Passivation‐Engineered Zinc Peroxide Nanoparticles as Th1‐Biased Adjuvant for Antitumor Immunity

Abstract

The efficacy of cancer vaccines depends significantly on adjuvants that can stimulate robust Th1 immune responses. However, conventional inorganic adjuvants like Alum generally elicit Th2‐biased immune response. Despite recent efforts in developing organic adjuvants to mimic bacterial or viral infections for generating Th1 immunity, excessive inflammation remains a major concern. Herein, a new class of Th1‐biased adjuvant is reported via passivation engineering of zinc peroxide nanoparticles (p‐ZPNs) for potentiating the immunogenicity of cancer vaccines. p‐ZPNs show improved biocompatibility compared to unpassivated ZPNs due to retarded zinc ion release and hydrogen peroxide production, as well as efficient cellular uptake, endosome escape, dendritic cell maturation and antigen cross presentation. Mechanistic study indicates the intrinsic activity of p‐ZPNs to activate toll‐like receptors (TLR)‐2, 4, and 6, triggering the TLR/Myeloid differentiation primary response 88 (MyD88)/Nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) axis for producing proinflammatory cytokines and therefore a robust Th1 immunity. Biosafety evaluation has shown the importance of the passivation engineering of ZPNs in reducing safety risks. Furthermore, in a p‐ZPNs‐based autologous cancer vaccine model, potent immune response is demonstrated in the inhibition of B16F10 tumors in both prophylactic and therapeutic vaccination settings. These findings suggest the great potential of p‐ZPN adjuvants for future Th1‐biased vaccine development.

This study demonstrates passivated zinc peroxide nanoparticles (p‐ZPNs) as a novel class of Th1‐biased adjuvants to boost the efficacy of cancer vaccines. The p‐ZPNs exhibit excellent cellular uptake, low cytotoxicity, efficient endosome escape and intrinsic capability for triggering TLR/MyD88/NF‐κB axis, leading to robust T cell immunity. A personalized p‐ZPN‐based cancer vaccine shows strong antitumor responses in both prophylactic and therapeutic settings.

1. Introduction

As a promising strategy of cancer immunotherapy, cancer vaccines typically leverage tumor‐associated antigens (TAAs) or neoantigens to prime antigen‐presenting cells (APCs), such as dendritic cells, to activate T cell‐mediated immune responses against tumors.^{[ 1} , ² , ³ , ^{4 ]} Given the limited vaccine immunogenicity, adjuvants are required to boost APCs maturation and therefore the downstream T cell activation. Organic adjuvants such as pathogen‐associated molecular patterns (PAMPs) mimicking microbial pathogens, e.g., CpG oligodeoxynucleotides (CpG ODNs) and monophosphoryl A (MPLA), can induce a strong Th1 response.^{[ 5} , ⁶ , ⁷ , ⁸ , ^{9 ]} However, there are major concerns associated with PAMPs, such as excessive inflammation and potential tissue damage.^{[ 10} , ¹¹ , ^{12 ]} Therefore, it remains challenging to develop novel adjuvants that can trigger strong Th1 immunity without relying on PAMPs to achieve both high efficacy and good biocompatibility.

Zinc is an essential micronutrient support for the immune system, regulating the structural function of various proteins and participating in multiple immunological signalling pathways.^{[ 13} , ¹⁴ , ^{15 ]} A number of studies have demonstrated the adjuvant activity of zinc‐based nanoparticles, such as zinc oxide and zeolitic imidazolate framework(ZIF)‐8 nanoparticles, which primarily induce a Th2‐biased immune response.^{[ 16} , ¹⁷ , ^{18 ]} To induce a Th1‐biased immunity, PAMPs are often used into the vaccine formulations.^{[ 14} , ¹⁹ , ^{20 ]} But the mechanism of zinc oxide induced immunity seems to be controversial. Zinc oxide has been reported to mimic the role of PAMPs in adsorbing protein corona for enhanced interaction with the TLRs, leading to NF‐kb activation.^{[ 21 ]} In other reports, ZnO induces adjuvant effects via TLR1/2 heterodimers, TLR4, and TLR6, coupled with Src kinase signaling (e.g., phosphorylation of Lyn, Syk, PLC‐γ), increasing intracellular Ca²⁺, cAMP, and IP3 levels, where TLR6‐mediated responses are essential for inflammation and cytokine release in immune cells.^{[ 18 ]} Compared to other forms of zinc‐based nanoparticles, zinc peroxide nanoparticles (ZPNs) with intact O─O bonds and intrinsic antimicrobial activity have been used in clinical settings.^{[ 22 ]} Recent studies have shown the promise of ZPNs as an effective anticancer agent to directly kill cancer cell, leveraging their high reactivity in producing toxic levels of zinc ions and hydrogen peroxide (H₂O₂) in acidic tumor microenvironment.^{[ 23} , ²⁴ , ^{25 ]} However, the high reactivity and the consequent cytotoxicity of ZPNs can lead to lethal effects to immune cells. Therefore, it is not surprising that few reports have explored the potential of ZPNs as immune adjuvants.

