater, 12.5% or greater, 13% or greater, 13.5% or greater, 14% or greater, 14.5% or greater, 15% or greater, 20% or greater, or 25% or greater). In some aspects, the delivery efficiency of the therapeutic agent to the tumor microenvironment is measured as a ratio between the amount of nanohydrogel in the tumor relative to the amount entrained in the liver. In some aspects, the tumor: liver ratio is 1 :1 or more (e.g., 1.5: 1 or more, 2:1 or more, 2.5:1 or more, 3:1 or more, 3.5:1 or more, 4:1 or more, 4.5:1 or more, 5:1 or more, 5.5: 1 or more, 6:1 or more, 6.5:1 or more, 7: 1 or more, 7.5: 1 or more, 8: 1 or more, 8.5:1 or more, 9: 1 or more, 9.5: 1 or more, 10: 1 or more, 10.5:1 or more, 15:1 or more, 20: 1 or more, or 25:1 or more). In some aspects, 1 % or more of the population of nanohydrogels is retained after 24 hours from the initial dose (e.g., 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 10% or more).

[0102] In some aspects, the administration of the composition comprises administering a first composition comprising nanohydrogels having a first therapeutic agent and a second composition comprising a second therapeutic agent (e.g., nanohydrogels having a second therapeutic agent). In some aspects, the first composition and second composition are administered concurrently. In some aspects, the first therapeutic agent and the second therapeutic agent are the same. In some aspects, the first therapeutic agent and the second therapeutic agent are different. In some aspects, the first therapeutic agent comprises an RNAi agent and the second therapeutic agent comprises a chemotherapeutic agent.

[0103] Examples of suitable chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines (e.g., altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine); acetogenins; delta-9-tetrahydrocannabinol (e.g., dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

[0104] In vivo application of the disclosed nanohydrogels and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed nanohydrogels can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitableroute known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrastemal administration, such as by injection. In certain aspects, the administration can include intravenous, intrathecal, intracranial, also intramuscular, intratumoral, intratracheal, subcutaneous application. Administration of the disclosed nanohydrogels or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

[0105] The disclosed compositions and methods can open other patient populations with difficult to drug or undruggable targets that have potential to improve clinical outcomes. The term “undruggable” generally describe proteins that historically could not be targeted pharmacologically (Cang, C. V., et al., Nature Reviews Cancer, 17:502-508 (2017)). Today, there are many cancer targets that are considered undruggable (Cang, C. V., et al., Nature Reviews Cancer, 17:502-508 (2017)). Examples of undruggable proteins include intracellular proteins and proteins that lack domains that are readily bound by small molecule drugs e.g., c- Myc. APC, BRAF, KRAS. Advantageously, the SANG platform can be active agnostic, meaning any siRNA or miRNA target (or combination thereof) can be loaded and delivered to solid tumor indications. This emergent property of the SANG platform is distinct from the classical EPR (enhanced permeability and retention) effect but utilizes it, amplify the resulting large concentration gradient in the TME to allow SANG particles to penetrate vessels, reaching cancerous cells. Moreover, because aberrant vasculature is a highly conserved feature among solid tumors cancers, the presently described nanohydrogels and composition offers an agonist platform to target cancers of multiple tissue origins and molecular profile. Thus, the selfagglomerating hydrogels are capable of targeting of both primary and metastatic loci and offer a much needed paradigm shift in the treatment of cancer.

[0106] The term “cancer” refers to a malignant tumor (Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990). Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), nonsmall cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essentialthrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrinetumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).EXAMPLES

[0107] The following examples are set forth below to illustrate the compositions, methods, and results according to the disclosed subject matter. The examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.Example 1:

