Tumor agnostic drug delivery with dynamic nanohydrogels

Domain: chemistry
Published: 2026-01-07
Editorial outcome: major_revision

Abstract
RNA interference (RNAi) holds unique potential as a clinically viable modality to pharmacologically regulate oncogenes in sequence-specific manner. However, systemic delivery of RNAi to tumors encounters myriad obstructions, and strategies to overcome such barriers have largely consisted of academic demonstrations with few approaches reaching patients. Here we report the development of a self-agglomerating nanohydrogel (SANGs) platform that selectively localizes to tumor tissue, is efficiently internalized by cancer cells, is agnostic to RNAi payload, and achieves functional suppression of multiple oncogene targets. After intravenous injection, SANGs preferentially accumulate and are retained in primary and metastatic loci in four aggressive cancer models in rodents. SANGs deliver multiple RNAi payloads that significantly suppress oncogene expression and sensitize previously resistant tumors while being safe and well tolerated in simulated clinical applications across three species. We propose, and provide the first direct evidence in support of, a mechanism featuring emergent material properties by which SANGs achieve durable solid-tumor delivery without attachment of cell- or tumor-targeting ligands. Overall, the SANGs platform is an enabling technology for RNAi-based cancer therapeutics and is poised for advanced pharmaceutical development with multiple solid-tumor indications.

Introduction
RNA interference (RNAi) uses small interfering RNA (siRNA) and microRNA (miRNA) to silence the expression of target genes in a sequence-specific manner1,2. RNAi molecules hold unique potential as clinically viable modalities for manifold diseases3,4. Oncological indications are of particular interest because the vast majority of cancer-promoting genes are “undruggable”4. In order to reach its potential, RNAi molecules must be delivered to tumors, preferably after systemic injection. However, successful delivery of oligonucleotides is challenged due to their rapid clearance, nuclease susceptibility, and inability to traverse plasma membranes, resulting in insufficient localization to, and inadequate penetration of, tumors5–7.

Numerous strategies have been developed to overcome these barriers, notably by incorporation of siRNA into lipid nanoparticles (LNPs), cationic polymers, or by chemical modification7–12. While these strategies have improved delivery, trafficking is poorly understood13 and adequate design rules do not yet exist14–17. For example, LNP doses often become trapped in the liver and are not efficiently taken up by cancerous cells after systemic injection18–20. This leaves the majority of cancers untreated, especially metastases18. Cationic polymers face different challenges: they bind nonspecifically to many cell types21,22 and interact with serum components, resulting in short circulation times and toxic side effects23,24. Surface modifications can improve stability, reduce nonspecific binding and clearance, and prolong circulation, but can also diminish target cell uptake25–28. While ligand functionalization can improve specificity, it increases manufacturing complexity and costs, reduces stability, and requires prior knowledge of the cell type of interest18,19,29. Carrier-free delivery of siRNA can be achieved through chemical modification, which drastically improves in vivo stability. While this has led to several FDA-approved drugs, none have oncological indications7. Collectively, the development of a systemic, tumor-agnostic delivery system that is effective, safe, and simple to manufacture remains an important missing link for therapeutic translation of RNAi.

Here, we report the development of self-agglomerating nanohydrogels (SANGs) as a platform that achieves durable solid-tumor delivery without direct functionalization with targeting ligands. We demonstrate that SANGs enable efficient delivery of RNAi molecules in vitro and exhibit preferential accumulation and retention in primary and metastatic loci in four cancer models in a species-agnostic manner, sensitizing previously drug-resistant tumors. SANGs were found to be safe and well-tolerated in simulated clinical applications across three species. We provide a provisional mechanistic proposal to explain how SANGs operate, which takes advantage of an emergent property of these materials in vivo. Finally, we provide the first direct observation of this emergent material property under biologically relevant conditions.

Results
SANGs synthesis and characterization
Poly(methacrylamide)-based core-shell nanohydrogels are formulated as soft and biocompatible particles using a simple, two-stage free-radical precipitation polymerization method first described by Lyon and coworkers (Fig. 1a, Supplementary Fig. 1)30–32. We previously observed that nanohydrogels functionalized with tumor-targeting peptides exclusively targeted ovarian tumors33. However, instead of functionalizing the shell layer with directing ligands, we 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. 1b, c, Supplementary Fig. 1) and independently validated by the Nanotechnology Characterization Laboratory under conditions involving different solvents, dilutions, and temperatures (Fig. 1d, Supplementary Fig. 2).

Loading of siRNA molecules into these materials is performed by swelling lyophilized particles in RNA-containing buffer. SANGs do not contain a large amount of amine groups, as APMA (the amine-containing monomer) is introduced only in the shell, and only at 0.5% of the total monomer concentration during that step. Thus, oligonucleotides are unlikely to be retained by strong charge complementarity with cationic groups on the component polymer: the N:P ratio of effective siRNA-carrying SANGs particles is only approximately 0.24. Indeed, labeling of the SANGs with a large excess of dye NHS ester reagent (and therefore acylating most or all of the available amine groups) induces little or no change in siRNA carrying or delivery properties. We have not characterized the spatial distribution of RNA cargo within the core-shell structure, but given the modest change in measured zeta potential after siRNA loading (Supplementary Fig. 1), RNA is likely internalized and not surface-bound or at the core-shell interface. The observed stability of siRNA function in complete media and after pretreatment with RNase (0.03 mM) prior to transfection further supports an internalized distribution of siRNA, which would be expected to shield the cargo from the full complement of ribonucleases in FBS supplements (Supplementary Figs. 2, 9). After removal of unencapsulated siRNA, release follows a two-stage process with much of the cargo remaining entrained in the particles for extended periods30. 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. 1e). 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. 1f).

SANGs undergo endosomal uptake and escape
SANGs were taken up readily by HEY-A8-F8 cells in dose- and time-dependent fashion (Fig. 1g, h, Supplementary Fig. 3) in standard cell culture media (10% FBS) and elevated (50% and 90% FBS) serum concentrations simulating physiological conditions (Supplementary Fig. 2). Application of SANGs for increasing lengths of time (ranging from 30 min to 24 h) revealed detectable intracellular uptake by 1 h, which increased until maximal fluorescent signals were reached at 18 h (Supplementary Fig. 3). Internalization was comparable for epithelial (OVCAR3) and mesenchymal (HEY-A8-F8) type ovarian cancer cells, as well as MCF7 breast cancer cells (Supplementary Fig. 4). Internalized SANGs remained unchanged in terms of cellular localization and fluorescence intensity for at least 24 h (Supplementary Figs. 3, 4). SANG cellular uptake was shown to be energy-, clathrin-, and macropinocytotic-dependent (Fig. 2a–c, Supplementary Fig. 5) and unperturbed by inhibition of caveolar mechanisms (Supplementary Fig. 5), suggesting a combination of clathrin-mediated endosomal and macropinocytotic pathways. This conclusion was further corroborated by live-cell imaging with an endosomal marker (wheat germ agglutinin, WGA)34,35) and SANGs containing dye-labeled siRNA, showing simultaneous co-localization of all components in HEY-A8-F8 cells at early time points up to six hours (Fig. 2h, Supplementary Fig. 6). After 18 h, however, siRNA and SANGs were observed to be distributed throughout the cytoplasm (Fig. 2d-f, Supplementary Figs. 2, 6, 7, and Supplementary Movies 1, 2), with low correlation (R=0.142±0.16, n=24) between siRNA and endosomes, whereas no siRNA signals were detected in siRNA-only experiments (Supplementary Fig. 8). These data suggest an efficient process of endosomal escape and release from the particles (Fig. 2g, Supplementary Movie 3).

