A dual-functional in situ hydrogel for delivering vitamin E–based lipid nanoparticles to enhance cancer immunotherapy

Abstract

Conventional mRNA lipid nanoparticles often fail to elicit robust antitumor immunity due to their limited capacity to overcome the immunosuppressive tumor microenvironment (TME). Rational design of ionizable lipids with intrinsic bioactivity presents a promising strategy to enhance mRNA-based cancer immunotherapy. Here, we synthesized a bioactive vitamin E–based ionizable lipid to formulate lipid nanoparticles co-loaded with IL-12 mRNA and the IDO1 inhibitor NLG919 (N@VEBLNP), which were subsequently embedded into polyether F127-diacrylate hydrogel (NVF Gel). This hydrogel enables thermosensitive gelation for intratumoral injection and photocrosslinkable curing for postoperative site retention in the treatment of triple-negative breast cancer (TNBC). Specifically, NVF Gel exerted a dual immunomodulatory function: sustained release of N@VEBLNPs activated migratory cDC1s and augmented antigen presentation in tumor-draining lymph nodes, while concomitant release of NLG919 inhibited IDO1 expression, reduced regulatory T cells, and reprogrammed M2 macrophages toward the M1 phenotype. In 4T1 murine models, NVF Gel transformed the tumor environment into a more “immune-hot” state, effectively suppressed tumor growth and delayed postoperative recurrence. Collectively, NVF Gel provides a versatile platform for in situ cancer immunization and tumor microenvironment modulation.

Graphical abstract

Scheme 1. (A) Structure of Vitamin E and Vitamin E-based ionizable lipid (VEBIL). (B) Schematic illustration of the composition and preparation of N@VEBLNP, along with the application of NVF Gel as a dual-functional hydrogel for both intratumoral and postoperative drug delivery. (C) Schematic illustration of NVF Gel–mediated TME reprogramming, inducing a cold-to-hot tumor transition and potent antitumor immunity.

1. Introduction

Cancer immunotherapy has revolutionized oncology by harnessing the immune system to eliminate malignant cells and establish lasting immunological memory that prevents recurrence [[1], [2], [3], [4], [5]]. Clinical advances such as immune checkpoint inhibitors and adoptive cell therapies have markedly improved survival, particularly in patients unresponsive to conventional chemotherapy [[6], [7], [8]]. In parallel, messenger RNA (mRNA) therapeutics have emerged as a versatile platform capable of encoding tumor antigens [9,10], cytokines [9,11,12], and tumor suppressors [13,14]. However, clinical translation of mRNA-associated formulation is impeded by its instability, negative charge, and poor cellular uptake. Lipid nanoparticles (LNPs), as exemplified by FDA-approved siRNA formulations, offer the most promising delivery strategy, with ionizable lipids serving as the critical determinant of transfection efficiency [[15], [16], [17]]. Yet, the biological functionality of these lipids remains largely untapped, highlighting opportunities to design multifunctional ionizable lipids that concurrently promote mRNA delivery and augment therapeutic efficacy.

Vitamin E (VE), a lipophilic compound best known for its antioxidant activity, has gained increasing attention for its immunomodulatory and antitumor properties [[18], [19], [20]]. Recent studies demonstrate that VE enhances dendritic cell (DC) activation and antigen presentation by inhibiting the intrinsic checkpoint SHP1, thereby potentiating T-cell-mediated immune responses [21]. It also promotes type I interferon signaling and CD8⁺ T cell differentiation. Despite these benefits, VE's clinical utility is limited in cancer treatment by poor oral bioavailability and low tumor accumulation. Structurally, VE's hydrophobic tocopherol backbone and modifiable hydroxyl group make it an ideal scaffold for constructing ionizable lipids [22]. These features not only support efficient mRNA encapsulation and endosomal escape but also endow LNPs with intrinsic immunoregulatory activity. Thus, VE-derived ionizable lipids offer a promising strategy to simultaneously enhance mRNA delivery and antitumor immunity in LNP-based cancer immunotherapy.