Herein, we report that chemically passivated ZPNs (p‐ZPNs) are a new class of potent adjuvants with strong capability to elicit a Th1‐biased immune response. To alleviate the cytotoxicity arising from the high reactivity of ZPNs, a crystallinity gradient‐driven selective etching (CGSE) strategy^{[ 26 ]} was employed for passivation engineering to selectively remove the more reactive components of ZPNs in the inner part of nanoparticles. The resultant p‐ZPNs with a hollow structure exhibited retarded release of zinc ions (Zn²⁺) and production of H₂O₂ under acidic endosomal conditions, enabling significantly lowered toxicity toward APCs compared to untreated ZPNs. As shown in Scheme  1 , p‐ZPNs can be efficiently internalized by APCs, promoting endosome escape and triggering the APC maturation through the Toll‐like receptor (TLR) / Myeloid differentiation primary response 88 (MyD88) / Nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) axis, leading to secretion of pro‐inflammatory cytokines for a robust Th1 immune response as illustrated in Scheme 1. By applying the cell membrane of melanoma cells B16‐F10 as the antigens, p‐ZPNs‐based vaccine formulation exhibited excellent anti‐tumor performance in both therapeutic and preventive vaccination models.

Scheme 1.

Schematic illustration of p‐ZNPs as an adjuvant for cancer vaccines, enhancing cellular immunity (Created in BioRender.com). After cell uptake, Zn²⁺ released from p‐ZNPs activate TLR2, TLR4, and TLR6, initiating Myd88‐TRAF6 signaling. This activation triggers MAPK and NF‐κB pathways, leading to the secretion of pro‐inflammatory cytokines. Meanwhile, ROS generation induces endosomal rupture, further amplifying TLR downstream signalling and reinforcing immune activation. The released cytokines drive the differentiation of CD4⁺ T cell into Th1 cells, which, along with activated CD8⁺ cytotoxic T cells, promotes a potent antitumor immune response.

2. Results and Discussion

2.1. Synthesis and Characterization of p‐ZPNs

The ZPNs were prepared using zinc acetate and H₂O₂ as precursors in the presence of poly(vinyl pyrrolidone) (PVP) in an aqueous system at room temperature (see details in Supporting Information).^{[ 23 ]} As shown in the transmission electron microscopy (TEM) image (Figure S1, Supporting Information), ZPNs exhibit a spherical morphology with an average particle size of ≈70 nm. For passivation engineering (Figure  1a), a modified CGSE method developed before^{[ 26 ]} was applied using hydrogen chloride (HCl) as the etchant. The pH and reaction temperature were carefully optimized to achieve passivation engineering without destroying the nanostructure. As shown in Figure S2a (Supporting Information), at the reaction temperature of 60 °C and the pH of 2.56, the morphology of ZNPs was preserved. When the pH decreased to 2.53, partially etched p‐ZPNs were obtained (Figure S2b, Supporting Information). However, when further lowering the pH to 2.50, ZNPs were nearly degraded (Figure S2c, Supporting Information). Then, pH 2.53 was chosen to further study the temperature impact (70, 80, and 90 °C, Figure S2d–f (Supporting Information), respectively), suggesting that uniform p‐ZPNs were obtained at 90 °C.

Figure 1.

Synthesis and characterizations of p‐ZPNs. a) A scheme of the passivation process (Created by Biorender); b) TEM image of p‐ZPNs, c) Raman spectrums, d) XPS, e) XRD of ZPNs and p‐ZPNs; f) in vitro time‐dependent zinc ions release from ZPNs and p‐ZPNs analyzed by ICP, g) in vitro time‐dependent release of H₂O₂. Data were presented as mean values ± SD, n = 3.

