Thermosensitive hydrogel releasing nitric oxide donor and anti-CTLA-4 micelles for anti-tumor immunotherapy

Exogenous GSNO promotes tumor growth and enriches CTLA-4-expressing immune cell milieus within lymphoid tissues

Given NO’s reported effects on tumor immunity-relevant regulatory networks^9,15, responses by immune cells located within lymph nodes (LNs) draining the site of subcutaneous (s.c.) injection (dLN) to administered GSNO were profiled (Fig. 1a), as well as cells within non-draining LNs (ndLNs) and spleens. Many changes within cells located within draining lymphoid tissue microenvironments were found to result from GSNO treatment (Fig. 1b–j and Supplementary Figs. 1–19). CD45⁺CD11b⁺CD11c⁺F4/80⁻ DCs [F4/80⁻ conventional DCs (F4/80⁻cDCs)] were expanded and activated in dLN and expanded in spleen (Fig. 1b–d and Supplementary Fig. 3, 4), while other subsets of CD11b, CD11c and/or F4/80 expressing immune cells were maintained at similar levels in dLN, spleen, and ndLN (Supplementary Figs. 3–6). CD11b⁺CD11c⁺ DCs (cDC type 2, cDC2) are known to preferentially direct CD4⁺ over CD8⁺ T cells, though their precise functions are largely unknown due to their heterogenicity^20,21,22. Nevertheless, frequencies of total (CD45⁺CD3⁺) and CD4 (CD45⁺CD3⁺CD4⁺) versus CD8 (CD45⁺CD3⁺CD4⁻) T cells were significantly decreased within LN draining the injection site (Supplementary Fig. 7), spleens and ndLNs (Supplementary Figs. 8–9). In addition, the overall levels of immunosuppressive cells, including regulatory T cells (T_regs, CD45⁺CD3⁺CD4⁺Foxp3⁺) and myeloid-derived suppressor cells (MDSCs, CD45⁺CD11b⁺Gr1⁺), in lymphoid tissues remained unchanged by GSNO treatment (Supplementary Figs. 10–11). These results imply that rather than expanding populations of immune suppressive cells (e.g., T_regs and MDSCs) within lymphoid tissues, GSNO may influence adaptive immune signaling through its enhancement of these cells’ regulatory functions.

Immune phenotyping of dLN leukocyte populations 1 day after treatment of GSNO (570 μg kg⁻¹) in 30 μL saline. Gating strategy can be found in Supplementary Fig. 1. a Schematic illustration to investigate the effects of subcutaneously injected GSNO on immune cells in dLN. b–d Number and frequency of (b) CD45⁺CD11b⁺CD11c⁺F4/80⁻ cDCs (F4/80⁻cDCs) (left p = 0.0321 and right p = 0.0046), (c) CD86⁺ activated F4/80⁻cDCs (CD86⁺ F4/80⁻cDCs) (left p = 0.0485 and right p = 0.0087), and (d) CD86⁺ and MHCII⁺ activated F4/80⁻cDCs (CD86⁺MHCII⁺ F4/80⁻cDCs) (left p = 0.0466 and right p = 0.0120) within dLNs. e–j Representative histograms (left) and number and frequency of cell subpopulation (right) of (e, g, i) surface and (f, h, j) intracellularly expressed CTLA-4 by various dLN leukocyte populations. e, f F4/80⁻cDCs (e left p = 0.0277, e right p = 0.0480, f left p = 0.0298, and f right p = 0.0321). g, h CD11b⁺CD11c^–F4/80⁺ macrophages (CD11c^–M) (g left p = 0.0772, g right p = 0.0377, h left p = 0.0396, and h right p = 0.0123). i, j CD45⁺CD11b⁺GR-1⁺ (MDSCs) (i left p = 0.0353, i right p = 0.0285, j left p = 0.0375, and j right p = 0.0192). Number and frequency data are presented as individual biological replicates and mean ± SEM. b–j n = 5 for control and n = 4 for GSNO. *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1 by two-tailed Student t-test. Source data are available in a Source Data file.