Blockade of epidermal growth factor receptor (EGFR) kinase activity has been used for cancer therapy. However, other kinase-independent functions of EGFR also mediate pro-survival and chemoresistance, so siRNA-mediated knockdown should be able to achieve levels of therapeutic efficacy in accessible to kinase inhibition alone. Consistent with the apparent escape of siRNA to the cytosol, efficient suppression of EGFR mRNA and protein (Supplementary Fig. 9) was observed in overexpressing HEY-A8-F8 cells after treatment with SANGs formulated with siRNA (20 nM) against EGFR and (separately) against KRAS, resulted in significant mRNA knockdown, in each case to a degree similar to that observed using the same concentration of siRNA delivered with lipofectamine (Fig. 2i). The same formulation substituting the miRNA-429 induced phenotypic epithelial-to-mesenchymal transition and target mRNA (ZEB1) suppression. SANGs containing scrambled siRNA had no effect (Supplementary Fig. 9).

SANG biodistribution and retention in mouse and rat
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 was engineered to express luciferase and thus could be readily followed by bioluminescence imaging (BLI).

Within 30 min following i.v. injection in murine ovarian and breast cancer models, SANGs accumulated in regions with higher BLI signal indicating presumptive tumor allocation (Fig. 3a, Supplementary Fig. 10). 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. 3a). By 24 h, the specificity of biodistribution improved out to 72 h (Fig. 3b) 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 targeted (YSA-functionalized) nanohydrogels33 and repeated in Supplementary Fig. 11, indicating that the targeting ligand is irrelevant. SANGs remained closely associated with BLI signals 7 days after i.v. delivery, indicating preferential retention in tumors (Fig. 3c).

Analysis of excised tissues confirmed SANGs rapidly reached tumors, reaching 5.1±1.2-fold increase by 4 h and a maximum (approximately 200-fold above background) at 72 h (Fig. 3c, Supplementary Fig. 12). While significantly reduced compared to peak, we find SANGs were retained in primary tumors 7 days after systemic delivery (Fig. 3c). In contrast, intravenous infusion of free siRNA (20 µM) gave significantly lower tumor localization at 3 and 7 days (1.14±0.02 and 2.29±0.17 fold above background, Fig. 3c)36. Quantitative analysis of SANG biodistribution to major organ systems confirmed that non-cancerous organs remained largely devoid of SANG signal out to one week, with the exception of a transient increase detected in the kidney (Fig. 3d, Supplementary Fig. 12b).

To test the metastatic localization capacity of SANGs, a limitation of existing RNAi delivery platforms37, we modeled late-stage ovarian cancer by IP administration of HEY-A8-F8 cells. After confirming tumor induction and extensive abdominal metastasis, we administered SANGs intravenously and quantified biodistribution as described above. We observed expansive tumor load across the spleen, GI-tract, liver (Fig. 3e, f), and occasionally epidural metastasis (Supplementary Fig. 13), and corresponding rapid (4 h) and highly specific accumulation of SANGs in diffuse metastatic lesions (Fig. 3e). The temporal biodistribution in metastatic loci closely mirrored that of primary tumors, independent of organ (Fig. 3g), while signal in the non-cancerous portions of organs remained largely devoid of SANG signal (Fig. 3d–f, Supplementary Figs. 12, 14).

Co-localization analysis of ex vivo SANG fluorescence and tumor bioluminescence (Fig. 3h) showed a strong association between SANGs and diffuse metastases (R=0.84, p<0.001, Supplementary Fig. 14) 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 localize to both primary and metastatic murine tumors, are retained for extended periods of time, and have minimal off-target accumulation (Fig. 3d).

To quantitatively assess SANGs biodistribution in off-target tissues, we compared the cellular distribution of SANGs to that of a well-documented liver-targeting LNP formulation based on the lipid LP01 (Fig. 3i)38. In separate cohorts of control (C57BL/6) mice, we intravenously injected SANGs or LNP-LP01 (n=4 and n=3 independent biological replicates, respectively), each formulated to carry AlexaFluor-647 labeled siRNA against EGFR. After three hours, CD31−CD45− (epithelial and stromal), CD31+CD45− (endothelial), and CD31-CD45+ (leukocyte) cell populations were isolated from liver and kidney, and the number of those cells carrying the AlexaFluor dye was quantitatively determined. As expected, significantly more cells extracted from liver in LNP-LP01 treated animals were dye labeled compared to PBS (1X, pH 7.4) controls (Fig. 3j). 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. 3h). In contrast, we found only 4% of CD31−/CD45− renal cells from SANGs treated animals to contain significant amounts of dye relative to PBS (1X pH 7.4) treated controls (Fig. 3j). Collectively, across all cell-types studied, we found 10–30 times fewer dye-labeled cells in SANGs treated animals compared to LNPs (Supplementary Fig. 15), thereby verifying whole organ and confocal microscopy biodistribution data (Supplementary Fig. 15). 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 (Supplementary Fig. 15).

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 distribution to the diffuse tumors (Fig. 4a, Supplementary Fig. 16). 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 elimination (Fig. 4a). Ex vivo analysis of colon with diffuse adenocarcinomas (Fig. 4b) showed detectable SANG signal at 4 h, increased signal at 24 and 48 h, and a maximal recorded value at 7 days (Fig. 4c); mouse biodistribution peaked at 72 h (Fig. 3c). As in mouse, SANGs remained largely absent from other major rat organs with the exception of a nominal increase detected in liver and in the kidney (Fig. 4c) which was confirmed by confocal microscopy (Fig. 4d). Intravenous delivery of SANGs (1 mg kg–1) to lung tumor (GFP-MDA-MB-231) and non-tumor-bearing SRG rats showed significantly higher SANG delivery to tumor-bearing animals (Fig. 4e). SANGs tumor biodistribution progressively increase over time (Fig. 4f), consistent with in vivo behavior in mice with ovarian cancer and rats with colorectal cancer and correlated with tumor fluorescent intensity, a proxy for tumor load (Fig. 4g, h).

In vivo pharmacokinetics and clearance studies revealed an apparent bi-exponential blood distribution half-life (α) of approximately 2.5 h for SANGs and an elimination half-life (β) of ~13.4 h (Supplementary Fig. 17) in rats. While this value is comparable to that of other nanoparticle formulations36,39, SANGs behave differently in at least one important respect. We found 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 tissues such as fat, to be released over time.

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. 5c, Supplementary Fig. 18). A similar result was observed in mice with orthotopically implanted ovarian carcinomas (HEY-A8-F8) and breast cancers (MDA-MB-231) (Supplementary Figs. 10, 11) as well as in rats with advanced colorectal cancer, where we saw strong co-localization with EGFR-overexpressing tumor cells (Supplementary Fig. 18). In all cases, the extravasated distribution was retained for at least 7 days. Across murine ovarian (HEY-A8-F8) and rat colorectal cancers, we found that a single intravenous infusion of SANGs loaded with siRNA against EGFR or KRAS resulted in dose-dependent reduction in mRNA (Fig. 5a, b) and protein expression (Fig. 5c–e) in tumor tissue. The therapeutic efficacy of SANGs was then tested in mice with tumors established from HEY-A8-F8 ovarian carcinoma cells in a single-dose efficacy study. Enhanced expression of EGFR by these cells is correlated with drug resistance, and is a well-established model40,41. We treated groups of mice with two independent doses of SANGs, 24 h 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. Intraperitoneal cisplatin was then delivered 24 h later. Cisplatin alone was used as the standard of care control. All animals were euthanized six days after treatment to measure tumor size. Improvements relative to drug alone were evident from significant inhibition of tumor growth and reduction in tumor weight (Supplementary Fig. 18). 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.