As shown in Figure 1b, p‐ZPNs exhibited a hollow structure with an average diameter of 70 nm. Both p‐ZPNs and ZPNs carried a negative surface charge in PBS, with zeta potentials of ≈−27 and −33 mV, respectively (Figure S3, Supporting Information). The formation of the hollow morphology could be explained by the selective etching of solid ZPNs with inherent inhomogeneity.^{[ 26} , ²⁷ , ^{28 ]} The formation of metal peroxide nanoparticles usually begins with the initial formation of primary nanocrystals and their aggregation to form spheres, serving as the secondary nucleation site for particle growth. Thus, the inner core formed at earlier reaction time point has a lower crystallization degree compared to the shell that is formed at prolonged time with enhanced chemical resilience. By carefully choosing the etching conditions, the core of ZPNs can be selectively removed, leading to the formation of hollow p‐ZPNs. To our knowledge, this is the first report of the synthesis of hollow zinc peroxide nanoparticles.

The chemical composition was characterized by Raman and X‐ray photoelectron spectroscopy (XPS). In the Raman spectra, both p‐ZPNs and ZPN exhibited the typical characteristic peak at 840 cm⁻¹, assigned to the O─O stretching vibration, suggesting the presence of peroxide ligands (O₂ ²⁻) (Figure 1c). The existence of O₂ ²⁻, Zn, C was further validated from XPS spectra (Figure 1d). Both particles exhibited the O 1s peak with the binding energy of 532 eV corresponding to O₂ ²⁻. Furthermore, in X‐Ray diffraction (XRD) patterns, both p‐ZPNs and ZPNs exhibited three characteristic peaks at 32.80, 37.18, 53.48, and 63.48° (2θ), assigned to the pure ZnO₂ phase (Figure 1e).^{[ 29 ]} Moreover, the full width at half maximum (FWHM) of the peak at 37.18° was measured. The p‐ZPNs exhibited a lower FWHM value compared to ZPNs (1.39 vs 1.65). Similarly, FWHMs of additional major peaks for p‐ZPNs and ZPNs, including 32.80° (1.40 vs 1.65), 53.48° (1.41 vs 1.84), and 63.48° (1.51 vs 2.68), showed the similar trend, suggesting a higher crystallinity of p‐ZPNs.^{[ 27} , ^{28 ]}

The release profiles of Zn²⁺ and H₂O₂ from these two NPs were then investigated under neutral and acidic conditions, simulating extracellular physiological and endo/lysosome environments, respectively. As shown in Figure 1f, at neutral pH 7.4, negligible Zn²⁺ was released from both particles. At acidic pH 5.0, ZPNs exhibited a significantly higher release of ≈80% within 24 h compared to p‐ZPNs (∼50%), suggesting a faster dissociation behavior at acidic pH. Similar acidic pH‐responsive release was observed for H₂O₂ release (Figure 1g). The above results indicate the higher stability of p‐ZPNs than ZPNs in intracellular acidic environments due to the higher crystallinity.

2.2. Cytotoxicity Assessment and Cellular Response to p‐ZPNs

Next, the cytotoxicity of the synthesized nanoparticles toward APCs was evaluated in DC 2.4 cells. As shown in Figure S4 (Supporting Information), p‐ZPNs and ZPNs displayed a dose dependent cytotoxicity after 24 h incubation. The cell viability of p‐ZPNs remained above 80% at the dosage up to 6.25 µg mL⁻¹, thus the following studies were conducted at 4 µg mL⁻¹ to ensure the cytocompatibility of both nanoparticles. To further assess the cytotoxicity, the live/dead assay was conducted by calcian AM/propidium iodide (PI) dual staining (Figure  2a), showing the least red fluorescence from p‐ZPNs in three treated groups (including lipopolysaccharide (LPS) and ZPNs), indicating the least cytotoxicity. Besides, lactate dehydrogenase (LDH), a marker of cellular damage, was measured at 24 h postincubation. As shown in Figure 2b and p‐ZPNs exhibited the lowest LDH production compared to ZPNs and LPS, suggesting the least membrane disruption. The viability of p‐ZPNs treated cells was significantly higher than ZPNs, which could be likely due to the differences in Zn²⁺ and H₂O₂ release profiles (Figure 1f,g). Collectively, the cytotoxicity data evidenced the advantage of p‐ZPNs as a safer platform compared to ZPNs and LPS.

Figure 2.