CTLA-4, which is expressed widely on T cells as well as various other cells, including cancer cells^23,24, DCs²⁵, and MDSCs²⁶, is an immune checkpoint whose major role is attributed to the modulation of T cell priming, differentiation, and function^{1,3,8,24,25,26}. When expressed by cancer cells, CTLA-4 has also been shown to suppress the maturation and functions of DCs^23,24. Nevertheless, the effects of NO-delivery on CTLA-4 expression have never been investigated, although NO donors were reported to control the activity of AP-1 (transcription factor as well as clathrin adaptor protein)²⁷ that governs both the metabolism and expression of CTLA-4²⁸. Accordingly, extracellular and intracellular expression of CTLA-4 by various immune cells was also profiled (Fig. 1e–j and Supplementary Figs. 12–19). Interestingly, the populations of extra- and intracellular CTLA-4 expressing F4/80⁻cDCs in dLN (Fig. 1e, f), and CD11b⁺CD11c^–F4/80⁺ (CD11c^–M) in dLN (Fig. 1g, h) were significantly expanded, while the CTLA-4 expression on/in other subsets of CD11b, CD11c and/or F4/80 expressing immune cells were negligibly changed in dLN, spleen, and ndLN (Supplementary Figs. 12–17). In addition, CTLA-4 expressing MDSCs (Fig. 1i, j) were significantly expanded in dLN, while there were no changes in spleen and ndLN (Supplementary Fig. 18). However, the expression of CTLA-4 by T_regs was reduced in dLN, spleen, and ndLN (Supplementary Fig. 19).

The therapeutic effects of GSNO on the growth of B16F10-OVA melanoma-inoculated C57BL/6 mice were evaluated. Repeated (3x) intravenous (i.v.) administration of GSNO at 600 μg kg⁻¹ resulted in prolonged animal survival (Supplementary Fig. 20). Contrastingly, intratumoral (i.t.) administration of 570 μg kg⁻¹ GSNO (Fig. 2) slightly accelerated tumor growth, effects seen both in the treated (primary, 1^o) tumor as well as in an untreated (secondary, 2^o) tumor implanted in the contralateral dorsal skin (Fig. 2b, c). Tumor growth effects were not associated with changes in animal weight or survival (Fig. 2d, e), nor was GSNO treatment found to induce in any direct cytotoxic or cytostatic effects on B16F10-OVA cells in vitro (Supplementary Fig. 21) or proliferation in vivo, as suggested by no change in frequencies of Ki-67⁺ CD45⁻ cells (Fig. 2h). These results imply that i.t. administration of GSNO may have protumoral effects that are immune-mediated, including but not limited to tumor cell immunogenicity or by expanding tolerogenic immune cells that foster immunosuppressive tumor microenvironments. However, except for expression of PD-1 by CD45⁻ cells in the contralateral (untreated) tumor, GSNO administered i.t. appeared to exert negligible effects on expression of tumor immunogenicity markers including calreticulin (CRT), CTLA-4, PD-1, and PD-L1 (Fig. 2f–j and Supplementary Fig. 22). These observations suggest that the protumoral effects of GSNO may be associated with the CTLA-4 mediated hindrance of antitumor immunity, rather than direct effects of NO on tumor cell immunogenicity or proliferation, a hypothesis consistent with GSNO’s expansion of CTLA-4 expressing DCs, macrophages, and MDSCs and within LNs draining the locoregional site of injection.

a Tumor model and treatment schedule. 1^o and 2^o tumors were formed in C57Bl/6 mice by inoculation of 10⁵ B16F10-OVA cells in 30 μL saline on day 0 and day 3, respectively. GSNO (570 μg kg⁻¹) in 30 μL saline was administered on day 6, 8, and 10. b, c Average and (B’, B”, C’, C”) individual volumes of (b) 1^o (directly injected) and (c) 2^o tumors (uninjected). d Relative body weight changes post treatment. e Kaplan–Meier survival curves. f Tumor model and treatment schedule. 1^o and 2^o tumors were formed by inoculation of 10⁵ B16F10-OVA cells in 30 μL saline on day 0 and day 4, respectively. GSNO (570 μg kg⁻¹) was administered i.t. on day 7. Gating strategy can be found in Supplementary Fig. 22. g–j Number or frequency of each population of the indicated parent gate in the (g, h) 1^o or (i, j) 2^o tumor. g, i CD45⁻; h, j Ki-67⁺ (j p = 0.0677), CRT⁺, CTLA-4⁺, PD-1⁺ (j p = 0.0144), and PD-L1⁺ of CD45⁻. Data are presented as individual biological replicates and mean ± SEM. b–e n = 6. g–j n = 5. *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1. b, c ANOVA using linear mixed-effects model. d Two-way ANOVA using Tukey post-hoc statistical hypothesis. e Log-rank using Mantel–Cox statistical hypothesis. g–j Two-tailed Student t-test. Source data are available in a Source Data file.