Systemic delivery of SANGs is minimally toxic
A variety of tests revealed no significant toxicity associated with high doses (40x anticipated starting human dose) of either empty or scrambled miRNA/siRNA-loaded SANGs. These included observations without bred mice and NOD-SCID mice with ovarian tumors including delivery of cisplatin (Supplementary Fig. 19). A repeat-dose tolerability study of high-dose SANG-siRNA and co-infused oxaliplatin in rats with advanced colorectal cancer42 produced no mortality or any clinical signs of distress while weights recovered back to baseline by study completion (Supplementary Fig. 20). A maximal tolerable dose study in rats revealed only minor changes in blood chemistry at 6 and 24 h (Supplementary Fig. 20) and no difference from controls in histopathology of major organs for the three escalating doses (Supplementary Fig. 20 up to 96x anticipated starting human dose). Finally, a single acute dose study in an adult female (73 kg) Yucatan swine showed no clinically meaningful deviances out to 6 h 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 min (Supplementary Fig. 20). Thus, SANGs induce 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.

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. Five 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 µM (Fig. 6a, b), which revealed an interesting dynamic behavior. At the lowest three concentrations studied, the number and size distribution of SANGs closely matched the values predicted by a linear dependence of concentration on the number of particles, expected for any solute that does not undergo changes in form with changes in concentration (Fig. 6b). At higher SANG concentrations, however, progressively smaller numbers of SANG particles were observed, with increasing sizes and size distributions (Fig. 6b, Supplementary Fig. 21). 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 concentration (52 nM), but clear evidence of larger and less regular aggregates at 156 nM (Fig. 6c, Supplementary Fig. 21). When concentrated SANGs samples were re-diluted, much of the material returned to the original size range on average as indicated with DLS (Supplementary Fig. 21) while larger aggregated states were detected with TEM (Fig. 6a).

Parallel diffusion-ordered NMR measurements allowed us to detect 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. 6d, Supplementary Fig. 22). The former parameter indicates slower particle movement in solution, and the latter reflects restricted molecular motion43–45, both consistent with nanogel agglomeration in solution. This phenomenon was directly visualized by liquid-phase transmission electron microscopy (LP-TEM), allowing us to directly measure SANG behavior with nanometer-scale and sub-second resolution in a liquid environment. Liquid cell chambers with 150 nm apertures were used to provide adequate room for particle motion. SANGs were readily detected above background and demonstrated clear evidence of nanogel agglomeration throughout every field of view studied. SANGs were observed to ‘snap’ together in long chains and islands similar to structures observed with negative stain TEM (Fig. 6a), much larger and less mobile at higher concentration and retaining their close association as they rotated across the field of view (Fig. 6e, Supplementary Movie 4). This real-time, nanoscale evidence of nanogel agglomeration in naturalistic conditions provides the first direct observations of the emergent SANG materials property.

To test if nanogel aggregation occurs in vivo, we sequentially intravenously infused two populations of SANGs conjugated to different fluorophores in rats with advanced colorectal cancer. If SANGs do not agglomerate in vivo (and if not every cell is flooded with particles), fluorescent signals from both populations would co-localize at a nominal frequency determined by chance. Conversely, co-localization to a greater degree would indicate that agglomeration does indeed occur prior to or during tissue/cell accumulation. Fluorescent signals from SANGS of both colors were readily detected (Fig. 6f, Supplementary Fig. 23) and found to be co-localized significantly more often than that expected by chance. Furthermore, colocalized deposits (both colors) were significantly larger (~74-fold) than non-colocalized (single color) SANGs (Supplementary Fig. 23). A very stable, non-aggregating comparator, the Qb virus-like particle (Qβ-VLP), was dye-labeled in the same manner (Supplementary Fig. 24) and was found in its expected location (liver)46 with random co-localization frequency (Fig. 6g). Collectively, results from these five methodologically independent experiments are consistent with the hypothesis that environmentally-responsive aggregation is at least partially responsible for the preferential tumor delivering property of SANGs (Fig. 6h).

Characterization of SANGs biomolecular corona
Given the role that biomolecular coronas can play in biodistribution, we tested if protein coronas form on SANGs (Fig. 7a). Quantitative proteomic study of the protein corona of SANGs in rat serum compared to coronas formed on LP01 nanoparticles, a well-known drug delivery vehicle38, revealed significant differences between the platforms. LP-01 coronas contained significantly more proteins, present in much greater abundance, than those associated with SANGs, which were present in amounts only marginally higher than background (nonspecifically bound proteins) (Fig. 7b). Figure 7c illustrates the distribution of distinguishing corona proteins detected after filtering out background plasma proteins (FDR<0.001 and >1.5-fold-change relative to plasma sample), with protein identities listed in Supplementary Data 1. Hierarchical clustering further emphasized the stark difference between LP01 and SANG protein coronas, showing shared abundances and compositions of only one of eight clusters containing 6 of 115 proteins (Fig. 7d, Supplementary Data 2). Thus, SANGs develop a significantly less dense (in number and abundance) and compositionally distinct protein corona relative to LP-01. Determining the role played by the protein corona on SANGs function will be the focus of future studies.

Methods
Animal studies
Six- to eight-week-old female NOD.CB17-Prkdcscid IL2rgtm1/BcgenHsd (B-NDG: Envigo), NOD.CB17-Prkdcscid/NCrCrl (NODSCID), C57BL/6, and CD-1 Mice (Charles River) were maintained at the Georgia Institute of Technology’s Physiological Research Laboratory in individually ventilated and watered cages kept at negative pressure. Immunodeficient animals were housed under pathogen-free conditions according to Association for Assessment and Accreditation of Laboratory Animal Care guidelines. The mice were kept in rooms on a 12 h light/dark cycle with ambient temperature between 22.8 and 23.9°C with 30–40% relative humidity. The experiments were only performed during the light phase. Sterile food (Alfalfa Free Imaging Lab Diet) and sterile water was provided to mice ad libitum. The mice were acclimatized for at least 6 days before the beginning of the experiments. The animals were randomly distributed among the experimental groups. Researchers were blinded to the animal group allocation during data acquisition. The animals were euthanized by isoflurane overdose (5%) and exsanguination.

Six-month male and eight-month-old F344/NTac-Apcam1137 (Pirc) and F344 rats (Rat Resource & Research Center), ten-to fourteen-week-old Sprague Dawley-Rag2em2heraIl2rgem1hera/HblCrl (SRG), Sprague Dawley (Charles River), Sprague Dawley HsdSage:SD-Rag2em1sage (Rag2) (Envigo), and were maintained at the Georgia Institute of Technology in individually ventilated and watered cages kept at negative pressure. Immunodeficient animals were housed under pathogen-free conditions according to Association for Assessment and Accreditation of Laboratory Animal Care guidelines. The rats were kept in rooms on a 12 h light/dark cycle with ambient temperature between 22.8 and 23.9°C with 30–40% relative humidity. The experiments were only performed during the light phase. Sterile food (Alfalfa Free Imaging Lab Diet) and sterile water were provided to rats ad libitum. The rats were acclimatized for at least 6 days before the beginning of the experiments. The animals were randomly distributed among the experimental groups. Researchers were blinded to the animal group allocation during data acquisition. The animals were euthanized by isoflurane overdose (5%) and exsanguination.

Ten-to fourteen-week-old Sprague Dawley rats with chronically catheterized left jugular veins (Charles River) were used for pharmacokinetic and toxicity studies and handled as described above, with the following exceptions. Rats were singly housed to avoid disruption of the Vascular Access Buttons™ (Instech), and catheter patency was maintained by flushing (heparinized saline 500 units/mL) and locking (heparinized dextrose: 500 units/mL in dextrose 50%) catheters every 5–7 days.

All animals were cared for according to the Georgia Institute of Technology Physiological Research Laboratory policies and under ethical guidance from the university’s Institutional Animal Care and Use Committee (IACUC: A100128) or Global Center for Medical Innovation (GCMI) T3 Labs with Institutional IACUC: GT94Rr/A100632) following National Institutes of Health guidelines.