In vitro cytotoxicity and immune‐stimulating activities. a) Calcein AM/PI Double Staining (scar bar 100 µm), b) LDH (24 h), c) the percentages of CD80+DC, d) CD86+ DC, e) pro‐inflammatory cytokines secretions including TNF‐α, f) IL‐12 and g) IFN‐γ; h) p‐ZPNs and ZPNs induced endosome escape in 24 h (scar bar 20 µm); i) MHC I OVA specific cross‐presentation; j) MHC II presentation. Data were presented as mean values ± SD. Statistical analysis was carried out via unpaired t‐test. n = 3; *p < 0.05, **p < 0.01, and ***p < 0.001.

2.3. Immunogenicity and Antigen Delivery Performance of p‐ZPNs

The maturation studies were conducted in DC2.4 cells by analyzing the expression of costimulatory surface markers (CD80 and CD86) using flow cytometry. As shown in Figure 2c, compared to untreated cells, p‐ZPNs and ZPNs exhibited dose‐dependent increase in CD80 expression. At both dosages, the CD80 positive cell percentage of p‐ZPNs treated group was ≈1.6‐fold compared to ZPNs. Similar upregulation was observed in CD86 expression (Figure 2d; Figure S5, Supporting Information). In addition, cytokine release from DC2.4 after incubation was analyzed, showing a significantly higher level of Th1 related cytokines (IL‐12, TNF‐α, and IFN‐γ) from p‐ZPNs group, compared to PBS, LPS, and ZPNs (Figure 2e–g).

Next, we evaluated the antigen delivery performance using ovalbumin (OVA) as a model antigen. Both p‐ZPNs and ZPNs exhibited a similar OVA loading capacity (≈0.4 mg mg⁻¹, Figure S6, Supporting Information), despite p‐ZPNs having a larger surface area. This is due to the reduction in surface hydroxyl (─OH) groups after HCl etching, which weakens OVA adsorption, counterbalancing the increased surface area.^{[ 30 ]} The release study (Figure S7, Supporting Information) at pH 7.4 and 5 further revealed acidic pH‐responsive antigen release property. Both nanoparticles exhibited a faster and higher percentage of OVA release at pH 5 compared to pH 7.4, likely due to the decomposition of ZnO₂ in acidic conditions.^{[ 31 ]} At pH 5, ZPNs released ≈82% of OVA within 24 h, higher than that of p‐ZPNs (≈70%), which could be explained by the higher OH density weakened association with antigen protein.^{[ 32 ]} At pH 7.4, both ZPNs and p‐ZPNs showed a more sustained release profile, reflecting ZnO₂’s stability under physiological conditions. However, a similar trend of faster OVA release from ZPNs than p‐ZPNs was observed. The above results indicated the unique structure of p‐ZPNs slowed down the antigen release under physiological conditions and enabled faster release in the acidic endosomal environment of antigen‐presenting cells (APCs). The adjuvant‐antigen formulations were denoted as p‐ZPNs (OVA) and ZPNs (OVA).

Endosomal escape performance was further evaluated using a confocal microscope at 10 h (Figure S8, Supporting Information) and 24 h (Figure 2h). Lysosomes and cell nuclei were stained with LysoTracker Red and Hoechst dye, respectively, while FITC‐labeled OVA was used to visualize intracellular antigen distribution. At 10 h, the free OVA control showed almost no green fluorescence, indicating poor cellular uptake, whereas both ZPNs (OVA) and p‐ZPNs (OVA) exhibited intracellular green fluorescence, confirming successful antigen internalization. However, ZPNs (OVA) displayed early‐stage endosomal escape, as evidenced by green fluorescence separating from the lysosomal marker, while p‐ZPNs (OVA) showed more yellow fluorescence, suggesting delayed escape due to slower ROS release. By 24 h, p‐ZPNs (OVA) exhibited intense cytoplasmic green fluorescence, confirming efficient and sustained endosomal escape. In contrast, ZPNs (OVA) showed only faint green fluorescence, suggesting that while escape occurred, excessive ROS generation may have induced cytotoxic stress. This observation is consistent with the MTT results (Figure S4, Supporting Information), Calcein AM/PI double staining (Figure 2a), and LDH release assays (Figure 2b), which indicate potential cytotoxicity associated with ZPNs at 24 h. The successful endosomal escape may be due to the lipid peroxidation induced by ROS generation and ion release caused by osmotic pressure.^{[ 33 ]} Similar mechanisms have been reported for calcium peroxide (CaO₂) nanoparticles, where ROS production induces oxidative damage to the endosomal membrane, leading to its destabilization and rupture.^{[ 34 ]}