Immunotherapeutic effects of GSNO and aCTLA-4 are enhanced in combination

The combination of intraperitoneally (i.p.) administered mAb antagonizing CTLA-4 signaling (aCTLA-4 mAb) with i.t. GSNO treatment was evaluated for its potential to unleash the functions of activated and mature DCs that appear to be restrained by CTLA-4 expressing tolerogenic DCs, macrophages, and MDSCs induced by GSNO treatment (Figs. 1, 2) using a dual B16F10-OVA mouse tumor model to reveal direct as well as abscopal therapeutic effects (Fig. 3a). The combination therapy, but not GSNO or aCTLA-4 when used as monotherapies, led to a significant slowing of the treated 1^o tumor’s growth, despite having no cytotoxic effects on B16F10-OVA cells in vitro (Fig. 3b, Supplementary Table 1, and Supplementary Figs. 21 and 23), suggestive of the therapeutic benefit not being associated with direct drug effects on the tumor. Treatment with the combination therapy furthermore resulted in substantial diminution in the growth of a contralateral tumor with no change in animal weight, indicating a strong abscopal effect, and animal survival was improved compared to treatment with GSNO alone (Fig. 3c–e and Supplementary Table 1 and 2). Consistent with these observed therapeutic benefits, the combination therapy was associated with an expansion of CD4⁺ T, CD8⁺ T, CD3^–NK1.1⁺ (NK), and CD3⁺NK1.1⁺ (NKT cells, NKT) cells in the blood day 13 post tumor implantation (Fig. 3a, f–i and Supplementary Table 2). Suggestive of robust priming of tumor antigen-specific T cells underlying these improvements in tumor control enabled by GSNO and aCTLA-4 when used in combination, the populations of CD4⁺ and CD8⁺ T cells that express activation markers CD25⁺ and LAG-3⁺, as well as antigen-experience marker PD-1⁺, were increased in the blood, as were tetramer-positive, tumor antigen-specific CD8⁺ T cells (Fig. 3g, h)^29,30. CTLA-4 antagonism with GSNO treatment thus appears to suppress the regulatory functions of CTLA-4 on immunosuppressive immune cells induced by GSNO, resulting in expansion of NK³¹ and NKT³¹ cells and improved priming of T cells (Fig. 3j).

a Tumor model and treatment schedule. 1^o and 2^o tumors were formed in C57Bl/6 mice by inoculation of 10⁵ B16F10-OVA cells in 30 μL saline on day 0 and day 4, respectively. GSNO (480 μg kg⁻¹) in 30 μL saline was intratumorally treated on day 7, and aCTLA-4 (100 μg mouse⁻¹) in 30 μL saline was intraperitoneally administered on day 8, 11, and 14. Blood was harvested from the facial vein on day 13 for the profiling of blood immune cells. b Average and individual volumes of 1^o (directly injected) tumors. c Average and individual volumes of 2^o (uninjected) tumors. d Relative body weight changes post treatment. e Kaplan–Meier survival curves. f–i Relative blood abundance of f CD45⁺, g CD45⁺CD3⁺CD4⁺ T (CD4⁺ T), LAG-3⁺ CD4⁺ T, PD-1⁺ CD4⁺ T, and CD45⁺CD3⁺CD4⁺CD25⁺Foxp3⁺ (T_reg), h CD45⁺CD3⁺CD8⁺ T (CD8⁺ T), CD25⁺ CD8⁺ T, LAG-3⁺ CD8⁺ T, PD-1⁺ CD8⁺ T, and tetramer⁺ CD8⁺ T, and i CD45⁺CD3^–NK1.1⁺ (NK) and CD45⁺CD3⁺NK1.1⁺ (NKT). Data are presented as individual biological replicates and mean ± SEM. b–e n = 5 for Control, Control+aCTLA-4, and GSNO + aCTLA-4, and n = 6 for GSNO. f–i n = 5 for Control, Control+aCTLA-4, and GSNO + aCTLA-4, and n = 4 for GSNO. *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1. Exact p-values for b, c and e–i are reported in Supplementary Table 1 and 2. b, c ANOVA using linear mixed-effects model. d Two-way ANOVA using Tukey post-hoc statistical hypothesis. e Log-rank using Mantel–Cox statistical hypothesis by comparing the GSNO + aCTLA-4 with control groups. f–i One-way ANOVA using Tukey post-hoc statistical hypothesis. j Proposed actions of combinational use of GSNO and aCTLA-4 on immune response. Blue arrows indicate the mechanism associated with GSNO. Red arrows and crosses represent mechanisms associated with aCTLA-4. Source data are available in a Source Data file.