[0108] Recognizing the need to unlock pan-cancer siRNA therapy and in particular for the vast array of cancers driven by EGFR overexpression, e.g. lung cancers (50-90%), OC (30- 70%), colorectal (80%), head and neck (90-95%), pancreas (65-95%), breast (27-90%), and glioblastoma (40%), two major nanohydrogel design decisions were made: 1) removing any functionalizing moiety and 2) modifying the surface topology. These steps were done to simplify the manufacturing process and increase stability / shelf-life, all the while imbuing thenanohydrogel with improved targeting and retention capability. The SANG particles can achieve desirable in vivo biodistribution following i.v. administration in mice with tumors (EphA2 positive OC cells). As described below, SANG can achieve this capability through a unique targeting mechanism. This emergent property of SANG as “self-agglomerating” enables its targeting mechanism. Moreover, SANG can achieve desirable siRNA delivery kinetics and tumor efficacy. SANG can further demonstrate favorable acute biodistribution to both primary tumor loci and remote metastatic lesions, high retention rates (detected >7 days), while being nearly completely absent from major organ system devoid of cancer by 24 hours (Figure 1) which undoubtedly contributes to its favorable toxicology profile.SANGs synthesis and characterization

[0109] Polyacrylamide-based core-shell nanohydrogels were made from inexpensive materials and were formulated as soft and biocompatible particles using a two-stage free- radical precipitation polymerization method first described by Lyon and coworkers (Fig. 1, panel a and Fig. 6) (Blackburn, 2009; Gan, 2001; Jones, 2000). siRNA can be loaded into these materials by swelling lyophilized particles in RNA-containing buffer; uptake of the RNA into the particles is complete and irreversible upon standing. It was previously observed that nanohydrogels functionalized with tumor-targeting peptides exclusively targeted ovarian tumors. (Satpathy, 2016). However, instead of functionalizing the shell layer with directing ligands, the present example explored the capabilities of untargeted nanohydrogels (SANGs), chemically functionalized only by the attachment of a small amount of fluorophore by amide bond formation. Physicochemical properties were characterized (Fig. 1, panels b-c) and independently validated by the National Characterization Laboratory under a variety of solvent, pH, dilutions, and temperature conditions (Fig. 1, panel d). Both siRNA-loaded and unloaded SANGs were found to be unchanged in size, morphology, and concentration upon storage at - 25°C, 4°C, and 25°C out to 60 days in aqueous conditions (Fig. 1, panel e). SANGs prepared with negative control (NC) and scrambled RNAi molecules were shown to have no significant effect on the viability of HEY-A8-F8 cells (75 nM in particles, Fig. 1, panel f).SANGs undergo endosomal uptake and escape

[0110] SANGs were taken up readily by HEY-A8-F8 in dose- and time-dependent fashion (Fig. 1, panels g-h and Fig. 8). Application of SANGs for increasing lengths of time (ranging from 30 minutes to 24 hours) revealed detectable intracellular uptake by 1 hour, which increased until maximal fluorescent signals were reached at 18 hours (Fig. 8). Internalization was comparable for epithelial (OVCAR3) and mesenchymal (HEY-A8-F8) type ovarian cancer cells, as well as MCF7 breast cancer cells (Fig. 9). Internalized SANGs remained unchangedin terms of cellular localization and fluorescence intensity for at least 24 hours (Figs. 8-9). Without wishing to be bound by theory, SANG cellular uptake was shown to be energy-, clathrin-, and macropinocytotic-dependent (Fig. 2, panels a-c, Fig. 10) and unperturbed by inhibition of caveolar mechanisms (Fig. 10), suggesting a combination of clathrin- mediated endosomal and macropinocytotic pathways. This was further corroborated by live-cell imaging with an endosomal marker (wheat germ agglutinin, WGA) (Liu, 2011; Raub, 1990) and SANGs containing dye-labelled siRNA, showing simultaneous co- localization of all components in HEY- A8-F8 cells at early time points up to six hours (Fig. 2, panel h). After 18 hours, however, siRNA and SANGs were observed to be distributed throughout the cytoplasm (Fig. 2, panels d-f and Fig. 11), with low correlation (R=0.142+0.16, n=24) between siRNA and endosomes, suggesting an efficient process of endosomal escape and release from the particles (Fig. 2, panel g).