Then antigen cross‐presentation was evaluated by analyzing fluorescein‐labelled anti‐mouse MHC I bound to the peptide SIINFEKL. As shown in Figures 2i and S9 (Supporting Information), p‐ZPNs (OVA) exhibited the highest positive cell percentage and mean fluorescence intensity (MFI) of MHC I among ZPNs (OVA) and free OVA group. MHC II expression was comparable among all treated groups, with a slight increase compared to PBS. (Figure 2j; Figure S9, Supporting Information). The enhanced MHC I expression by p‐ZPNs can be attributed to their lower cytotoxicity and larger surface area compared to ZPNs, which not only facilitate more favorable cellular uptake but also boosts antigen delivery and presentation. This is further supported by Figure  3b, which clearly demonstrates that cell uptake of p‐ZPNs is significantly higher than that of ZPNs. Additionally, differences in endosomal escape efficiency also contribute to the observed difference in MHC I expression. The differential MHC I expression, driven by variations in endosomal escape efficiency, positions p‐ZPNs as effective carriers for MHC I cross‐presentation, crucial for eliciting CD8⁺ T cell responses in cancer immunotherapy.

Figure 3.

Proposed mechanisms for p‐ZPNs enhanced DC maturation and induced Th1 immune response as an adjuvant for cancer vaccine (Created by Biorender) a). Cellular uptake b); Intracellular fluo‐3/zinc MFI c); Positive cells fluo‐3/zinc MFI d); Positive cells percentage of fluo‐3/zinc; e); Intracellular DCFDA MFI f); Positive cells DCFDA MFI g); Positive cells percentage of DCFDA h); Signaling pathway study after 24 h treatment with LPS, ZPNs, and p‐ZPNs; flow analysis of TLR2 i), TLR4 j), TLR6 k); western blot analysis l), MyD88 (≈36 kDa) m), TRAF6 (≈65 kDa) n), MAPK (≈40 kDa) (p38) o), NF‐κB (≈68 kDa) (p65) p), Active MAPK (≈38 kDa) (p‐p38) q), and Active NF‐κB (≈70 kDa) (p‐p65) r). Data was presented as mean ± SD. Statistical analysis was carried out via an unpaired t‐test. n = 3; *p < 0.05, **p < 0.01, and ***p < 0.001.

2.4. Mechanistic Insights into Immune Stimulation by p‐ZPNs

Next, mechanistic study on the p‐ZPNs‐mediated immune simulation was carried out. Initially, the intracellular uptake of both NPs was studied by analyzing the intracellular zinc content through inductively coupled plasma‐optical emission spectrometry (ICP‐OES). As shown in Figure 3b, p‐ZPNs treated group exhibited higher intracellular zinc content compared to ZPNs (8.81 pg/cell and 2.91 pg/cell, respectively), suggesting higher uptake efficiency, which could be explained by the increased number of particles due to the low density of the hollow‐structured p‐ZPNs. Further assessments using Fluo‐3/Zinc, analyzed by flow cytometry (Figure 3c; Figure S10a, Supporting Information), revealing that free zinc ion levels in the p‐ZPNs group were lower than those in the ZPNs from 2 to 24 h, consistent with the slower release rate observed in in vitro studies under acidic conditions (Figure 1f). A similar trend was noted in the positive cell mean fluorescence intensity (MFI) (Figure 3d) and positive percentage (Figure 3e) due to the slower release rate from p‐ZPNs. The intracellular ROS levels analysed using 2′,7′‐Dichlorofluorescin Diacetate (DCFDA) and flow cytometry (Figure 3f; Figure S10b, Supporting Information), showing no significant difference in ROS generation between p‐ZPNs and ZPNs within the first 4 h; however, ROS levels were elevated from the 8th hour in both groups. p‐ZPNs generated significantly higher ROS compared to ZPNs, likely due to higher cellular uptake. However, the MFI of DCFDA‐positive cells in the ZPNs group was significantly higher than in the p‐ZPNs group (Figure 3g) due to the rapid release of ROS during the first 8 h, leading to ROS levels that exceeded the cells' tolerance and resulted in cell death, aligning with previous Calcein AM/PI double staining and LDH release data. Furthermore, the lower quantity of ZPNs may have caused a rapid increase in ROS within a limited number of dendritic cells (DCs), ultimately leading to cell death rather than activation. Studies have shown that excessive ROS can induce cell death instead of activating DCs.^{[ 35 ]} While ROS plays an important role in regulating DC maturation, excessive ROS can impair their ability to present antigens, leading to cellular dysfunction and potentially apoptosis. This is also supported by our observation: In the first 8 h, the percentage of ROS‐positive cells was almost the same between two groups, but after 24 h, the p‐ZPNs group exhibited a significantly higher proportion of ROS‐positive cells that ZPNs group since the latter would cause excessive ROS that eventually led to extensive cell death.