A sexually mature (6-month) female Domestic Yorkshire Crossbred Swine weighing 73 kg was studied at Global Center for Medical Innovation (GCMI) T3 Labs with Institutional IACUC approval. The Test Facility is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and registered with the United States Department of Agriculture to conduct research in laboratory animals (T311P/A1004210). Before treatment, a physical exam and clinical assessment were performed. After the swine was deeply anesthetized, an acutely placed jugular catheter was placed with ultrasound guidance for slow infusion of SANGs and serial blood sampling. At the conclusion of the study, the swine was euthanized via intravenous administration of KCL 1–2 meq/kg under a deep plane of anesthesia.

Tumor cell lines and cancer model induction
HEY-A8 gifted by Dr. Gordon B. Mills, (MD Anderson Cancer Center, Houston, TX) were stably transfected with pGL4.51 Luciferase Reporter Vector (Promega). The resulting HEY-A8-F8 cell lines and MDA-MB-231-luc2 (SC059-Puro) TNBC cell lines (GeneTarget) were maintained in RPMI 1640 (Mediatech) supplemented with 10% FBS (Fetal Bovine Serum; Atlanta Biologicals) and 1% antibiotic-antimycotic solution (Mediatech). Media were supplemented with Geneticin G418 sulfate (GIBCO) and Puromycin (GIBCO) selective agents to maintain Luciferase expression. Adherent monolayer cultures were maintained at 37°C in 5% CO2.

For the establishment of the mouse TBNC model, 1×104 cells/50 µL saline were subcutaneously injected into the fourth mammary fat pads using a 27 gauge × 1/2 inch needle. For the establishment of the mouse primary OC model, 4×105 cells/100 µL saline were injected into the right ovary. For the establishment of the mouse metastatic OC model, 4×105 cells/100 µL saline were injected into the right lower quadrant of the abdominal cavity using a 27 gauge × 1/2 inch needle.

For the establishment of rat metastatic lung cancer (MDA-MB-231) and OC models, between 1–2×106 cells/100 µL were injected in the right lower quadrant of the abdominal cavity using a 27 gauge × 1/2 inch needle.

SANG synthesis
SANGs were synthesized via emulsion precipitation polymerization. In a typical core nanogel synthesis, N-iso-propylmethacrylamide (NIPMAm) monomer (117.8 mM), N, N’-methylene-bis (methacrylamide) (BIS) cross-linker (6.2 mM) and sodium dodecyl sulfate (SDS) surfactant (8 mM) were dissolved in deionized H2O. The solution was stirred under nitrogen, brought to 70°C, and treated with 0.5 mL of a 10 mg/mL aqueous ammonium persulfate (APS) solution to initiate polymerization (800 mM). The reaction was carried out at 70°C for 4 h with vigorous stirring under nitrogen. The resulting core particle reaction solution was passed through a 0.2 µm filter. Core particle synthesis was verified by DLS as having an unimodal distribution between 47–57 nm. The core nanogels were used as a seed for the addition of a shell layer. A mixture of NIPMAm (48.75 mM), BIS (1.0 mM), and aminopropyl methacrylamide (APMA, Polysciences, 0.25 mM) prepared in deionized H2O (dH2O) was added to the core seed. When the temperature stabilized at 70°C, the reaction was initiated by adding a 0.5 mL aliquot of 0.05 M APS, and was allowed to proceed for 4 h under N2 atmosphere. After cooling to room temperature, the reaction mixture was filtered through a 0.2 µm filter and the size of the crude core-shell nanogels was measured by multi-angle dynamic light scattering. SANGs were then purified and concentrated using a VivaFlow 50 100 kDa MWCO tangential-flow filtration (TFF) cartridge (Sartorius, circulation flow rate 10 mL/min, transmembrane pressure ≈2 bar), retaining the SANGS within the retentate while allowing small-molecule reagents to be removed. During the initial cleaning phase, the circulating volume was maintained by continual addition of dH2O, corresponding to 10x volume of washing (for example, a 50 mL reaction volume was washed with 500 mL of dH2O). After cleaning, no additional dH2O was added, and the solution was allowed to concentrate by ~10-fold. Purified SANGs were re-checked with DLS, and then concentrated SANG solutions were flash frozen and lyophilized under vacuum. When reconstituted in aqueous media, SANGS swell back to their original size.

SANGs labeling
SANGs that were used for in vitro delivery and in vivo biodistribution studies were labeled with AF-488-NHS, Texas Red-NHS, or LiCOR IRDye® 800RS NHS ester following the manufacturer’s protocol for labeling high molecular weight molecules, depending on the study needs. For example, SANGs were reconstituted in pH 8.4 PBS (1X) at 52 nM and were incubated with 1 mM LiCOR IRDye® 800RS NHS ester overnight at room temperature. Unreacted dyes were then removed by Pierce® ZebaTM Desalting Spin Columns following the manufacture’s protocol.