[0111] Blockade of epidermal growth factor receptor (EGFR) kinase activity can be used for cancer therapy. However, other kinase-independent functions of EGFR also mediate prosurvival and chemo-resistance, so siRNA mediated knockdown should be able to achieve levels of therapeutic efficacy inaccessible to kinase inhibition alone. Consistent with the apparent escape of siRNA to the cytosol, efficient suppression of EGFR mRNA and proteins was observed in overexpressing HEY-A8-F8 cells after treatment with SANGs formulated with siRNA against EGFR (20 nM) (Fig. 7). The same formulation substituting the appropriate siRNA induced similar knockdown of KRAS, Glutl, and miRNA-429, all known regulators of cancer cell proliferation, migration, and invasion. SANGs containing scrambled siRNA had no effect. The degree of reduction in protein expression was similar to that observed using the same concentration of siRNA delivered with lipofectamine (Fig. 7).SANG biodistribution and retention in mouse and rat

[0112] The in vivo biodistribution of SANGs was explored with xenograft and orthotopic murine models of ovarian and breast cancer and a genetically-engineered rat model of colorectal cancer to survey different tumor types, induction methods, and species, all in the context of a functioning immune system. Each of the cancer strains employed were engineered to express luciferase and thus could be readily followed by bioluminescence imaging (BLI).

[0113] Within 30 minutes following i.v. injection in murine ovarian and breast cancer models, SANGs accumulated in regions with higher BLI signal indicating presumptive tumoral targeting (Fig. 3, panel a and Fig. 12). In vivo distribution of SANGs was distinct from unloaded siRNA and free dyes, which were detected in regions devoid of BLI signal in lungs (within 5 min) and in liver shortly thereafter (30 min) (Fig. 3, panel a). By 24h, the specificity ofbiodistribution improved out to 72 h (Fig. 3, panel b) where little (if any) SANGs were detected outside BLI inferred tumors. The in vivo biodistribution and siRNA delivery of SANGs was equivalent to that previously observed for the targeted (YS A-functionalized) nanohydrogels (Fig. 13). SANGs remained closely associated with BLI signals 7 days after i.v. delivery indicating preferential retention in tumors.

[0114] Analysis of excised tissues confirmed SANGs rapidly targeted tumors, reaching 5.1+1.2-fold increase by 4 hours and a maximum (198-fold) at 72 hours (Fig. 3, panel c and Fig. 14). While significantly reduced compared to peak, it was shown that SANGs were retained in primary tumors 7 days after systemic delivery (Fig. 3, panel c). In contrast, intravenous infusion of free siRNA (20 pM) gave significantly lower tumor localization at 3 and 7 days (1.14+0.02 and 2.29+0.17 fold above background, Fig. 3, panel c). (Chen, 2023).

[0115] To test the metastatic targeting capacity of SANGs, a limitation of existing RNAi delivery platforms (Subhan, 2021), the study modeled late-stage ovarian cancer by IP administration of HEY-A8-F8 cells. After confirming tumor induction and extensive abdominal metastasis, the study i.v. administered SANGs and quantified biodistribution as described above. It was observed that expansive tumor load across the spleen, GLtract, liver, and occasionally epidural metastasis (Figs. 14-15). Surprisingly, rapid (4h) and highly specific accumulation of SANGs in diffuse metastatic lesions were also observed. The temporal biodistribution in metastatic loci closely mirrored that of primary tumors independent of organ while signal in the non-cancerous portions of organs remained largely devoid of SANG signal (Fig. 3, panel d and Figs. 12-14).

[0116] Colocalization analysis of ex vivo SANG fluorescence and tumor bioluminescence (Fig. 3, panel f) showed strong association between SANGs and diffuse metastases (R=0.84, cO.OO l , Fig. 16) with >96% of the SANG signal residing within 15% of the line of identity. Few if any (<0.1%) metastatic loci were untargeted by SANGs (below line of identity) while only <3.5% of SANGs were detected in regions without any BLI confirmed metastasis. These results indicate a low level of off-target accumulation and corroborate biodistribution data observed in less metastatic murine models. Taken together, these results indicate that SANGs rapidly target both primary and metastatic murine tumors, are retained for extended periods of time, and have minimal off-target accumulation (Fig. 3, panel g).