Moreover, the immunosuppressive tumor microenvironment (TME) remains a key barrier limiting the efficacy of conventional LNP-based immunotherapies [[23], [24], [25]]. In immune “cold” tumors such as triple-negative breast cancer (TNBC), high expression of inhibitory mediators—particularly PD-L1 and the metabolic checkpoint enzyme indoleamine 2,3-dioxygenase 1 (IDO1)—impairs antigen presentation, T cell activation, and downstream cytokine signaling [[26], [27], [28]]. On the other hand, systemically administered LNPs often suffer from nonspecific biodistribution and transient transgene expression, resulting in suboptimal tumor accumulation and a narrow window of immune activation. In this context, hydrogel-based delivery platforms have garnered attention for their ability to provide localized and sustained release, making them well-suited for intratumoral administration of LNPs [[29], [30], [31]]. Therefore, engineering multifunctional LNPs that can deliver mRNA efficiently and actively reprogram the immunosuppressive TME—combined with in situ release via hydrogel—may offer a breakthrough strategy to overcome immune resistance in hard-to-treat tumors.

Here, we present a rationally engineered mRNA delivery system that addresses the challenges of insufficient immune activation and the immunosuppressive TME commonly associated with conventional LNPs. We synthesized a vitamin E–based ionizable lipid (VEBIL) that not only facilitates efficient mRNA encapsulation and cytosolic delivery, but also promotes conventional dendritic cell 1 (cDC1) immunostimulation. By co-loading IL-12 mRNA and the IDO1 inhibitor NLG919 into VEBIL-based LNPs (N@VEBLNP) and embedding them within a thermosensitive and photocrosslinkable F127-diacrylate hydrogel (NVF Gel), we achieved spatiotemporally controlled release at both primary and postoperative sites. This platform reprograms the TME by sustaining the activation of cDC1s, thereby enhancing antigen presentation within tumor-draining lymph nodes and promoting the priming and infiltration of cytotoxic CD8⁺ T cells. Simultaneously, the localized inhibition of IDO1 by NLG919 suppresses immunosuppressive TME by limiting regulatory T cells and inducing M2-to-M1 macrophage polarization, collectively transforming immune “cold” tumors into “hot” immune niches (Scheme 1). In 4T1 murine models, we demonstrated that NVF Gel elicited potent antitumor immune responses, resulting in marked tumor regression and delayed postoperative recurrence. Together, this work introduces a multifunctional, bioactive mRNA delivery platform for in situ immunomodulation and durable antitumor immunity in refractory solid tumors.

Scheme 1.

(A) Structure of Vitamin E and Vitamin E-based ionizable lipid (VEBIL). (B) Schematic illustration of the composition and preparation of N@VEBLNP, along with the application of NVF Gel as a dual-functional hydrogel for both intratumoral and postoperative drug delivery. (C) Schematic illustration of NVF Gel–mediated TME reprogramming, inducing a cold-to-hot tumor transition and potent antitumor immunity.