F127-g-Gelatin thermosensitive hydrogel facilitates the sustained and targeted delivery of GSNO and aCTLA-4

The potential for sustained release technology to benefit immunomodulatory and/or immunotherapy applications is now established^{32,33,34,35,36,37}. In addition, considering the potential side effects of systemic NO delivery in blood pressure³⁸, local NO delivery systems have also attracted significant attention in biomedical applications¹⁰. To this end, injectable thermosensitive hydrogels offer numerous advantages, including their simple, simultaneous loading of diverse drug types, facile administration without surgery, and prolonged drug release from the polymer matrix^37,39.

Lower critical solution temperature (LCST) polymer F127 is widely utilized because it is cheap, biocompatible, and renal clearable. Despite its FDA approval, however, its practical hydrogel application is limited due to short residence in aqueous and physiological conditions^39,40. Accordingly, we hypothesized that the grafting F127 onto the also FDA-approved polymer gelatin, which is likewise biocompatible and exhibits low antigenicity, is compositionally diverse to provide sufficient functional groups amenable for easy chemical modifications, and is biodegradable and responsive to matrix metalloproteinases (MMPs) that are overexpressed by melanoma as well as various metastatic tumors,^41,42,43 would yield a biocompatible and biodegradable thermosensitive hydrogel that would facilitate the sustained delivery and therapeutic effects of aCTLA-4 mAb and GSNO in vivo. Such an approach would reduce the number of injections needed to elicit the therapeutic effects of GSNO and aCTLA-4 when used in combination for melanoma immunotherapy.

F127-grafted gelatin (F127-g-Gelatin) was synthesized by conjugation of 4-nitrophenyl chloroformate-activated hydroxyl groups of F127 to gelatin amine groups (Supplementary Figs. 24–26). The grafted F127-g-Gelatin polymer formed thermosensitive hydrogels at very low concentrations (4.0–7.0 wt.%), a surprising result given gelatin’s upper critical solution temperature (UCST) behavior (Fig. 4a, Supplementary Figs. 27,28 and Supplementary Table 3,4). This enhanced thermosensitive behavior was not observed in the mixture of F127 and gelatin (Supplementary Fig. 28c, and Supplementary Table 4), in contrast to previously reported F127-Gelatin copolymers that show sol-gel transition behavior (>10–15 wt.%) similar to that of bare F127 (> ~15 wt.%)^44,45. F127-g-Gelatin showed a reduced peak for crystalline structure of triple-helix (2θ = 8.4)⁴⁶ and an increased peak for amorphous phase (2θ = 21.1)⁴⁷ of gelatin in X-ray diffraction (XRD) compared to gelatin alone (Supplementary Fig. 29), negating potential contributions of coil-to-helix conversion to the observed thermosensitive gelation behavior⁴⁵. In addition, the crystalline peak for F127 (2θ = 19.2 and 23.4)⁴⁶ was reduced in F127-g-Gelatin, compared to F127 (Supplementary Fig. 29). Furthermore, F127-g-Gelatin exhibited no additional crystalline peaks in differential scanning calorimeter (DSC), compared to gelatin and F127 (Supplementary Fig. 30,31). On the other hand, F127-g-Gelatin showed significantly decreased critical micellar concentration (CMC) with the increase of temperature-dependency in CMC compared to F127 and the mixture of F127 and gelatin (Supplementary Fig. 32, and Supplementary Table 5). These results indicate that enhanced amorphous hydrophobic interactions contribute to the improved thermosensitivity of F127-g-Gelatin. The resultant F127-g-Gelatin hydrogels exhibited sheet-like microstructures (Fig. 4b) capable of solvent entrapment, which might enhance the hydrogel’s swelling property to contribute to the diffusion-mediated release of drugs. In addition, F127-g-Gelatin hydrogel showed the concentration-dependent rheology at 37 °C (Fig. 4c, and Supplementary Fig. 33).