siRNA species and labeling
Pre-miR™ miRNA Precursor for miR-429 (Cat #: AM17101

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_3": "The authors proposed the self-agglomerating nanohydrogel (SANGs) as a platform for RNA delivery. They demonstrated that SANGs preferentially accumulated and were retained in primary and metastatic loci in three aggressive cancer models in rodents. It is noteworthy that this was achieved without the aid of targeting ligand. As a proof of concept, the authors demonstrated suppression of oncogene expression and sensitized previously resistant tumors applying SANGs with RNAi payloads. The safety profile was also evaluated. Specifically in this revised manuscript, the authors have also addressed most issues I and other reviewers commented on. I suggest a Minor Revision before it can be published for Nature Communication.\n\nThe authors should show Kaplan–Meier survival curves for all therapeutic studies they have performed. “Dead time” should be defined as the time point after tumor inoculation when the mice were actually found dead or euthanized according to veterinary guidelines (e.g., they were moribund/unable to reach food and/or water, or when the tumor reached a diameter of more than euthanization point or when the enlarging tumor ulcerated.).", "reviewer_4": "In the manuscript entitled ‘Tumor Agnostic Drug Delivery with Self-Agglomerating Nanohydrogels (SANGs)’ by Housley et al., the authors have designed self-agglomerating nanohydrogels (SANGs) for the systemic delivery of RNAi to solid tumors. They show that the SANGs are taken up by cancer cells and preferentially accumulate in primary and metastatic loci in three different cancer models after IV injection. SANGs can deliver a variety of RNAi payloads and achieve functional suppression of multiple oncogenes.\n\nThe manuscript is nicely designed and well-written. However, I express my concerns about relevant topics below:\n\nComment 1\nThe authors add pre-formed SANGs with siRNA. Do SANGs entrap the siRNA, or is it only absorbed on the surface? Did the authors test the stability of the siRNA-loaded SANG with RNase? This will help understand the formulation stability.\n\nComment 2\nThe authors answered reviewer #1 as follows: ‘High dose was selected based upon allometric scaling to the anticipated first human dose’. I have a few relevant comments on this topic:\n1) Is the manufacturing of SANGs scalable? Can they produce GMP SANGs? Are the materials used for SANGs FDA-approved? This is a relevant step in facilitating the translation of the formulation into humans. Not sure about the toxicity and biodegradability of permanent cationic polymers. Considering the selected materials and manufacturing process, I am unsure if this system will ever be able to enter clinical studies.\n\nComments 3\nIt would be fantastic if a nanoparticle could accumulate only in the tumor, avoiding completely healthy tissues. I do not believe it.\nI fully agree with the reviewer. The EPR is an archaic view of delivery to the TME. Why should these particles accumulate only in tumors? This is not fully clarified. Please, provide a concrete explanation.\n\nComment 4\nIn the table of Supplementary Figure 1C, which solvent is used for the size, PDI, and ζ-potential measurements of the SANGs? I see a relevant difference between these values (Table 1C) and the DLS size distribution reported in supplementary Figures 2A and 2B. Here, size and PDI look much larger; such values are not ideal for IV injections. Further, the curve reported in Figure 2C (ζ-potential) shows multiple populations of the nanohydrogels. Are siRNA-loaded SANGs stable in isotonic saline solutions?", "reviewer_5": "Specific Comments:\nOverall, the authors have generally addressed the reviewer’s concerns with thoughtful responses. However, several critical issues remain unresolved and require clearer, data-supported explanations to enhance the scientific rigor of the manuscript.\n\n1. (Refer to previous Q18) The simple assumption that the encapsulation efficiencies of three different siRNA molecules are similar seems to be made without supporting data. Experimental validation or, at least, citation of relevant literature is necessary to provide a more robust and scientifically grounded justification.\n\n2. (Refer to previous Q24, Q28, and Q30) The tumor accumulation mechanism of SANGs appears not to be clearly supported. The authors claim concentration-dependent “self-agglomeration” drives tumor retention, but the aggregation rate of particles both in buffer and in vivo seems not to be provided. Evaluating aggregation behavior under serum-like or tumor-relevant conditions over time is necessary to reliably predict the kinetics of tumor localization and retention. Without such data, the claim regarding the mechanism may remain speculative.\n\n3. (Refer to previous Q33) The authors’ explanation regarding the use of the term “environmentally-responsive” in the Discussion section appears not to be adequately supported. Claiming environmental responsiveness without identifying the mechanism of self-agglomeration, while speculating that TME factors may be involved without supporting evidence, could be overly presumptive. Clarification of the material’s specific responsiveness under tumor-relevant conditions or use of more accurate terminology is essential.", "reviewer_1": "Housley et al. have designed self-agglomerating nanohydrogels (SANGs) that overcome the myriad obstructions associated with systemic delivery of RNAi to solid tumors. They show that the SANGs platform is efficiently internalized by cancer cells, is amenable to a variety of RNAi payloads and achieves functional suppression of multiple oncogenes. They show that SANGs preferentially accumulate in primary and metastatic loci in three different cancer models after IV injection. The SANGs were found to be safe and well tolerated across three species, and a new mechanism for this system is proposed. Overall, this is a very comprehensive, well-designed study and well-written manuscript that is an exceptional example of tumor targeting particles with efficacy in a variety of cancer models. I have the following queries and suggestions:\n\nPage 3: What is the mechanism for which the SANGs remain co-localized with the endosome, but the siRNA disperses through the cytoplasm? Did you use an siRNA-only control in these experiments to confirm that it’s not diffusing from the SANGs prior to cellular entry?\n\nPage 5: Need to define ‘high-dose’ and how this was decided for the toxicity study.\n\nPage 6: Was the co-localization of SANGs observed in healthy tissues, or was this uniquely observed in tumors? Suggest characterizing this in other tissues in these mice to confirm this mechanism. Is the agglomeration observed in the blood vessels as proposed in the schematic? Unclear if this is based off speculation or data, but should be possible to quantify the distribution in blood vessels in the tumor.\n\nPage 6: Suggest including experiments in vitro to confirm that the agglomerated SANGs are indeed taken up by clathrin-mediated endocytosis and micropinocytosis as indicated in the discussion.\n\nPage 6: The EPR is an archaic view of delivery to the TME (e.g. https://www.nature.com/articles/s44222-024-00154-9 and https://www.nature.com/articles/s41563-023-01630-0); suggest reviewing recent literature and reframing this postulation.\n\nFigure 4: Need to add/label descriptions for b-c).", "reviewer_2": "1. In the introduction, the authors use LPNs and cationic polymers as examples of other nanoparticle delivery systems of RNAi and list their drawbacks. Many of these references are not within the past 5 years and do not represent the work of many key groups that study these materials. For example, the reference listed for cationic polymers (ref 19) is a paper that studies liposomes prepared soley with lipids. Additionally, the authors equate liposomes to lipid nanoparticles in the introduction, which suggests a cursory understanding of the field or a hastily written manuscript. The groups of Hai-Quan Mao (JHU), Paula Hammond (MIT), Jordan Green (JHU), Kathryn Whitehead (CMU), and Kazunori Kataoka (Univ. Tokyo) (to list a few) study these types of materials whose works are not cited here.\n\n2. Authors describe the formulation of nanogels using an emulsion precipitation polymerization technique. On the shell of the final particles are free amines that are used for subsequent fluorophore NHS ester labeling. What percent of free amines are exist after the reaction? This is not quantified (but potentially could be as methods detail a wash step to remove unreacted dyes). The free amine on the particle surface is impacts the described mechanism of siRNA loading. What is the driving force for siRNA to diffuse into nanogel particles as they are swelling? If a high percentage of free amines exist on the particle surfaces, the siRNA could be simply binding to these amines and siRNA would only be coating the particle surface and not actually be loaded inside. This is consistent with the zeta potential measurements provided in the SI, which measure neutral to slightly anionic for the loaded particles. A zeta potential measurement of the particles before and after siRNA loading could serve as evidence to whether siRNA molecules have or have not coated the nanogel surfaces. However, no zeta potential measurement before loading is provided. Related, why report an N:P ratio of the particle amines to RNA phosphates if charge neutralization and complexation are not factors in siRNA encapsulation?\n\n3. Relating to the previous point, the Supporting Figure 2 shows intensity distributions of empty and siRNA-loaded nanogel particles by DLS. The size of the particles measures larger on average after siRNA loading, which would support siRNA binding to the surface. It is unclear whether the data presented in SI Figure 2 is for a singular batch or averaged across several batches, but regardless, the extremely low PDI (~0.02) would suggest that the differences measured in size is likely not simply due to measurement error. Please elaborate on the siRNA loading mechanism and whether or not electrolyte complexation is occurring for the loading of RNAi cargo.