2. Results and discussions

2.1. Rational development of N@VEBLNP and dual-functional NVF Gel

As shown in Fig. S1, a vitamin E-derived ionizable lipid (VEBIL) was synthesized via a modular approach involving esterification of α-tocopherol with succinic anhydride, followed by amide coupling with a tertiary amine. The resulting structure comprises a hydrophobic tocopherol tail, a biodegradable linker, and a protonatable amine headgroup, conferring pH-responsive ionization for efficient nucleic acid complexation and endosomal escape. To formulate NLG919-loaded lipid nanoparticles (NLG919@VE-based LNPs, termed N@VELNPs) for tumor microenvironment (TME) modulation, we synthesized three VEBIL variants bearing distinct tertiary amine headgroups—VEBIL1, VEBIL2, and VEBIL3 (Fig. 1A, Fig. S2–S4). Lipid compositions were systematically optimized using an orthogonal design (Table S1), and EGFP mRNA was encapsulated as a reporter for in vitro screening. Notably, three representative formulations—VEBIL1K, VEBIL2O, and VEBIL3N—exhibited favorable compatibility and were selected for further characterization (Fig. 1B). We then assessed the physicochemical properties of these N@VELNPs, including particle size, zeta potential, and encapsulation efficiency. Among them, the VEBIL3N-based LNPs showed the most uniform size distribution, highest mRNA encapsulation efficiency, and near-neutral surface charge (Fig. 1C and D). Further optimization of the NLG919 loading revealed that a VEBIL3:NLG919 wt ratio of 20:1 maximized mRNA encapsulation (Fig. S5). Transmission electron microscopy (TEM) and cryo-TEM confirmed the formation of monodisperse, spherical nanoparticles with well-defined bilayer structures (Fig. 1E). To investigate intracellular trafficking, Cy5-labeled mRNA-loaded VEBIL3N LNPs were incubated with 4T1 cells and visualized by confocal microscopy. Progressive dissociation from LysoTracker-positive endo/lysosomes indicated efficient endosomal escape, as quantified by a time-dependent decrease in Pearson's correlation coefficient (Fig. 1F–I). Functionally, VEBIL3N LNPs enabled potent mRNA transfection in 4T1 cells, achieving EGFP expression levels comparable to Lipofectamine 3K at 0.8 μg/mL (Fig. 1G).

Fig. 1.

Rational development of VEBIL-based LNPs (N@VEBLNP) and dual-functional NVF Gel. (A) Schematic of N@VELNP formulation comprising VEBIL, cholesterol, DSPC, DMG-PEG₂₀₀₀, NLG919, and mRNA via microfluidic mixing. (B) Heatmap of EGFP expression in 4T1 cells treated with LNPs of different compositions (n = 3). (C, D) Characterization of VEBIL1K, VEBIL2O, and VEBIL3N formulations: particle size, PDI, zeta potential, and mRNA encapsulation efficiency. (E) TEM and cryo-TEM images showing spherical morphology and bilayer structure of N@VELNPs. Scale bars: 300 nm (TEM), 200 nm (Cryo-TEM). (F) Confocal images and Pearson's coefficients showing time-dependent endosomal escape of Cy5-mRNA-loaded N@VELNPs in 4T1 cells. Scale bar: 20 μm. (G) Flow cytometry analysis of EGFP expression in 4T1 cells transfected with N@VELNPs at increasing doses (n = 3). (H) ¹H NMR spectrum confirming the structure of diacrylate-functionalized Pluronic F127 (F127DA). (I) Schematic of NVF Gel formation via blending N@VELNPs with F127DA precursor, followed by thermal or UV crosslinking. (J) Photographs showing sol–gel transition of NVF Gel under heat or UV. (K) SEM image of lyophilized NVF Gel revealing porous structure with embedded N@VELNPs (yellow arrow). Scale bar: 25 μm. (L, M) Rheological assessment showing temperature-responsive gelation (L) and stable storage modulus across frequencies (M). (N) Cumulative release profiles of N@VELNPs from NVF Gels under different pH and crosslinking conditions over 72 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To enable localized and sustained delivery of N@VELNPs, we engineered a dual-responsive hydrogel system based on diacrylate-modified Pluronic F127 (F127DA). The successful synthesis of F127DA was confirmed by the characteristic vinyl proton resonances of the terminal acrylate groups (–OCO–CH=CH₂) in the ¹H NMR spectrum (Fig. 1H). To identify the optimal formulation for thermoresponsive gelation, a range of F127DA concentrations was screened (Table S2). A minimum concentration of 20 wt% was required to induce sol–gel transition at ∼33 °C, which was selected as the final concentration for subsequent hydrogel fabrication. Upon mixing with N@VELNPs, the F127DA precursor solution rapidly formed NVF Gel upon mild heating (32 °C) or 405 nm UV irradiation (Fig. 1I and J). Scanning electron microscopy revealed that UV-crosslinked NVF Gel exhibited a densely interconnected porous network with well-embedded nanoparticles (Fig. 1K), while the thermally crosslinked counterpart showed a more irregular and loosely packed structure (Fig. S6). To further characterize the mechanical robustness of NVF Gel, we performed rheological analyses under different crosslinking conditions. The hydrogel displayed temperature-induced gelations at ∼30 °C (G'＞G'') and maintained a stable, frequency-independent storage modulus thereafter (Fig. 1L and M). Frequency sweep and time sweep profiles confirmed the formation of a solid-like hydrogel under UV irradiation with high storage modulus (G′ ≫ G″) (Fig. S7A and C). Strain sweep analysis revealed that G′ was well-maintained under increasing deformation, indicating strong gel integrity (Fig. S7B). Viscosity measurements revealed that NVF Gel exhibited a faster gelation rate upon heating, whereas gelation under 405 nm UV irradiation resulted in a higher final viscosity (Fig. S7D). Next, we evaluated the drug release behavior of NVF Gel under different physiological conditions. In vitro release studies revealed that N@VELNPs embedded in NVF Gel exhibited sustained release profiles over 72 h, with slower release observed under UV crosslinking and at neutral pH (Fig. 1N). Moreover, direct quantification of NLG919 release from N@VELNPs demonstrated time-dependent diffusion kinetics, supporting the feasibility of controlled delivery from both nanoparticle and hydrogel compartments (Fig. S8).