[0117] To quantitatively assess SANGs apparent avoidance of liver and kidney entrainment, the study compared the cellular distribution of SANGs to that of a well- documented liver-targeting LNP formulation based on the lipid LP01 (n=3) (Finn, 2018). In separate cohorts of control mice, the study intravenously injected SANGs or LNP-LP01, eachformulated to carry AlexaFluor-647 labeled siRNA against EGFR (n=4). After three hours, CD31-CD45-, CD31+CD45-, and CD31-CD45+ cell populations were isolated from liver and kidney using fluorescenceactivated cell sorting (FACS) and the number of those cells carrying the AlexaFluor dye was determined. It was noted that significantly more cells extracted from liver in LNP-LP01 treated animals were dye labeled compared to PBS controls (Fig. 3, panel h). With the exception of presumptive renal endothelial cells, significantly more CD31- / CD45+ and CD31- / CD45- cells extracted from kidneys also were labeled with the dye, presumably by LNP uptake (Fig. 3, panel h). In contrast, the study found only 4% of CD31 - / CD45- renal cells from SANGs treated animals to contain significant amounts of dye relative to PBS treated controls (Fig. 3, panel i). Collectively, across all cell-types studied, it was found that 10-30 times fewer dye-labeled cells in SANGs treated animals compared to LNPs (Fig. 17), thereby verifying whole organ and confocal microscopy biodistribution data (Fig. 18).

[0118] Ovarian tumor bearing mice were intravenously injected simultaneously with LNP- LP01 formulated to carry AlexaFluor-647 labeled siRNA against EGFR and SANGs formulated to carry Texas-Red labeled siRNA against EGFR. Analysis of excised tissues at one and four hours after injection demonstrates orthogonal biodistribution between SANGs and LNP-LP01 (Fig. 19). While the use of different fluorophores precludes quantitative comparisons, these data qualitatively showcase the ability of SANGs to target solid tumors compared to a state-of-the-art delivery platform.

[0119] Intravenous delivery of SANGs to rats genetically engineered to spontaneously develop colorectal cancer resulted in immediate diffuse SANG signal across the abdominal cavity which began to coalesce in small punctate intensities around the perimeter of the abdomen, suggestive of preferential targeting to the diffuse tumors (Fig. 3, panel j and Fig. 20). SANG distribution remained localized in this fashion for 7-days with increasing signal-to-noise ratio over time, indicating preferential tumor retention. In contrast, free siRNA distributed mostly to the liver followed by rapid elimination (Fig. 3, panel j). Ex vivo analysis of colon with diffuse adenocarcinomas (Fig. 3, panel k) showed detectable SANG signal at 4 hours, increased signal at 24 and 48 hours, and a maximal recorded value at 7 days (Fig. 3, panels k- i); mouse biodistribution peaked at 72 h (Fig. 3, panel c). As in mouse, SANGs remained largely absent from other major rat organs with the exception of a nominal increase detected in liver and a transient increase (at 4 hours) in the kidney (Fig. 3, panels k-i and Fig. 18).

[0120] In vivo pharmacokinetics and clearance studies revealed an apparent bi-exponential blood distribution half-life (a) of approximately 2.5 h for SANGs and an elimination half-life (P) of -13.4 h (Fig. 21) in rat. While this value is comparable to that of other nanoparticleformulations (Chen, 2023; Benezra, 2011), SANGs behave differently in at least one important respect. The study saw relatively high SANG concentrations in blood at 24 and 48 h with between 2.8-5.6% of the initial dose remaining in circulation, a factor that likely played a role in the favorable tumor biodistribution and retention. Analysis of urine and feces showed rapid and near complete (-92%) excretion of naked siRNA by 24 h which was dominated by renal metabolism whereas only 16% of the SANG dose was excreted during the first 24 h. These data show that a large fraction of SANGs leaves systemic circulation rapidly, partitions efficiently to tumors, and perhaps is also sequestered in low concentrations in tissue such as fat, to be released over time.

[0121] In vivo functionality of SANGs. When examined by confocal fluorescence microscopy, SANGs were found distributed throughout ovarian carcinoma (HEY-A8-F8) tumor sections and deep within tumor parenchyma, exhibiting an extravasated distribution into the tumor interstitium (Fig. 4, panel a and Fig. 22). A similar result was observed in mice with orthotopically implanted ovarian carcinomas (HEY-A8-F8) and breast cancers (MDA-MB- 231) (Fig. 12) as well as in rats with advanced colorectal cancer, where strong colocalization with EGFR-overexpressing tumor cells (Fig. 4, panel f) was observed. In all studied cases, the extravasated distribution was retained for at least 7 days.