\n\n4. Authors detail that physicochemical properties of the nanogels were separately validated by the National Characterization Lab whose data are reported in Supporting Figure 4; do the authors mean Nanotechnology Characterization Lab? In the main text, the authors state that particle properties were validated “under a variety of solvent, pH, dilutions, and temperature conditions.” However, the data provided only detail one temperature (25°C), one pH value (~7.25), two dilution factors (10- and 100-fold), and only two solvents (PBS buffer and 10 mM NaCl). This seems shallow compared to the statement given in the main text. In Figure SI4, what is the difference between the 385-1 and 385-2 formulations? No description is provided. At any rate, to make the claim that a variety of conditions were examined, the authors should an extensive set that truly test their hypothesis.\n\n5. Figure 1E details long-term stability of nanogel particles over the course of 60 days in aqueous conditions at different temperatures. Authors need to be specific about the solvent conditions (salt concentration, pH, etc.) used for this study. Is Figure 1E monitoring size over time or something else? The figure caption states ‘chemical stability’ which is extremely vague and there is no methodology provided to indicate what is actually being measured. What concentrations were the particles incubated during the study? Later, the authors show that the particles aggregate at high concentrations. Can these suspensions be considered a stable colloid? The zeta potential is almost zero, so little to no electrostatic repulsion likely occurs between neighboring particles. What prevents particles from aggregating if production of the particles were to be scaled-up for clinical production? This information is important in the context of translation, which is expounded in the Discussion.\n\n6. Given that these particles are to be administered by IV injection, the serum stability of the particles should be monitored. Do serum protein adsorb to the surface of the particles? Would that contribute to their aggregation/’self-agglomerating’?\n\n7. Why is there an observed increase in SANG fluorescence in endosomes after treatment of cells with MβCD (up to 2 mM) in SI Figure 7?\n\n8. Live cell imaging shows that SANGs themselves are trapped in endosomes (punctate fluorescence and overlap of signals with endosome labels). The punctate fluorescence of the SANGs does not diminish across the time points studied, so it appears that they remain trapped in the endosomes. The main text states that “After 18 hours, siRNA and SANGs were observed to be distributed throughout the cytoplasm (Fig. 2d-f and Supplementary Fig. 6)”. However, imaging reveals that siRNA does in fact enter the cytoplasm, but not the SANGs. What is the mechanism of siRNA release from the particles and then endosomal escape if the particles themselves remain trapped? More work needs to be done to investigate this mechanism, especially given only one particle formulation is studied here.\n\n9. The main text describes that siRNA showed ‘similar knockdown of KRAS, Glut1, and miRNA-429’. Did the authors mean ZEB1 here as is reported in SI Fig 10? Moreover, the lipofectamine positive control mentioned in the main text is missing from this data set. The reduced mRNA expression induced by the siRNA delivered by SANGs needs this comparison to better reflect the efficacy of this delivery vehicle. The gene silencing data is a very important reflection of the in vitro efficacy of the SANG platform and should be included in the main Figure 2.\n\n10. Almost all panels in Figures 3-5 and supporting figures detailing in vivo experiments lack information regarding animal species, cancer models used, administration route of SANGs, and location of tumor. Many figure captions lack any labeling at all. This severely complicates understanding the data and interpreting the results of the experiments appropriately, given several different models are used. It is highly recommended that the authors thoroughly review all figure labels and captions and update them with this information (and also include the number of replicates for each data set).\n\n11. With respect to the biodistribution data (Fig 3A), the manuscript states that ‘SANGs accumulated in regions with BLI signal indicating presumptive tumoral targeting’ in the main text (paragraph 2 of that section). This phrasing is misleading since there is a lack of tumor-targeting moiety. This is actually stated as an advantage of their delivery platform compared to other materials. This should be rephrased.\n\n12. In vivo biodistribution and siRNA delivery of untargeted and targeted (YSA-functionalized) SANGs was equivalent (SI Fig 12). Is this because protein adsorption onto the corona of the functionalized particles renders the targeting moiety ineffective, making these particles equivalent to the non-functionalized particles? This is related to the previous point above regarding particle serum stability.\n\n13. In paragraph 5 of section ‘SANG biodistribution and retention in mouse and rat’, the final sentence states “[…] SANGs rapidly target both primary and metastatic murine tumors, […]”. ‘Targeting’ is misleading as the particles lack a targeting moiety; maybe replace with ‘accumulate’.\n\n14. In comparing SANGs and LNP formulation for kidney and liver accumulation, why were CD31-CD45-, CD31+CD45-, and CD31-CD45+ cell populations selected to demonstrate particle accumulation? This is unclear. Also, it appears that SI Fig 17 is mistakenly referenced in the discussion of this data (paragraph 6 of this section, last sentence).\n\n15. The manuscript compares ovarian tumor accumulation of SANGs and LNP-LP01 formulations, showing accumulation of SANGs and little accumulation of LNP-LP01. Authors state that “these data qualitatively showcase the ability of SANGs to target solid tumors compared to a state-of-the-art delivery platform.” However, in the previous paragraph, the authors emphasize that this LNP is a “well-documented liver-targeting LNP formulation.” The comparison here seems biased as these LNP formulations are formulated for targeting the liver and would not be used in a tumor delivery context. To make comparison of SANGs tumor accumulation to a ‘state-of-the-art’ delivery system, a different LNP formulation should serve as a comparison that is actually used for delivery to solid tumors.\n\n16. Figure 3L is never referenced in the text.\n\n17. Figure 4 quality appears degraded.\n\n18. Figure 4d shows mRNA expression in vivo. Is it one singular siRNA that reduces expression of these mRNAs (EGFR, KRAS, Glut1) or is it three different siRNA molecules? Most likely, it is the former. However, this should be explained at least once in the text. If this data is reflected of 3 separate formulations, how does encapsulation efficiency compare across these different siRNA molecules into SANGs? For these studies, do the SANGs have fluorophore-labeling on their surfaces? This is related to a previous point about siRNA loading mechanism and free amine content. Without the fluorophores, the surface chemistry of the particles is different and may behave differently. This is also relevant for the miRNA loading as well. For all the efficacy studies described in the section ‘In vivo functionality of SANGs’, are the particles fluorescently labeled?\n\n19. What is ‘EMT’ (in relation to miR-429)? This is not defined in the text.\n\n20. In the context of in vivo efficacy experiments, why not load SANGs simultaneously with siRNA-EGFR and miRNA-429 and deliver as one dose? Is miRNA-429 activity prerequisite for siRNA-EGFR to induce biological effect? Do neither of these RNAi molecules suppress tumor growth/metastasis on their own? These studies appear to be missing siRNA/miRNA only treatment groups as a control.\n\n21. If these RNAi molecules serve to sensitive tumors to cisplatin, why not also incorporate cisplatin into the SANG design? Small molecule drugs like cisplatin would also benefit from delivery in nanoparticles to enhance tumor accumulation and retention while mitigating off-target effects and rapid renal clearance. This would be a more interesting particle platform and also require less repeat IV injections over the course of 3 days.\n\n22. SI Fig 22 has no labeling for the swine data.\n\n23. In the first of four methods to determine the mechanism through which the SANGs exhibit enhanced in vivo performance, the authors use negative-staining TEM to study particle aggregation in terms of size and number. Conventional TEM imaging can induce drying artifacts that influence observed particle size/morphology; for example, during drying of particle-containing solution atop a TEM grid, the local concentration can increase and promote particle aggregation that may not occur in solution.\n\n24. Does concentration-induced aggregation still happen in the presence of serum? An aqueous solution is not the same environment as that experienced in blood, so making a connection between aggregation behavior in buffer and how the particle behaves in vivo is tentative at best.\n\n25. The masking used to count particles in the TEM image analysis appears to be lumping discrete particles into a “single” particle for counting. TEM is not ideal for this type of analysis. Moreover, particle solutions at 394 nM in SI Fig 26 appear less aggregated than the image in Figure 5A which depicts particles at 156 nM. This discrepancy needs to be addressed.\n\n26. The Methods section mentions that cryogenic TEM imaging was conducted on particle samples; however, no cryoTEM data is shown in the manuscript. CryoTEM would be the better technique to analyze particle size/morphology changes with concentration/time as samples are imaged in vitrified ice.\n\n27. DLS measurements confirm particle aggregation (Figure 5C). However, how does PDI change over these concentration ranges? Are the data presented in SI Fig 27 showing average and SD of 3 separate batches? A representative size distribution by intensity would be informative to include for each concentration studied and after the re-dilution step. Moreover, it appears that the two different DLS instruments yield highly different results. Why are these results so different?