2.2. NVF Gel activates cDC1 to enhance antigen presentation in vitro

To evaluate the bioactivity of the VEBIL-based ionizable lipid, Fluc-mRNA was used as a non-immunogenic reporter, and Dlin-MC3-DMA–based LNPs incorporated into an F127DA hydrogel (NLF Gel) served as the control group. Given the central role of dendritic cells (DCs) in initiating antitumor immunity, we next examined the intratumoral DC landscape following NVF Gel administration. In 4T1 tumors, DCs-particularly conventional type 1 DCs (cDC1s)-are typically sparse, peripherally localized, and functionally impaired, limiting effective T cell priming [32,33]. Immunofluorescence analysis revealed that NVF Gel markedly increased cDC1 (CD103⁺CD11c⁺) infiltration, especially at the tumor center, whereas NLF Gel treatment failed to enhance cDC1 accumulation in the central tumor region (Fig. 2A–D and Fig. S9). Flow cytometry confirmed a ∼3.8-fold increase in cDC1s following NVF Gel treatment compared with the untreated and NLF Gel groups (Fig. 2B and C). To evaluate antigen presentation, BMDCs were incubated with various OVA formulations. NVF Gel co-loaded with OVA significantly elevated SIINFEKL–MHC I (H-2K^b–SIINFEKL) expression, exceeding levels induced by conventional LNP (provided in the Supplementary Information) and NLF Gel, confirming cross-presentation capacity (Fig. 2E). Moreover, NVF Gel treatment led to robust upregulation of co-stimulatory molecules CD80 and CD86 on CD11c⁺ DCs (Fig. 2F and G), as well as elevated MHC II expression (Fig. 2H), indicating maturation of DCs. The slightly reduced activity compared to the VE group likely results from incomplete ester hydrolysis of VEBIL, delaying VE-mediated immune activation [34,35]. Notably, although the antigen presentation efficiency induced by NVF Gel was slightly lower than that of N@VEBLNPs, this difference is likely attributable to the need for nanoparticle release from the hydrogel matrix prior to cellular uptake. In contrast, NLF Gel did not enhance antigen presentation, indicating that this effect derives from the VEBIL-based ionizable lipid rather than the hydrogel carrier itself.

Fig. 2.

NVF Gel activates cDC1s to enhance antigen presentation in virto. (A) Representative immunofluorescence images of 4T1 tumor sections at the tumor edge and center, stained for CD103 (green), CD11c (red), and DAPI (blue). Scale bars: 200 μm. (B) Flow cytometry plots showing CD103⁺CD11c⁺ cDC1s in tumors following the indicated treatments. (C) Quantification of cDC1s (CD103⁺CD11c⁺) among CD11c⁺ cells (n = 5). (D) High-magnification images highlighting the spatial enrichment of CD103⁺ DCs at the tumor margin after NVF Gel treatment. Scale bars: 100 μm. (E) Representative flow cytometry histograms of BMDCs showing SIINFEKL–H-2K^b + peptide–MHC I complexes after treatment with OVA or OVA-loaded formulations. (F) Representative flow cytometry plots of BMDCs showing CD80 and CD86 expression after different treatments. (G) Quantification of mature CD86⁺CD80⁺ DCs among CD11c⁺ cells (n = 3). (H) Mean fluorescence intensity (MFI) of MHC II on CD11c⁺ BMDCs (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.3. NVF Gel repolarizes M2-like macrophages to remodel immunosuppression in virto