The expression levels of TLR2, TLR4, and TLR6 were analysed by flow cytometry (Figure 3i–k; Figure S11, Supporting Information), showing that p‐ZPNs markedly upregulated these TLRs. Compared to the control group, TLR2 expression increased by ≈11‐fold, TLR4 by 8.78‐fold, and TLR6 by 2.2‐fold. This upregulation is presumably due to zinc‐based nanoparticles binding to TLRs, similar to LPS^{[ 18} , ^{36 ]} or the sustained release of Zn²⁺ within the endosome indirectly activating DCs. Subsequently, TLR activation initiated downstream signalling pathways (Figure 3l). Myd88 expression increased by ≈1.6‐fold compared to ZPNs and 10‐fold compared to the control (Figure 3m). TRAF6 further amplified the signal, activating the MAPK and NF‐κB pathways, promoting the release of more inflammatory factors (Figure 3n). The immunostimulatory effect of p‐ZPNs was enhanced by the combined action of sustained ROS generation ^{[ 35 ]} and zinc ions, which boosted MAPK activity, with phosphorylated MAPK levels increasing by ≈16.93‐fold over the control (Figure 3o,q). NF‐κB, as the terminal signalling molecule, initiated the expression of immune and inflammatory genes, further enhancing immune responses (Figure 3p,r). Based on above results, an intracellular molecular pathway for the adjuvant effect of p‐ZPNs was proposed (Figure 3a), which involves primarily activation of TLR signalling pathway, subsequently enhancing MyD88 and TRAF6 expressions. This activation initiates a cascade effect, amplifying signalling through the MAPK and NF‐κB pathways, ultimately boosting the release of inflammatory mediators.

2.5. In Vivo Evaluation of p‐ZPN Adjuvanticity

To evaluate the in vivo adjuvanticity of p‐ZPNs, zinc oxide nanoparticles (ZnO NPs) were used for comparison, which were prepared by calcination of ZPNs at 300 °C for 3 h. The TEM image of ZnO NPs (Figure S12, Supporting Information) exhibited the hollow spherical structure with an average diameter of 70 nm. The typical characteristic peaks from XRD analysis, indexed to stimulated pattern, confirmed the crystal structure of ZnO (Figure S13, Supporting Information).^{[ 37 ]} MPLA was selected as the positive control, and OVA was selected as the model antigen. Female C57BL/6 mice received three subcutaneous (s.c) injections at one week interval (Figure  4a). The CD11c⁺ DCs from lymph nodes and spleen were gated to determine CD80⁺CD86⁺ expression (Figure S14, Supporting Information).

Figure 4.

In vivo DC maturation and T cells analysis. Timeline of injection and cell collection (Created by Biorender) a); the percentages of CD80⁺CD86⁺DC in Lymph Nodes b), the percentages of CD80⁺CD86⁺DC in Spleen c); the percentages of CD3⁺CD4⁺T cells in Lymph Nodes d), the percentages of CD3⁺CD8⁺T cells in Lymph Nodes e);the percentages of CD3⁺CD4⁺T cells in Spleen f), the percentages of CD3⁺CD8⁺T cells in Spleen g); the percentages of IFN‐γ⁺CD3⁺CD4⁺T cells in Spleen h); the percentages of IFN‐γ⁺CD3⁺CD8⁺T cells in Spleen i).; OD 450 of total IgG j), IgG2c k), IgG1 l) and titer of total IgG m), IgG2c n), IgG1 o), IgG2c/IgG1 ratio p);cytokines secretions in blood IL‐12 q), TNF‐α r)and IFN‐γ s) Data were presented as mean values ± SD. Statistical analysis was carried out via unpaired t‐test. n = 4 *p < 0.05, **p < 0.01, and ***p < 0.001.