\n\n28. How quickly does the particle aggregation occur that was observed with DLS? This aggregation behavior could be very problematic in the context of scale-up for clinical studies.\n\n29. DOSY NMR results are consistent with particle aggregation with increasing formulation concentration. In the main text, authors state that this aggregation is “caused at least in part by cross-linking between SANG particles as concentration increases.” By what mechanism would the particles be crosslinking?\n\n30. For the in vivo testing of nanogel aggregation, two different fluorophore-labeled SANGs are IV injected and co-localization of fluorescence is assessed in tissue slides. The manuscript states “If SANGs do not agglomerate in vivo, fluorescence 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.” It appears that this experiment lacks an appropriate control that would reflect “random” colocalization for comparing their SANG particles. In the experimental design, are these two populations injected in the same syringe or at different times? And are siRNA molecules loaded into the particles used for this experiment (see previous points above regarding RNA binding to particle surface)? As presented, this experiment does not clearly support the proposed SANG mechanism of tumor accumulation.\n\n31. In the Discussion section, authors cite references regarding tumor vasculature & microenvironment and its relation to the ‘EPR effect’ that are dated (published more than 10 years ago, ref 38-43). More recent literature discussing recent advancements in our understanding of nanoparticle trafficking to solid tumors and the mechanisms through which this happens include: Nguyen et al. Nature Reviews Bioengineering, 2024, 2, 201-213; Kingston et al. ACS Nano, 2021, 15, 14080-14094; Sindhwani et al. Nature Materials, 2020, 19, 566-575; Wilhelm et al. Nature Reviews Materials, 2016, 1, 16014].\n\n32. Of concern is that Figure 5g of the submitted paper looks almost identical to Figure 3a of Wilhelm et al. Nature Reviews Materials, 2016, 1, 16014.\n\n33. Authors state in the Discussion that SANGs are “environmentally-responsive nanostructures” but in what way? As designed, the particles appear to possess no such property. Moreover, authors state SANGs exhibit “substantial therapeutic index”, however, no therapeutic index is calculated.\n\n34. Supplementary Materials section states that there are SI Movies S1 to S3. These data files were not included with manuscript submission nor referenced in the main text.", "reviewer_3_v0": "The authors proposed the self-agglomerating nanohydrogel (SANGs) as a platform for RNA delivery. They demonstrated that SANGs preferentially accumulated and were retained in primary and metastatic loci in three aggressive cancer models in rodents. It is noteworthy that this was achieved without the aid of targeting ligand. As a proof of concept, the authors demonstrated suppression of oncogene expression and sensitized previously resistant tumors applying SANGs with RNAi payloads. The safety profile was also evaluated. Specifically in this revised manuscript, the authors have also addressed most issues I and other reviewers commented on. I suggest a Minor Revision before it can be published for Nature Communication.\n\nThe authors should show Kaplan–Meier survival curves for all therapeutic studies they have performed. “Dead time” should be defined as the time point after tumor inoculation when the mice were actually found dead or euthanized according to veterinary guidelines (e.g., they were moribund/unable to reach food and/or water, or when the tumor reached a diameter of more than euthanization point or when the enlarging tumor ulcerated.)."}, "author_response": "- We appreciate the opportunity to clarify the details of this study. It was intended as an initial investigation quantifying the efficacy of single doses on tumor size (weight as a proxy). As a result, we designed a single time point when all animals were to be euthanized to measure wet weight of tumors. Future studies will investigate the effects of SANGs (as a single agent and as a chemotherapy adjuvant) on survival. These details are included in the revised manuscript.\n\n- The process of loading SANGs with an siRNA cargo, as this reviewer indicates, relies on reconstituting lyophilized pre-formed SANGs in a solution of siRNA. The siRNA cargo is entrapped (internalized) inside the core of the polymer matrix during the reconstitution process. While we cannot completely exclude the possibility that siRNA cargos are absorbed on the surface (or at the core-shell interface) or partially exposed on the surface, we find that functional delivery of siRNA in vivo (sufficient to knockdown RNA and protein products) a good indication that the siRNA cargos are sufficiently shielded from circulating nucleases that would otherwise inactivate them, supporting the hypothesis that they are largely internalized. We have added additional discussion in the revised manuscript to ensure that we accurately convey this important point to readers.\n\nAll in vitro cell culture studies described here were performed in the presence of 10% FBS, exposing siRNA-loaded SANGs to the full complement of RNases in cell culture media. As the siRNA-loaded SANGs retained the ability to knockdown RNA targets such as EGFR, we inferred that siRNA-loaded SANGs were stable in this functional assay and therefore that siRNA is protected in or on the particles.\n\nTo explicitly test this conclusion, we pre-incubated siRNA-loaded (EGFR) SANGS with RNase (0.03 mM) for 1 hour before performing a standard transfection and knockdown assay in HEY cells. Forty-eight hours after transfection, we find no difference in EGFR knockdown relative to siRNA-loaded SANGs not pretreated with RNase. Treatment of siRNA (not entrained in SANGs) with RNase under these conditions before loading into SANGs resulted in almost complete loss of efficacy, indicating RNAse treatment was sufficient to degrade the siRNA-EGFR oligonucleotide. These results are summarized in the revised Supplemental Figure 2f.\n\n- Like the reviewer, we naturally have also considered the regulatory path for SANGs as their specific primary components have yet to be used in an FDA-approved drug, nor does a Drug Master File (DMF) exist. However, as described below, we did not receive push-back from the FDA about the SANGs components, which are generally considered to be biocompatible; similar monomers are used in other approved materials.\n\nWe also share this reviewer’s concern for the importance of safety and tolerability of SANGs. This is why we performed a substantial number of toxicity studies in mice, rats, and pig. All studies to date (which were reviewed in full by the FDA) identified little if any evidence of adverse events, even when SANG doses were escalated to ~100x anticipated starting human doses. While we acknowledge that SANGs have a minor surface charge (as indicated by zeta-potential), we consider SANGs largely neutral given the low (0.5%) amine containing content in the shell, which may help explain their relative ‘bio-inertness’ relative to cationic polymers.\n\nWhile not the subject of this manuscript, we have also explored the issues of scalability and GMP manufacture. In short, SANGs manufacturing is scalable and will require only minor additional adaptations to be fully cGMP compliant. The polymerization method is standard and essentially identical to that used for many FDA-approved polyacrylate and polyacrylamide materials. The key difference for SANGs is post-polymerization processing. In preparation for discussions with the FDA to plan a regulatory path to a first-in-human trial, we changed this step from post-synthesis ultracentrifugation to tangential flow filtration (TFF) in order to be ready for scale-up and GMP compliance. The remaining required modifications (shifting reaction vessels from glass flasks heated with mineral oil baths to water-jacketed stainless steel reaction vessels and the inclusion of data logging devices to monitor reaction conditions, processes which are currently manually performed) are anticipated to be innocuous.\n\nWe have engaged a cGMP CMO partner (nanoComposix, a division of Fortis Life Sciences based on San Diego, CA) for upcoming translational activities and have begun the tech transfer process. This involved formally vetting their facility (Quality Audit) and establishing a pipeline for transitioning the synthesis to their facility. While we have yet to perform the full cGMP synthesis, their polymer chemists report that there are no obvious roadblocks that would prevent SANG scale-up. These activities were informed in large part by our discussions with the FDA (PIND 162383) where we outlined our translational plan and received largely positive feedback about the nonclinical efficacy and safety data and clinical trial design.\n\nWe have added these comments and details about the potential scalability of SANGs to a new Supplementary Discussion section entitled: “Scalability and Translational Potential of SANGs”.\n\n- We agree with this reviewer’s hesitation and apologize for any confusion that emerged from our description of the data. We did not intend to suggest that SANGs do not go to other major organ systems; in fact, the quantification of the in vivo and ex vivo biodistribution studies (Fig 3d and Fig 4c) indicate that SANGs do go to kidneys and liver, but to a much lesser extent then that detected in tumors. We suspect that that initial biodistribution is proportional to the blood perfusion of the tissue, but then biased accumulation in tumor takes place by preferential retention in the TME. We have made these ideas clearer in the revised manuscript, in part by shifting descriptions away from the notion that SANGs “accumulate only in the tumor, avoiding completely healthy tissues” to the statement that they “accumulate in tumors while minimizing exposure to healthy tissues”.