M2-polarized tumor-associated macrophages (TAMs), which dominate the immunosuppressive tumor microenvironment (TME), play a key role in promoting tumor growth, angiogenesis, and T cell exclusion [36,37]. Then, we selected therapeutic IL-12 mRNA as the payload for encapsulation into N@VEBLNP, and further embedded the formulation into NVF Gel, to evaluate their capacity to reverse immunosuppression in vitro. As shown in Fig. S10, all cell types maintained over 90 % viability after 24 h incubation with NVF Gel, confirming its favorable biocompatibility for in vitro applications. In a co-culture system mimicking the immunosuppressive TME—composed of IDO1⁺ 4T1 tumor cells and M2-polarized RAW264.7 macrophages—both N@VEBLNP and NVF Gel markedly suppressed kynurenine production, indicating effective IDO1 inhibition. Importantly, the drug-free F127DA hydrogel control did not exhibit any measurable inhibition of kynurenine production or macrophage repolarization, demonstrating that the immunological effects are not attributable to the hydrogel carrier itself. Compared to traditional LNPs, both N@VEBLNP and NVF Gel demonstrated markedly superior immunomodulatory effects. Specifically, conventional LNPs and F127DA Gel failed to significantly repolarize M2-like macrophages, as indicated by their modest increase in CD80⁺ cells and insufficient downregulation of CD206 expression (Fig. 3C and D). In contrast, NVF Gel induced a clear M2-to-M1 transition, with a 3.5-fold increase in CD80⁺ cells and a substantial decrease in CD206⁺ populations, closely approaching the levels observed in the N@VEBLNP group. This phenotypic shift was functionally validated by enhanced phagocytosis (Fig. 3E and F), which remained negligible in LNP-treated macrophages. Additionally, NVF Gel treatment elicited robust inflammatory cytokine release, including TNF-α and IL-6, both of which were significantly elevated compared to LNPs and blank F127DA Gel (Fig. 3G). Notably, IL-12p70 levels in the NVF Gel group were over 2.1-fold higher than those induced by LNPs, further confirming the superior immunostimulatory capacity of VEBIL-based formulations. Although peak IL-12 expression occurred earlier in the N@VEBLNP group, NVF Gel maintained a sustained release profile, supporting prolonged macrophage activation (Fig. 3H). Furthermore, NVF Gel and N@VEBLNP treatment significantly reduced the secretion of anti-inflammatory cytokines, including IL-10 and TGF-β, compared to conventional LNPs and the hydrogel control, indicating a potent reversal of the M2-like phenotype and attenuation of the suppressive TME.

Fig. 3.

NVF Gel repolarizes M2-like macrophages to remodel immunosuppression in virto. (A) Schematic illustration of the in vitro co-culture model for assessing macrophage repolarization and phagocytic function. M2-like macrophages were generated by IL-4 stimulation of RAW264.7 cells, followed by co-culture with IDO1⁺ 4T1 tumor cells and treatment with various formulations. (B) Kynurenine (Kyn) inhibition assay showing reduced IDO1 activity in co-culture supernatants after different treatments (n = 3). (C) Representative flow cytometry plots of CD80 and CD206 expression on macrophages post-treatment. (D) Quantification of CD80⁺ and CD206⁺ macrophage subsets (n = 3). (E) Quantification of phagocytic activity in macrophages following different treatments (n = 3). (F) Fluorescence microscopy of FITC-labeled 4T1 cells (green) engulfed by macrophages (red); yellow arrows indicate phagocytosed tumor cells. Scale bar: 100 μm. (G) Secretion of pro-inflammatory cytokines TNF-α, IL-6, and IL-12p70 from macrophages measured by ELISA (n = 3). (H) Time-dependent IL-12p70 release from macrophages over 48 h, comparing N@VEBLNP and NVF Gel. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.4. NVF Gel exhibits prolonged intratumoral retention and suppresses orthotopic 4T1 tumor growth