a Concentration-dependent sol-gel transition properties of F127-g-Gelatin. b Representative SEM image of F127-g-Gelatin hydrogel (n = 3). c Concentration-dependent storage (G’) and loss (G”) modulus of F127-g-Gelatin at 37 °C (n = 3 for 4.5 wt.%, n = 4 for 5.5, 6.0, 6.5, and 7.0 wt.%, and n = 5 for 4.0 and 5.0 wt.%.). d, e Cumulative release of (d) NO₂⁻ + GSNO (n = 4) and (e) Alexa Fluor^TM 647 labeled aCTLA-4 (n = 3) from 4.5 wt% F127-g-Gelatin into PBS with or without MMP9. f, g In vitro residence stability of (f) GSNO (n = 4) and (g) Alexa Fluor^TM 647 labeled aCTLA-4 (n = 3) containing 4.5 wt% F127-g-Gelatin in PBS with or without MMP9. h DLS size distribution of aCTLA-4 and supernatants released from F127-g-Gelatin hydrogel (F127-g-Gelatin) or F127-g-Gelatin hydrogel containing aCTLA-4 (aCTLA-4/F127-g-Gelatin) (n = 12). The left and right insets represent the average size (n = 12) and zeta potentials (n = 6 for aCTLA-4, and n = 12 for the other) of materials, respectively. i Representative TEM image of F127-g-Gelatin micelles in situ released from F127-g-Gelatin thermosensitive hydrogel (n = 3). j FRET analysis with aCTLA-4-TRITC and F127-g-Gelatin-FITC at FITC excitation and TRITC emission (n = 4). k Competitive assay to verify the activity of aCTLA-4 released from F127-g-Gelatin hydrogel (n = 3 for None+None, and n = 4 for the other). l ALT/AST activities of blood taken from mice 2 d after subcutaneous administration of 4.5 wt.% F127-g-Gelatin hydrogel (n = 5). m In vivo residence stability of 4.5 wt.% F127-g-Gelatin hydrogel quantified by time-resolved volume of hydrogel remaining at the injection site (n = 4). n In vivo quantification of aCTLA-4-AF647 remaining at the injection site by using IVIS^® resulting from formulation in the 4.5 wt.% F127-g-Gelatin hydrogel (n = 4). Data are presented as mean ± SD for c–h and j, k, and mean ± SEM for l–n. *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1. Exact p-values for d–h, k, m, and n are reported in the source file. d–g, m, n Two-way ANOVA using Tukey post-hoc statistical hypothesis. h, k One-way ANOVA using Tukey post-hoc statistical hypothesis. l Two-tailed Student t-test. Source data are available in a Source Data file.

The potential for the resultant F127-g-Gelatin hydrogels for sustained drug release was next evaluated. Total levels of nitrite (NO₂⁻) and GSNO or Alexa Fluor^TM 647-labeled aCTLA-4 (aCTLA-4-AF647) (Supplementary Fig. 34) were released in a sustained manner from GSNO- or aCTLA-4-loaded 4.5 wt.% F127-g-Gelatin hydrogels (Fig. 4d, e), a process accelerated by enzymatic degradation with MMP9 (Fig. 4d, e) that is commonly overexpressed in melanomas^41,42,43. Interestingly, F127-g-Gelatin hydrogels containing aCTLA-4-AF647 mAb exhibited prolonged residence times as well as release half-lives in vitro compared to F127-g-Gelatin hydrogels containing GSNO (Fig. 4d–g), implicating the association of the loaded aCTLA-4 mAb in the formation of F127-g-Gelatin hydrogels. Indeed, aCTLA-4 mAb (d = 9.3 ± 0.6 nm) was not detected separately in the supernatants released from aCTLA-4 mAb loaded F127-g-Gelatin hydrogels in dynamic light scattering (Fig. 4h). In particular, the hydrogel released spherical micelles (Fig. 4i). The size of the in situ-formed micelles released from aCTLA-4 mAb loaded F127-g-Gelatin hydrogels (d = 30.0 ± 1.8 nm) was significantly larger than those of bare F127-g-Gelatin hydrogels (d = 26.8 ± 3.1 nm) (Fig. 4h), implying the loading of aCTLA-4 on the F127-g-Gelatin micelles. The in situ release of F127-g-Gelatin micelles would be attributed to Pluronic^® F127 components which self-assemble into the micelles via the dehydration of hydrophobic blocks with the increase in the entropy of the system at above critical micelle concentration (CMC)⁴⁸. Nevertheless, the size of aCTLA-4-loaded F127-g-Gelatin micelles was in a size range appropriate for efficient lymphatic uptake (10–100 nm)^49,50, which raised an expectation of efficient aCTLA-4 functions in both the dLN as well as tumor microenvironment site of injection³⁴.