As shown in Figures 4b,c and S15 (Supporting Information), p‐ZPNs (OVA) exhibited the highest CD80⁺ CD86 ⁺ CD11c⁺ DCs percentage in spleen (≈20%) and lymph nodes (≈55%) compared to free OVA, ZPNs (OVA) and ZnO NPs (OVA), which was comparable to MPLA(OVA), indicating the efficient DC maturation. In addition, T cell immunity was studied by examining T cell population in spleen and lymph node (Figure 4a; Figure S16, Supporting Information). The CD4⁺ cells and CD8⁺ cells populations in lymph nodes and spleens were the highest in p‐ZPNs (OVA) treated group among all nano‐formulations, comparable to MPLA(OVA) (Figure 4d,e; Figure S17, Supporting Information). Similar trends were observed in IFN‐γ produced OVA‐specific CD4⁺ (figure 4f; Figure S18, Supporting Information) and CD8⁺T cells (figure 4g; Figure S19, Supporting Information). Subsequently, the adaptive immune response was studied. As shown in Figure 4j,m and p‐ZPNs (OVA) showed the highest production of OVA‐specific total IgG in the serum among all groups. Notably, IgG1 and IgG2c are widely recognized as markers for Th2‐ and Th1‐type immune responses, respectively.^{[ 1} , ^{38 ]} It is worth noting that MPLA (OVA) elicited the highest IgG2c titers (Figure 4k,n), followed by p‐ZPNs (OVA), which still showed a 1.6‐fold increase compared to ZPNs (OVA). In contrast, the ZnO (OVA) treatment group induced the highest level of IgG1 production, while other treatment groups, including p‐ZPNs and MPLA, also promoted IgG1 expression but to a lesser extent, showing only modest increases compared to the OVA and PBS groups (Figure 4l,o). Furthermore, p‐ZPNs (OVA) exhibited a higher IgG2c/IgG1 ratio than all other groups except MPLA, and the ratio remained above 1(Figure 4p). These findings support the conclusion of a Th1‐biased immune response induced p‐ZPN adjuvant.

To further evaluate the T cell immune response, the Th1 related cytokine levels in splenocytes were investigated. To be noted, the mice group immunized with p‐ZPNs (OVA) exhibited the highest secretion of IL‐12, compared to mice groups treated with ZnO (OVA), ZPNs (OVA), and OVA, which was nearly two‐fold increase compared to the benchmark adjuvant, MPLA (OVA) (Figure 4q). A similar trend in the increase of TNF‐α and IFN‐γ levels was also observed in p‐ZPNs (OVA) immunized group (Figure 4r,s). In addition, no obvious weight loss was observed in all groups (Figure S20, Supporting Information). H&E staining results of major tissues suggested no detectable damage after p‐ZPNs (OVA) treatment, implying the good biosafety profile (Figure S21, Supporting Information). The biodistribution study indicated no significant difference in zinc biodistribution in major organs for NPs immunized mice (Figure S22, Supporting Information).

2.6. Enhanced Antitumor Immunity with p‐ZPNs in Melanoma Model

The adjuvant effect in inducing T cell response was evaluated in a B16‐F10 melanoma tumor model. B16‐F10 derived cell membranes (M) were collected as antigens and formulated with nanoparticles to fabricate an autologous vaccine. An immune checkpoint blockade, programmed death 1 antibody (anti‐PD‐1), were used in combination with p‐ZPNs‐M to enhance T cell immunity. C57BL/6 mice were challenged with B16‐F10 cells on day 0, followed by three s.c injections of vaccine formulations on days 3, 7, and 14, as depicted in Figure  5a. In the p‐ZPNs‐M+anti‐PD‐1 group, anti‐PD‐1 was administered on days 4, 8, and 15. Tumors were harvested on day 21 for T cell immune analysis (Figure S23, Supporting Information), and the gating strategy for identifying tumor‐infiltrating CD4⁺, CD8⁺, and IFN‐γ⁺ T cells is provided in Figure S24 (Supporting Information). Mice immunized with p‐ZPNs‐M and p‐ZPNs‐M + anti‐PD‐1 elicited dramatically high percentages of CD8⁺ cells (≈20% and 28%, respectively), (Figure 5b–d) compared to ZPNs‐M and MPLA‐M. Similar trends were observed in IFN‐γ‐produced B16‐F10‐specific CD4⁺ (Figure 5e,g) and CD8⁺ T cells (Figure 5f,h).

Figure 5.

Evaluation of T cell response in a melanoma tumour model. Schedule of immune cell analysis in the TME (Created by Biorender) a), flow cytometry dot plots gated on CD3⁺ b), the percentage of CD3⁺CD4⁺T cells in tumour c) and CD3⁺CD8⁺T cells d); the percentage of IFN‐γ⁺CD3⁺ CD4⁺T cells e); the percentage of IFN‐γ⁺CD3⁺ CD8⁺T cells f); flow cytometry dot plots gated on CD3⁺ CD4⁺T cells g); flow cytometry dot plots gated on CD3⁺ CD8⁺T cells h). Data were presented as mean values ± SD. Statistical analysis was carried out via unpaired t‐test. n = 3; *p < 0.05, **p < 0.01, and ***p < 0.001.