To investigate the intratumoral retention and antitumor efficacy of NVF Gel in vivo, we first evaluated the transfection kinetics of Fluc-mRNA-loaded nanoparticles using bioluminescence imaging. Compared with free N@VEBLNP, NVF Gel enabled significantly prolonged and enhanced luciferase expression within the tumor site, attributed to the sustained release of nanoparticles from the gel matrix (Fig. 4A). Quantitative analysis revealed that luminescence from N@VEBLNP rapidly declined after peaking at 8 h, while NVF Gel maintained a steadily increasing signal up to 48 h, indicating continuous nanoparticle release (Fig. 4B). Consistently, gel degradation analysis showed gradual in situ dissociation over 12 days, supporting its capability for long-term intratumoral retention (Fig. 4C). Next, biodistribution analysis using ex vivo imaging demonstrated predominant accumulation of NVF Gel in the tumor tissue up to 72 h, with negligible off-target distribution to major organs, confirming its intratumoral confinement (Fig. 4D and E). Based on this prolonged local retention, we performed a single intratumoral administration of each formulation (workflow in Fig. 4F) and monitored tumor progression in an orthotopic 4T1 model. Tumor growth curves and tumor weight measurements revealed that NVF Gel exhibited the strongest tumor suppression compared to all other groups, including free N@VEBLNP and conventional LNPs, underscoring the therapeutic advantage of hydrogel-mediated sustained delivery (Fig. 4G–I). Survival analysis further showed improved lifespan in the NVF Gel group (Fig. 4J). H&E staining of tumor sections revealed a marked reduction in tumor cell nuclei in the NVF Gel group, indicating effective tumor cell clearance (Fig. 4K). In addition, although H&E staining of major organs from mice in all treatment groups did not reveal any obvious tissue damage, serum biochemical analysis showed that intratumoral delivery of IL-12 mRNA by conventional LNPs led to marked elevations in ALT and AST levels, indicating systemic inflammation (Fig. S12 and Fig. S13). In contrast, both the N@VEBLNP and NVF Gel groups exhibited normal serum biochemical parameters, demonstrating the favorable systemic safety.

Fig. 4.

NVF Gel exhibits prolonged intratumoral retention and suppresses orthotopic 4T1 tumor growth. (A) In vivo bioluminescence imaging of 4T1 tumor-bearing mice after intratumoral injection of Fluc-mRNA-loaded N@VEBLNP or NVF Gel. (B) Quantitative analysis of photon radiance at tumor sites over time (n = 3). (C) In situ degradation profile of NVF Gel in the tumor over 14 days (n = 3). (D) Ex vivo imaging of major organs and tumors at 24, 48, and 72 h post-injection. (E) Quantification of average radiant efficiency in each organ (n = 3 per time point). (F) Schematic of treatment workflow. (G) Individual tumor growth curves for each treatment group (n = 5). (H) Average tumor volume growth curves. (I) Final tumor weight collected on day 21 (n = 5). (J) Survival curves of mice in a period of 60 days (n = 5). (K) Representative H&E staining of tumor sections from each group. Scale bar: 200 μm.