[0122] Across murine ovarian (HEY-A8-F8) and rat colorectal cancers, it was shown that single intravenous infusion of SANGs loaded with siRNA against EGFR, KRAS, or GLUT1 resulted in dose-dependent reduction in mRNA (Fig. 4, panels d-e) and protein expression (Fig. 4, panels a-c) in tumor tissue. Therapeutic efficacy of SANG-siRNA was then tested in mice with tumors established from HEY-A8-F8 ovarian carcinoma cells. Enhanced expression of EGFR by these cells is correlated with drug resistance, and is a well-established model. (Sheng, 2011 ; Teplinsky, 2015). The study treated groups of mice with two independent doses of SANGs, 24 hours apart - the first loaded with mir-429 and the second with siRNA against EGFR - because mir-429 driven EMT reversal requires time to induce the necessary phenotypic change. Following this treatment, i.p. cisplatin was delivered 24 hours later. Cisplatin alone was used as standard of care control. Six days after treatment, dramatic improvements relative to drug alone were evident from significant inhibition of tumor growth (Fig. 4, panel g) and reduction in tumor weight (Fig. 4, panel h). These data indicate that SANGs successfully extravasate, penetrate tumor microenvironments, gain access to the specific cells of interest in a species- and tumor-agnostic manner, and sensitize previously resistant tumors in vivo.

[0123] Systemic delivery of SANGs is minimally toxic. A variety of tests revealed no significant toxicity associated with high doses of either empty or scrambled miRNA / siRNA- loaded SANGs. These included observations with outbred mice (Fig. 18, panels a,b) and NOD- SCID mice with ovarian tumors including delivery of cisplatin. A repeat-dose tolerability study of high-dose SANG-siRNA and co-infused oxaliplatin in rats with advanced colorectal cancer (Housley, 2020) produced no mortality or any clinical signs of distress while weights recovered back to baseline by study completion. A maximal tolerable dose study in rats revealed only minor changes in blood chemistry at 6 and 24 hours (Fig. 18, panel c) and no difference from controls in histopathology of major organs for the three escalating doses. Finally, a single-acute dose study in an adult female (73 kg) Yucatan swine showed no clinically meaningful deviances out to 6 hours following i.v. infusion of SANGs-siRNA at the same high dose (7 mg*kg-1) with the exception of a transient increase in AST at 5 minutes (Fig. 18, panel d). Thus, SANGs induced minimal, if any, toxicity in a variety of animals and experimental conditions. Taken together, these data establish a wide preliminary safety profile and strongly support the systemic use of SANG to deliver RNAi to solid tumor cancers.

[0124] Cancer specific delivery achieved through emergent self-agglomerating mechanism. The results described here set SANGs apart from most nanoparticulate delivery vehicles, their most striking property being selective homing to tumors without the use of designed ligands for cell-surface markers. Four complimentary methods were used to probe the mechanism(s) responsible for this in vivo performance. We sought to distinguish between mechanisms involving cell binding vs. physical properties of the particles that may promote selective distribution to tumor tissue. First, TEM was used to measure the size and number of SANG particles over concentrations ranging from 25.5 nM to 1.6 pM (Fig. 5, panels a,b). Results revealed an interesting dynamic behavior. At the lowest three concentrations studied, the number and size distribution of SANGs closely matched the predicted values (Fig. 5, panel b). At higher SANG concentrations, however, this relationship no longer held. Instead, progressively smaller numbers of SANG particles were observed, with increasing sizes and size distributions (Fig. 5, panel b). High magnification views showed different representative forms of tightly clustered SANGs that emerged above 156 nM. Similarly, aqueous dynamic light scattering (DLS) on two different instruments showed strongly monodispersed hydrodynamic size distributions at low (52 nM) concentrations, but clear evidence of larger and less regular aggregates at 156 nM (Fig. 5, panel c, Fig. 23). When concentrated SANGs samples were re-diluted, much of the material returned to the original size range while a portion remained in a larger aggregated state (indicated by DLS and TEM, Fig. 5, panel a).