The interactions of aCTLA-4 mAb with F127-g-Gelatin in situ-formed micelles were further verified with the appearance of additional CMC (CMC₂) in F127-g-Gelatin solutions containing aCTLA-4 (Supplementary Fig. 35 and Supplementary Table 6) and the fluorescence resonance energy transfer (Fig. 4j) between TRITC-labeled aCTLA-4 (aCTLA-4-TRITC) (Supplementary Fig. 36a) and FITC-labeled F127-g-Gelatin (F127-g-Gelatin-FITC) (Supplementary Fig. 36b) at FITC excitation and TRITC emission. Considering that F127 has an ability to bind human serum albumin via hydrogen bonding and hydrophobic interactions⁵¹ and aCTLA-4 mAb in F127 micelles was not detected solely in dynamic light scattering measurements (Supplementary Fig. 37), the F127 blocks in F127-g-Gelatin may contribute to the formation of aCTLA-4 mAb loaded in situ F127-g-Gelatin micelles. In addition to the larger molecular size of aCTLA-4 than GSNO, this affinity of aCTLA-4 with F127-g-Gelatin would also allow the dependency of aCTLA-4 release on the hydrogel degradation (Fig. 4e, g). On the other hand, the weaker dependence of GSNO release on the degradation of F127-g-Gelatin matrix may be attributed to its higher rate of diffusion through the F127-g-Gelatin hydrogel (Fig. 4d, f). When supernatants of in vitro pre-incubated aCTLA-4-loaded F127-g-Gelatin hydrogels were pretreated onto B16F10-OVA cells, staining with aCTLA-4-BV605 was blocked to similar extents as the treatment that seen with free aCTLA-4 (Fig. 4k). These results demonstrate that despite the interactions between aCTLA-4 mAb and F127-g-Gelatin polymer, the activity of aCTLA-4 mAb is not diminished by incorporation into the F127-g-Gelatin in situ-formed micelles.

As an LCST polymer, F127-g-Gelatin solution (4–7 wt.%) existed in a sticky liquid state inside the syringe and underwent gelation after administration. In line with negligible cytotoxicity in vitro (Supplementary Fig. 38), the F127-g-Gelatin hydrogel administered i.d. in vivo had no effect on body weight (Supplementary Fig. 39) nor systemic liver toxicity, as measured by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (Fig. 4l). When the injected tissue volume and fluorescent signal of aCTLA-4-AF647 mAb loaded F127-g-Gelatin hydrogels were measured at the site of mouse dorsal skin injection, F127-g-Gelatin hydrogels were found to impart significantly longer residence times and sustained release of aCTLA-4 mAb in vivo compared to bolus delivery of free mAb (Fig. 4m, n and Supplementary Fig. 40). When administered i.t., aCTLA-4-AF647 mAb was retained at the injected tumor site to greater extents in its hydrogel form (aCTLA-4/Hydrogel) for as long as 11 d compared to free aCTLA-4-AF647 and aCTLA-4-AF647 loaded micelles formed from F127-g-Gelatin (Fig. 5a and Supplementary Table 7). Administration of F127-g-Gelatin micelles resulted in accumulation of aCTLA-4-AF647 in LNs draining the tumor injection site to greater extents compared to bolus delivery but at levels similar to that seen for aCTLA-4-AF647 delivered via the administered F127-g-Gelatin hydrogels (Fig. 5b and Supplementary Table 7). Considering aCTLA-4 mAb loaded in situ micelles that exhibit a hydrodynamic size amenable for efficient lymphatic uptake (10–100 nm)^49,50 are released from aCTLA-4 mAb containing F127-g-Gelatin hydrogels (Fig. 4i, j), efficient lymphatic delivery of aCTLA-4-AF647 with F127-g-Gelatin hydrogels can be attributed to the formation of in situ of aCTLA-4 loaded F127-g-Gelatin micelles. F127-g-Gelatin hydrogels thus facilitate not only the sustained i.t. accumulation of aCTLA-4 mAb, but also enable its efficient delivery into dLN. F127-g-Gelatin hydrogels also reduced the exposure of aCTLA-4-AF647 to other tissues (Fig. 5c–i and Supplementary Table 7) compared to aCTLA-4-AF647 in its free or F127-g-Gelatin micelle loaded form.