2.5. NVF reverses immunosuppression to activate potent in situ antitumor immunity

NVF Gel treatment initiated a cascade-like reversal of the immunosuppressive tumor microenvironment. Within the spectrum of immunosuppressive populations in the tumor microenvironment, myeloid-derived suppressor cells (MDSCs) are recognized as key mediators, as they attenuate T cell–driven antitumor responses, drive the polarization of macrophages from an M1-like to an M2-like phenotype, and facilitate the recruitment of regulatory T cells [38,39]. Immunofluorescence and metabolic analysis confirmed significant downregulation of IDO1 and a reduced Kyn/Trp ratio (Fig. 5A and B), thereby disrupting tryptophan metabolism–mediated expansion of MDSCs and Tregs. In line with this, flow cytometry revealed marked reductions in CD25⁺Foxp3⁺ Tregs and CD11b⁺Gr-1⁺ MDSCs, indicating effective dismantling of the IDO1–Kyn–MDSC/Treg axis (Fig. S14 and 15 and Fig. 5C). The alleviation of this suppressive circuit created a permissive niche for innate reprogramming. Specifically, TAMs underwent a pronounced shift from the M2 to the M1 phenotype, as reflected by a decreased M2/M1 ratio and increased CD86 expression (Fig. 5D and Fig. S16). This macrophage repolarization enables efficient phagocytosis of tumor debris and promotes dendritic cell maturation, particularly of the CD103⁺ cDC1 subset (Fig. 5E). In the TDLNs, NVF Gel led to the highest proportion of CD80⁺CD86⁺ mature DCs among all treatment groups (Fig. S17), and immunofluorescence quantification confirmed enhanced cDC1 and cDC2 infiltration (Fig. 5G). These professional antigen-presenting cells effectively cross-present tumor antigens to naïve T cells, initiating a potent adaptive immune response. UMAP-based spatial clustering demonstrated that NVF Gel treatment markedly increased the proportion of activated T cell subsets (CD69⁺CD4⁺ and CD69⁺CD8⁺) within the tumor microenvironment (Fig. 5F). This shift reflects enhanced T cell activation and immune infiltration. Consistently, flow cytometric quantification confirmed a significant elevation in both CD69⁺CD4⁺ and CD69⁺CD8⁺ T cells following NVF Gel administration, underscoring its superior capacity to stimulate effector T cell activation compared to all control groups (Fig. 5H). The observed activation of effector T cells thus reflects the cumulative output of a cascade-like series of immune interactions, orchestrated by IDO1 suppression and subsequently strengthened by TAM and DC remodeling.

Fig. 5.

NVF reverses immunosuppression to activate potent in situ antitumor immunity. (A) Representative immunofluorescence images showing IDO1 expression (green) in tumors from different treatment groups (G1–G5). Scale bar: 100 μm. (B) The intratumoral ratio of kynurenine (Kyn) to tryptophan (Trp) quantified by high-performance liquid chromatography (HPLC) (n = 5). (C) Flow cytometry analysis (left) and quantification (right) of intratumoral CD25⁺Foxp3⁺ regulatory T cells (n = 5). (D) Flow cytometry analysis of CD86 and CD206 expression (left) to assess M1/M2 macrophage polarization (right) in tumors (n = 5). (E) Immunofluorescence staining of CD103⁺ cDC1 (green) and CD172a⁺ cDC2 (magenta) in tumor sections. Scale bar: 100 μm. (F) UMAP plots displaying tumor-infiltrating immune cell subsets, including CD45⁺, CD3⁺, CD69⁺CD4⁺, and CD69⁺CD8⁺ T cells across treatment groups. (G) Quantification of CD103⁺ and CD172a⁺ dendritic cells in tumor sections (n = 5). (H) Quantification of activated CD69⁺CD4⁺ and CD69⁺CD8⁺ T cells in tumors (n = 5). (I) Heatmap showing expression levels of immune-related genes in saline and NVF Gel–treated tumors. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To capture the integrated and synergistic immunomodulatory effects of NVF Gel on the tumor microenvironment, we conducted transcriptomic analysis of 4T1 tumors subjected to different treatments. As shown in the Venn diagram, 1870 genes were uniquely upregulated in the NVF Gel group compared to saline, indicating a distinct gene expression signature (Fig. S18A). Heatmap analysis revealed elevated transcription of proinflammatory cytokines (e.g., IFN-γ, TNF, IL-12), chemokines (e.g., CXCL9, CXCL10), and cytotoxic mediators (e.g., GZMB), consistent with broad immune activation (Fig. 5I). KEGG enrichment analysis showed significant enrichment in immune-related pathways, including cytokine–cytokine receptor interaction, Toll-like receptor signaling, and IL-17 signaling pathways (Fig. S18B). Gene Ontology analysis further highlighted upregulation of biological processes related to antigen presentation, T cell-mediated cytotoxicity, cytokine biosynthesis, and immune effector responses (Fig. S18C). Together, these data demonstrate that NVF Gel reshapes the TME toward a proinflammatory and immunostimulatory phenotype, underpinning its potent antitumor activity.