[0125] Parallel diffusion-ordered NMR measurements allowed the detection of diffusional changes caused by aggregation. A significant decrease in diffusion coefficient (21.5%) and T2 relaxation time was observed over a 10-fold range of SANG concentration (Fig. 5, panel d). The former parameter indicates slower particle movement in solution and the latter reflects restricted molecular motion (Bornstein, 2020; Saito, 2021), both consistent with nanogel agglomeration in solution caused at least in part by cross-linking between SANG particles as concentration increases.

[0126] To test if nanogel aggregation occurs in vivo, the study intravenously infused two populations of SANGs conjugated to different fluorophores in rats with advanced colorectal cancer. If SANGs do not agglomerate in vivo, fluorescent signals from both populations would co-localize at a nominal frequency determined by chance. Colocalization to a significantly greater degree would indicate that agglomeration does indeed occur. Fluorescent signals from SANGS of both colors were readily detected (expanded views, Fig. 5, panels el -2) and found to be colocalized to a degree significantly greater than that expected by chance (Costes randomization-based colocalization R=0.85+0.02, <0.00 l , n=3 animals, 3 sections each). Furthermore, deposits showing both colors were significantly larger (~74-fold) than SANGs of a single color (Fig. 5, panel f). Collectively, results from these four methodologically independent experiments demonstrate that concentration-dependent aggregation effectuate preferential tumor targeting (Fig. 5, panel g).Discussion

[0127] Formulated to contain positively charged groups to facilitate the carrying of oligonucleotides, these nanogels were shown to efficiently package and stabilize RNAi molecules and to deliver siRNA and miRNA to three cancer models, equally well in both mouse and rat across primary and metastatic loci. The RNAi cargo successfully sensitized drug-resistant tumors, allowing subsequent delivery of chemotherapeutic agents to have a dramatically enhanced effect in arresting tumor growth in vivo. The nanogel platform was found to be minimally toxic and well tolerated in mice, rats, and swine in a variety of simulated clinical applications. Remarkably, SANGs were shown to exhibit a number of beneficial properties such as: 1) preferential targeting of primary and metastatic tumors (Rosenblum, 2018) relative to healthy tissue, 2) prolonged tumor retention, 3) extravasation into the tumor interstitium, gaining access to cancerous cells, and 4) delivery of sufficient RNAi payloads to silence oncogene mRNA and protein expression and result in efficient tumor growth suppression. This combination of properties is not present in any of the current delivery platforms - polymeric, liposomal, proteinacious, or viral.

[0128] At the whole animal level, systemically administered SANGs distribute to all organs proportional to the perfusion rate. However, elevated resistance, sluggish blood flow and increase tortuosity, which are overrepresented in the maladaptive vasculature of tumors (Less, 1997; Sevick, 1989), allows time for SANG particles to interact, coalesce (Chauhan, 2011 ; Fukumura, 2007) and become entrained in tumors rather than other tissues or organ systems. This responsive behavior results in the establishment of high concentration of SANGs in tumor vasculature, favoring extravasation down the concentration gradient. Once in the interstitial space (Fig. 4, panels a-c and f), cellular uptake mechanisms such as clathrin- mediated endocytosis and macropinocytosis mediate cellular internalization. In essence, the emergent aggregating behavior of SANGS - the existence of which is supported by the self- consistent results of four complimentary methods of analysis - amplifies the enhanced permeability and retention (EPR) effect to achieve tumor delivery, penetration, and retention to the entire tumor parenchyma regardless of carcinoma type (Whitehead, 2009; Ren, 2012; Ruoslahti, 2010). Other minor involvement may be attributed by receptor mediate processes such as multivalent glycoprotein recognition that might enable cells (e.g. CD31- / CD45- populations) to recognize surface features of unfunctionalized SANGs. However, the degree to which these processes affect the cellular uptake of SANGs is likely minimal.

[0129] Overall, environmentally-responsive nanostructures of the SANG type represents an important therapeutic paradigm shift for systemic delivery of RNAi to solid tumors, as it exhibits exceptional in vivo performance and substantial therapeutic index that qualify it for advanced development in preparation for clinical applications. Furthermore, the presently described compositions and methods can be further adapted to incorporate other therapeutic payloads.