a–i Biodistribution of free aCTLA-4-AF647, aCTLA-4-AF647 with 0.45 wt.% F127-g-Gelatin micelles (aCTLA-4 micelle), and aCTLA-4-AF647 with 4.5 wt.% F127-g-Gelatin hydrogel (aCTLA-4 dose equivalent to 162 µg mouse⁻¹) administered into the 1^o tumor of C57Bl/6 mice bearing 1^o and 2^o tumors inoculated with B16F10-OVA 10⁵ cells in 30 μL saline on day 0 and day 4, respectively. a 1^o (directly injected) tumor; b LN draining the 1^o tumor (1^o dLN); c 2^o (uninjected) tumor; d LN draining the 2^o tumor (2^o dLN); e blood; f spleen; g liver; h kidney; i lung. Data are presented as mean ± SEM. n = 4 except (a-i) Free aCTLA-4 groups on day 11 (n = 3) and (a) aCTLA-4/Hydrogel on day 7 (n = 3). *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1 with one-way ANOVA using Tukey post-hoc statistical hypothesis. Exact p-values for a–i are reported in Supplementary Table 7. Source data are available in a Source Data file.

Sustained GSNO + aCTLA-4 combination therapy using F127-g-gelatin augments antitumor immunotherapy

The benefit afforded by the dual tissue (tumor and tumor-dLN) delivering F127-g-Gelatin hydrogel on combination GSNO and aCTLA-4 therapy was assessed in the dual B16F10-OVA tumor model (Fig. 6a). Although no significant changes in body weight or ALT/AST activity were observed in any group (Fig. 6b, c), animal survival in response to a single i.t. injection of GSNO and aCTLA-4 co-formulated within the F127-g-Gelatin hydrogel was prolonged (Fig. 6d and Supplementary Table 8). This survival benefit was associated with the antitumor effects (Fig. 6e, f and Supplementary Table 9). In line with our other results (Fig. 3), i.t. administration of GSNO in both free and hydrogel formulations showed negligible effects on tumor growth. aCTLA-4 formulated within F127-g-Gelatin hydrogel showed limited improvement of aCTLA-4’s therapeutic index with respect to 1^o tumor growth and animal survival compared to free aCTLA-4. However, unexpectedly, i.t. administration of free GSNO + aCTLA-4 exhibited similar therapeutic effects with that of free aCTLA-4. These results may suggest that locally high levels of antibody achieved by i.t. administration enable aCTLA-4 to better exert its therapeutic effects due to the aCTLA-4’s actions on the tumor microenvironment and dLN³⁴. Nevertheless, combination of GSNO and aCTLA-4 co-formulated within F127-g-Gelatin hydrogel led to the decreased growth of the treated (1^o) tumor with efficacies being slightly superior to that of the free drugs in combination. In addition, GSNO + aCTLA-4/HG also substantially diminished the contralateral untreated tumor (2^o). In particular, one-time i.t. administration of GSNO + aCTLA-4/HG showed significantly higher antitumor effects than combined one-time i.t. administration of GSNO and three times i.p. administration of aCTLA-4 (Fig. 3, Supplementary Fig. 41, and Supplementary Table 10). These results suggest that prolonging the action of GSNO and aCTLA-4 mAb through i.t. administration of an F127-g-Gelatin hydrogel formulation through a single administration improves the combination therapy’s therapeutic index.