\n\n- We appreciate the opportunity to clarify this experimental detail which we failed to include in the original submission. Data in the main Figure 1 and in Supplementary Figure 1C were generated in house on our Wyatt DLS instrument in PBS. Data in the Supplementary Figure 2 was generated at the NCL with their Malvern Zetasizer Instrument. Variations in diameter of 5-10% are standard in batch-to-batch preparations. The apparent appearance of multiple populations in Supplementary Figure 2C likely results from the fact that each data point is an average of three replicate measurements at each value of zeta potential; the overall width of the distributions is similar to that of most nanoparticulate hydrogels. Instrument level details are now included in the revised main and supplementary figure legends and methods. We have not studied the stability of siRNA-loaded SANGs in isotonic saline but have done so in PBS. Data is reported in Figure 1e.\n\n- We cite the previously published report that documents encapsulation efficiencies. It should be noted that studies of loading kinetics were performed with fluorescently labeled siRNAs that were otherwise not specific to an RNA target. As siRNA are largely similar in their molecular weight and charge, their loading behavior into SANGs should be very similar to a scrambled siRNA.\n\n- We previously provided the results of multiple experiments in buffer (PBS): using DLS, NMR, and TEM documenting self-agglomeration in Figure 6, and, in the latest revision, a study of SANG dynamics under liquid-phase TEM conditions. In this set of experiments (Figure 6e), we showed with single particle resolution the agglomeration of SANGs in real time. Included supplementary movies show clear evidence of SANG particles ‘snapping together’ and staying closely associated through bulk aggregate motion (Movie S4).\n\nAgglomeration behavior was then studied in vivo with a set of experiments that utilized the statistics of random association of two populations of SANGs labeled with distinct fluorophores. The colocalization of SANG particles was then compared to a nanoparticle system that does not aggregate (virus like particles - VLP). We found that SANGs did colocalize to a greater extent than behavior driven by random chance (0.5) which is the rate of colocalization observed for VLPs.\n\n- Our use of the term “environmentally-responsive” was meant to describe the hypothesis that properties of the TME enable SANG particles to reach a sufficient threshold of concentration and collisional frequency to promote self-agglomeration, and that such coalescence favors accumulation and retention in tumor tissue. To avoid the reviewer’s confusion, which is likely to be shared by others, we have replaced the term “environmentally responsive” with more descriptive language."}, {"round": 2, "reviewer_comments": {"reviewer_4": "The authors have addressed some of the reviewers’ questions with appropriate comments and data. However, a few concerns remain unresolved and require more precise explanations and data to make the manuscript more robust and the research more reliable.\n\nComment 3\nThe mechanism by which SANG should preferentially accumulate in the tumor is not clear to me yet. The authors did not give an exhaustive explanation to this question.\n\nComment 4\nThe authors refer to the data as ‘stability in PBS’. What does it mean exactly? Does the suspension refer to which pH and salt concentration? Is it isotonic? This information should be clarified in the manuscript.\n\nComment 5\nSince the authors inject the SANG formulation intravenously, they should investigate and report the empty and RNAi-loaded SANG size stability under 90% serum conditions over time (for a few days). Are these nanoparticles stable under these conditions?\n\nFurther, in vitro transfection efficacy of the formulations might drastically decrease, passing from 10% to 90% serum-free cell media. The cell uptake might radically change, too. These results, together with size stability in serum, can support the in vivo efficacy/investigation of the formulation and make the study more robust. Did the authors carry out this set of experiments?", "reviewer_5": "The authors have adequately addressed all of my previous concerns. The revised manuscript is clearer and well-supported by additional data and analysis. I recommend acceptance of the current version."}, "author_response": "Response: We share the reviewer’s desire for greater clarity about the mechanism of tumor accumulation, a desire that is motivating follow-up studies in our laboratories. We describe in the text and Figure 5g our current conceptual framework and clearly identify assumptions or conclusions that remain to be further tested. We believe that the experimental results described in the paper provide enough new insights to be useful to the field, even if we have not answered every question. Our theoretical framework under understanding how SANGs achieve their in vivo functionality is based on the following.\n\n1. At the macro-level (whole animal), upon iv administration, SANGs distribute everywhere proportional to perfusion.\n2. When SANGs encounter a normal capillary bed, they enter at low concentration, transit the capillary bed at low concentration and exit at low concentrations.\n3. This is in contrast to what happens in the altered vasculature of the TME, where the tortured nature of the blood vessels creates slower and more turbulent blood flow. This may allow more time for SANGs particles to aggregate in regions of very slow flow or impose shear forces or increase the collisional frequency of SANGs particles in regions of flow discontinuity, each of which is likely to stimulate self-agglomeration. An analogy may be found in the pronounced tendency of blood clots to form at tubing junctions in extracorporeal blood circulation devices (citation added). Furthermore, agglomeration is a nucleated process rather than a linear one: once aggregates start to form, increased agglomeration is more and more likely. This is manifested in vitro by the threshold relationship between particle size and concentration.\n4. Agglomeration favors retention in the TME relative to other non-malignant capillary beds and results in the de novo generation of SANG depots in tumor vessels. This progressive accumulation process is supported by the slow accumulation in SANGs up to 72 hours after iv administration in mice and to 1 week in rats.\n5. SANG depots are then the driving force for extravasation down a concentration gradient into the intestinal space which is likely due to both active and passive processes.\n6. Once in the interstitial space (see Figure 5a-c &f), SANGs enter cells via clathrin-mediated endocytosis and macropinocytosis. While we observe functional siRNA delivery, we do not yet know the mechanism by which SANGs escape endosomal/micropinocytosis entrapment, which will be the focus of future studies.\n\nBecause accumulation occurs with multiple tumor types in multiple species, we believe it unlikely that a specific receptor-mediated process is responsible for cancer cell targeting. It is more likely that the dynamic behavior of SANGs enables them to respond to and become entrained in the tumor microenvironment because of aspects of its physical nature common to different solid tumors. Indeed, poly(N-isopropyl methacrylamide)s and related polymers are known to exhibit a wide variety of environmentally-responsive bulk properties, including changes in volume, solubility, and aggregation, as a function of changes in factors such as temperature, ionic strength, and concentration. Exploration of these phenomena from a physical chemistry and materials point of view is beyond the scope of this manuscript, but is the subject of ongoing studies in our laboratories.\n\nWe have included additional discussion of this provisional mechanism in the revised discussion.\n\nResponse: We used commercially purchased 1X PBS solutions which are at pH 7.4 for all use cases of PBS with the exception of NHS fluorescence decoration which required pH 8.4 for optimal reaction conditions. We have clarified these experimental details in the revised manuscript.\n\nResponse: We agree that 90% serum is more representative of the in vivo conditions encountered by i.v.-administered SANGs. As requested, we incubated SANGs in 90% serum (FBS) and collected DLS data relative to blank serum. Unfortunately, the complex nature of serum composition inhibits our ability to detect SANGs in the presence of large amounts of nanosized components heterogeneously distributed throughout the 10-500 nm size range, as shown below. These overlapping size distributions make it impossible to reliably distinguish SANGs from serum and therefore to determine their stability under these conditions, as shown below. Our in vivo data, bolstered by the additional functional study described below (also performed at the reviewer’s suggestion), provides sufficient support for SANG stability under physiologically relevant conditions.\n\nResponse: As suggested, we tested SANGs internalization and siRNA delivery in vitro in 90% and 50% serum, the latter included in case cell viability was impaired under the former conditions. As shown in the revised Supplementary Figure 2 (excerpted here), unloaded SANGs were internalized in both 50% (panel h) and 90% (panel k) serum conditions. SANGs loaded with a Cy3-labeled siRNA were internalized and released their siRNA cargo to cytosolic distribution in both media as well (panels I and l). conditions. Experiments were performed in duplicate in Hey-A8-F8 ovarian cancer cells and images were collected 18 hours after incubation, matching the conditions of Figures 1-2. These data, together with in vivo biodistribution, target engagement and functional studies support the notion that SANGs are stable in simulated physiologic and in vivo conditions to both enter cells and deliver siRNA cargo."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have addressed all the reviewers’ questions with appropriate comments and data. Therefore, I suggest the publication of this paper in Nature Communication Journal."}, "author_response": ""}]

Decision letter