[0130] Those skilled in the art will appreciate that numerous changes and modifications can be made to the examples described herein and that such changes and modifications can be made without departing from the spirit of the disclosure. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the disclosure.

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Claims

CLAIMSWhat is claimed is:

1. A nanohydrogel comprising a crosslinkable core / shell polymer; wherein the nanohydrogel is configured to self- agglomerate in aberrant vasculature environments.

2. The nanohydrogel of claim 1, wherein the crosslinkable core / shell polymer comprises a gel-forming polymer.

3. The nanohydrogel of claim 2, wherein the gel-forming polymer comprises a biocompatible polymer.

4. The nanohydrogel of any one of claims 1-3, wherein the crosslinkable core / shell polymer comprise polyacrylamide.

5. The nanohydrogel of any one of claims 1-4, wherein the crosslinkable core / shell polymer comprises a copolymer of N-isopropylmethacrylamide and N,N'- methylenebis(acrylamide).

6. The nanohydrogel of any one of claims 1-5, wherein a shell of the nanohydrogel comprises a cationic polymer ligand (e.g., poly(aminopropyl methacrylate)).

7. The nanohydrogel of claim 1-6, wherein the crosslinkable core / shell polymer is unfunctionalized.

8. The nanohydrogel of any one of claims 1-7, wherein the nanohydrogel has a hydrodynamic size of from about 50 nm to about 250 nm prior to self-agglomeration.

9. A population of the nanohydrogels of any one of claims 1-8, wherein the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to self-agglomerate to form an agglomerated cluster, wherein theagglomerated cluster has an average hydrodynamic size larger than an average hydrodynamic size prior to self-agglomeration.

10. The population of nanohydrogels of claim 9, wherein self- agglomeration of the population of nanohydrogels is concentration-dependent.

11. The population of nanohydrogels of any one of claims 9-10, wherein a concentration of nanohydrogels causing self-agglomeration is 10 nM or greater (e.g., 20 nM or greater, 30 nM or greater, 40 nM or greater, 50 nM or greater, 60 nM or greater, 70 nM or greater, 80 nM or greater, 90 nM or greater, 100 nM or greater, 110 nM or greater, 120 nM or greater, 130 nM or greater, 140 nM or greater, 150 nM or greater, 156 nM or greater, 200 nM or greater, 250 nM or greater, 500 nM or greater, 1 pM or greater).

12. The population of nanohydrogels of any one of claims 9-11, wherein a concentration of nanohydrogels causing self-agglomeration is from about 10 nM to about 1 M (e.g., from about 10 nM to about 500 nM, from about 30 nM to about 500 nM, from about 60 nM to about 500 nM, from about 60 nM to about 250 nM, from about 100 nM to about 250 nM, from about 100 nM to about 200 nM, or about 156 nM).

13. A composition comprising: the population of nanohydrogels of any one of claims 9-12; and an agent (e.g., a therapeutic agent).

14. The composition of claim 13, wherein the agent is encapsulated within a core of the nanohydrogel.

15. The composition of any one of claims 13-14, wherein the agent is a therapeutic agent selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, nanobodies, antibody fragments, cholesterol, hormones, peptides, proteins, chemo therapeutics, antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, polynucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys, analogs, plasmids, expression vectors, small nucleic acid molecules, mRNA, RNAi agents,short interfering nucleic acid (siNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), peptide nucleic acid (PNA), locked nucleic acid ribonucleotides (LNA), morpholino nucleotides, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (assymetrical interfering RNA), and siRNA with 1, 2, or more mismatches between the sense and anti-sense strand to relevant cells or tissues, or a combination thereof.

16. The composition of any one of claims 13-15, wherein the agent comprises an RNAi agent.

17. The composition of any one of claims 13-16, wherein the agent comprises an anticancer agent.

18. The composition of any one of claims 13-17, wherein the agent comprises a nucleic acid.

19. The composition of any one of claims 13-18, wherein the agent comprises small interacting RNA (siRNA) and / or microRNA (miRNA).