a Tumor model and treatment schedule. 1^o and 2^o tumors were formed in C57Bl/6 mice by inoculation of 10⁵ B16F10-OVA cells in 30 μL saline on day 0 and day 4, respectively. GSNO (570 μg kg⁻¹) and aCTLA-4 (50 μg mouse⁻¹) were administered intratumorally on day 7 in a total volume of 30 μL in saline or 4.5 wt.% F127-g-Gelatin hydrogel. Blood was harvested from the facial vein on day 9 for assessment using the ALT/AST assay (n = 4 for HG and aCTLA-4/HG, and n = 5 for the other). b Relative body weight changes post treatment (n = 5). c ALT/AST activities of blood taken from mice 2 d after treatment (n = 5). d Kaplan–Meier survival curves (n = 5). e 1^o (directly injected) tumor size (n = 5). f 2^o (uninjected) tumor size (n = 5). Data are presented as individual biological replicates and mean ± SEM. *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1. Exact p-values for d–f are reported in Supplementary Tables 8 and 9. b Two-way ANOVA using Tukey post-hoc statistical hypothesis. c One-way ANOVA using Tukey post-hoc statistical hypothesis. d Log-rank using Mantel–Cox statistical hypothesis. e, f ANOVA using linear mixed-effects model. Source data are available in a Source Data file.

Antitumor immunotherapeutic effects of GSNO + aCTLA-4/HG were also explored in the 4T1 model of mammary carcinoma (Balb/C mouse strain) using an aCTLA-4 (clone 9D9) that is of mouse origin (Fig. 7) in order to demonstrate the relevance of this immunotherapeutic synergy and drug delivery approach to another cancer and tissue type, a different mouse strain, and when employed using a therapeutic mAb of the same species as the host to replicate the human scenario. Despite the negligible survival benefit (Fig. 7c and Supplementary Table 11) and therapeutic effects on the 1^o tumor (FIg. 7d and Supplementary Table 12), i.t. administration of free GSNO + aCTLA-4 led to the slight antitumor effects on 2^o 4T1 tumors, compared to saline (Fig. 7d, e and Supplementary Table 12). The combination of GSNO and aCTLA-4 co-formulated within the F127-g-Gelatin hydrogel exhibited significantly stronger antitumor effects than saline, bare hydrogel, and free GSNO + aCTLA-4. These results not only indicate that the synergistic therapeutic effects of GSNO and aCTLA-4 in combination are maintained with a different antibody clone and species as well as in another mouse strain, but also imply the potential of locoregional sustained release platforms with combinational GSNO and aCTLA-4 therapy to cancers in different tissue sites and underlying biologies. Histological analysis of the mammary fat pad tumor injection site also revealed no effect of bare F127-g-Gelatin hydrogels compared to saline (Supplementary Fig. 42). Nor were any substantial changes in body weight measured for any treatment group (Fig. 7b). These results support the conclusion that the F127-g-Gelatin hydrogel exhibits no overt toxicity or inflammatory response and is generally biocompatible.

a Tumor model and treatment schedule. 1^o and 2^o tumors were formed in Balb/C mice by inoculation of 3 × 10⁵ 4T1 cells in 30 μL saline to left mammary fat pad on day 0 and at right mammary fat pad on day 4, respectively. GSNO (570 μg kg⁻¹) and aCTLA-4 (50 μg mouse⁻¹) were administered intratumorally on day 7 in a total volume of 30 μL in saline or 4.5 wt.% F127-g-Gelatin hydrogel. b Relative body weight changes post treatment. c Kaplan–Meier survival curves. d 1^o (directly injected) tumor size. e 2^o (uninjected) tumor size. Data are presented as individual biological replicates and mean ± SEM. n = 11 for saline, HG, and Free GSNO + aCTLA-4. n = 12 for GSNO + aCTLA-4/HG. *****p < 0.0001, ****p < 0.001, ***p < 0.01, **p < 0.05, and *p < 0.1. Exact p-values for c–e are reported in Supplementary Tables 11 and 12. b Two-way ANOVA using Tukey post-hoc statistical hypothesis. c Log-rank using Mantel–Cox statistical hypothesis. d, e ANOVA using linear mixed-effects model. Source data are available in a Source Data file.