2.6. NVF Gel delays postoperative 4T1 tumor growth

To assess the therapeutic efficacy of NVF Gel in preventing postoperative tumor recurrence, we established a 4T1-luciferase orthotopic breast tumor model followed by surgical resection and intratumoral administration of NVF Gel into the postoperative cavity (Fig. 6A). Bioluminescence imaging revealed rapid tumor regrowth in the saline group, whereas NVF Gel markedly suppressed tumor recurrence throughout the observation period, with significantly reduced photon flux observed on days 21 and 31 (Fig. 6B and C). Correspondingly, NVF Gel significantly prolonged overall survival, highlighting its potential to prevent local relapse (Fig. 6D).

Fig. 6.

NVF Gel delays postoperative 4T1 tumor growth. (A) Schematic of the therapeutic protocol in a 4T1-luc orthotopic tumor resection model. (B) Representative bioluminescence images of tumor-bearing mice at the indicated days post-surgery. (C) Quantification of tumor bioluminescence intensity over time (n = 5 per group). (D) Survival curves showing mice survival in a period of 60 days (n = 5). (E) UMAP plots of single-cell suspensions from spleen analyzed by high-dimensional flow cytometry, showing T cell subset distribution. (F) Quantification of CD8⁺ and CD4⁺ central memory (T_cm) and effector memory (T_em) T cell subsets in CD3⁺ T cells.

To further investigate whether NVF Gel elicited durable immune memory, we performed high-dimensional flow cytometric profiling of spleen T cells. UMAP analysis demonstrated an expansion of both CD8⁺ and CD4⁺ memory T cell subsets (T_cm and T_em) in the NVF Gel group compared to saline (Fig. 6E). Quantitative analysis confirmed significantly increased proportions of CD8⁺ T_cm, CD8⁺ T_em, and CD4⁺ T_em cells within total CD3⁺ T cells (Fig. 6F). These findings suggest that NVF Gel not only inhibits residual tumor growth but also facilitates the generation of long-lasting memory T cells, offering sustained immunological protection against tumor recurrence.

3. Conclusion

In summary, this study presents a rationally engineered vitamin E-based ionizable lipid nanoparticle system embedded within hydrogel (NVF Gel) for localized mRNA immunotherapy. Mechanistically, NVF Gel initiates a coordinated immunological cascade by enhancing cDC1-mediated antigen presentation, which in turn promotes CD8⁺ T cell priming. Concurrently, it downregulates IDO1 expression, mitigates the immunosuppressive effects of MDSCs and Tregs, and reprograms tumor-associated macrophages toward a pro-inflammatory M1 phenotype. These concerted effects collectively remodel the tumor immune microenvironment to support robust cytotoxic T cell responses and durable immune memory—an integrated immunomodulatory capacity not achievable with conventional LNP formulations. Looking forward, the rational design of bioactive ionizable lipids may unlock new avenues for precise and synergistic immunotherapies across a wide range of solid tumors.

CRediT authorship contribution statement

Chaozhu Zheng: Writing – original draft, Supervision, Methodology, Formal analysis, Data curation. Dekang Nie: Methodology, Investigation. Zhao Wang: Methodology. Nanjun Li: Methodology. Xiaolu Jin: Methodology. Ya Zhou: Methodology. Jun Wang: Methodology. Jun Xu: Methodology. Zhengqing Cai: Methodology. Binbin Xu: Methodology. Zizhuo Wei: Methodology. Feng Zhou: Methodology. Yiming Qi: Writing – review & editing, Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Data availability

Data will be made available on request.