20. The composition of any one of claims 13-19, wherein the agent comprises an inhibitor of epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus oncogene homolog (KRAS), Glul, and / or Zinc Finger E-Box Binding Homeobox 1 (ZEB1).

21. The composition of any one of claims 13-20, wherein the agent comprises an inhibitor of EGFR.

22. The composition of any one of claims 13-21, wherein the agent comprises an inhibitor of Glul.

23. The composition of any one of claims 13-22, wherein the agent comprises an inhibitor of ZEB 1.

24. The composition of any one of claims 13-23, wherein the agent comprises an inhibitor of KRAS.

25. A method of delivering an agent (e.g., a therapeutic agent) to a tumor of a subject, the method comprising: administering the compositions of any one of claims 13-24 to the subject; wherein the population of nanohydrogels is passively inducible to self-agglomerate to form an aggregated cluster of nanohydrogels via the aberrant microvasculature of a tumor microenvironment.

26. The method of claim 25, further comprising: allowing the agent to be released from the aggregated cluster of nanohydrogels.

27. A method of treating cancer in a subject, the method comprising: administering the compositions of any one of claims 13-26 to the subject; wherein the population of nanohydrogels is passively inducible to self-agglomerate to form an aggregated cluster of nanohydrogels via the aberrant microvasculature of a tumor microenvironment; and allowing the agent to be released from the aggregated cluster of nanohydrogels.

28. The method of claim 27, wherein the cancer is ovarian cancer, colorectal cancer, lung cancer, or breast cancer.

29. The method of any one of claims 27-28, wherein the cancer is ovarian cancer.

30. The method of any one of claims 27-28, wherein the cancer is colorectal cancer.

31. The method of any one of claims 27-28, wherein the cancer is breast cancer.

32. The method of any one of claims 27-28, wherein the cancer is lung cancer.

33. The method of any one of claims 27-32, wherein the cancer is a cancer having an undruggable cancer-promoting gene.

34. The method of claim 33, wherein the undruggable cancer-promoting gene comprises one or more of c-Myc. APC, BRAF, and / or KRAS.

35. The method of any one of claims 27-34, wherein a tumor selectivity of the nanohydrogel is 1% or greater (e.g., 1.5% or greater, 2% or greater, 2.5% or greater, 3% or greater, 3.5% or greater, 4% or greater, 4.5% or greater, 5% or greater, 5.5% or greater, 6% or greater, 6.5% or greater, 7% or greater, 7.5% or greater, 8% or greater, 9.5% or greater, 10% or greater, 10.5% or greater, 11% or greater, 11.5% or greater, 12% or greater, 12.5% or greater, 13% or greater, 13.5% or greater, 14% or greater, 14.5% or greater, 15% or greater, 20% or greater, or 25% or greater).

36. The method of any one of claims 27-35, wherein a ratio of relative delivery of the nanohydrogels between the tumor and a liver of the subject is 1: 1 or more (e.g., 1.5: 1 or more, 2: 1 or more, 2.5:1 or more, 3: 1 or more, 3.5: 1 or more, 4: 1 or more, 4.5:1 or more, 5:1 or more, 5.5: 1 or more, 6: 1 or more, 6.5:1 or more, 7:1 or more, 7.5:1 or more, 8:1 or more, 8.5: 1 or more, 9:1 or more, 9.5:1 or more, 10:1 or more, 10.5:1 or more, 15:1 or more, 20:1 or more, or 25:1 or more).

37. The method of any one of claims 27-36, wherein 2.5% or more of the population of nanohydrogels is retained after 24 hours from an initial dose (e.g., 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 10% or more).

38. The method of any one of claims 27-37, wherein the administration of the composition comprises administering a first composition comprising nanohydrogels having a first therapeutic agent and a second composition comprising a second therapeutic agent.

39. The method of claim 38, wherein the first composition and second composition are administered concurrently.

40. The method of any one of claims 38-39, wherein the first therapeutic agent and the second therapeutic agent are the same.

41. The method of any one of claims 38-39, wherein the first therapeutic agent and the second therapeutic agent are different.

42. The method of any one of claims 38-40, wherein the first therapeutic agent comprises an RNAi agent and the second therapeutic agent comprises a chemotherapeutic agent.

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