The preventive and therapeutic effects of the autologous nanovaccine against melanoma tumors were further evaluated. As shown in Figure  6a, in the prophylactic model, mice were immunized three times at a 7‐day interval and then inoculated with B16‐F10 cells. Tumor growth was monitored. As shown in Figure 6b‐g and p‐ZPNs‐M significantly suppressed tumor growth compared to other groups without obvious weight loss (figure S25, Supporting Information). Furthermore, the survival rates showed p‐ZPNs‐M group could extend the survival up to 30‐days (Figure 6h). The efficacy was further evaluated in a therapeutic model (Figure 6i). Mice were first challenged with B16‐F10 cells on day 0, followed by three s.c. injections of nanovaccines on days 3, 7, and 14, while anti‐PD‐1 was injected one day post p‐ZPNs‐M injection. Both p‐ZPNs‐M and p‐ZPNs‐anti‐PD1‐M exhibited remarkable tumor growth (Figure 6j–p) without obvious change in body weight (figure S26, Supporting Information). Besides, 83.3% of p‐ZPNs‐anti‐PD1‐M treated mice achieved the survival rate over 30 days (figure 6q). These results collectively evidenced the superior immunological adjuvant effect of p‐ZPNs for stimulating antitumor immunity.

Figure 6.

Antitumor efficacy of the autologous nanovaccine in preventive and therapeutic models. Schematic illustration of the experimental protocol for B16‐F10 Preventive tumor model (Created by Biorender) a), average tumor growth curve b); each group tumor growth curve for preventive model PBS c), MPLA ‐M d), ZnO‐M e), ZPNs‐M f), p‐ZPNs‐M g) and percentage of mouse survival after vaccination three times(n = 6) h); Schematic illustration of the experimental protocol for B16‐F10 Therapeutic tumor model (Created by Biorender) i), average tumor growth curve j) each group tumor growth curve for therapeutic model PBS k), MPLA(B16‐F10) l), ZnO‐M m), ZPNs‐M n), p‐ZPNs‐M o), p‐ZPNs ‐PD‐1‐M p) and percentage of mouse survival after vaccination three times(n = 6) q). Data was presented as mean values ± SEM. Statistical analysis was carried out via unpaired t‐test.

3. Conclusion

In conclusion, a new class of Th1‐based adjuvant, p‐ZPNs, has been developed via the passivation engineering of zinc peroxide nanoparticles. The controlled release of Zn²⁺ and H₂O₂ effectively activated TLR pathways and robust MyD88/MAPK /NF‐κB signalling cascades, driving enhanced dendritic cell activation and a potent Th1 immune response. Preclinical studies using murine melanoma models demonstrated that p‐ZPNs, particularly in combination with PD‐1 inhibitors, effectively suppressed tumour progression, reduced tumour burden, and improved survival rates, underscoring their potential as a promising strategy for future cancer vaccine development. It is envisioned that this nano‐adjuvants can be extended to developing Th1‐focused vaccine technology that not only target tumors, but also intracellular pathogen infections.

4. Experimental Section

Chemicals and Biological Reagents

The following chemicals and biological reagents were purchased from the specified suppliers: Zinc acetate dihydrate, Hydrogen peroxide, Polyvinylpyrrolidone (Mw: 10 000), ethanol, ovalbumin (OVA) (5503‐1G), hydrogen chloride, RIPA (Radio‐Immunoprecipitation Assay) Buffer, and Protease Inhibitor Cocktail were purchased from Sigma‐Aldrich. Monophosphoryl Lipid A from S. minnesota R595 (MPLA) (cat tlrl‐mpla) was purchased from InvivoGen. Dulbecco's modified eagle medium (DMEM) was obtained from Life Technologies. RPMI 1640 Medium, B16‐F10 and DC 2.4 were acquired from the American Type Culture Collection (ATCC). Antibodies for cell staining and blocking, including anti‐mouse bound to SIINFEKL Antibody MHC I (Clone 25‐D1.16, cat 141 603), MHC II (Clone M5/114.15.2 cat 115 006), FITC anti‐mouse CD11c (Clone N418 cat 117 306), PE anti‐mouse CD80(Clone N418, cat 104 708), APC anti‐mouse CD86 (Clone GL‐1, cat 105 114),

Brilliant Violet 421 anti‐mouse CD3 (Clone 17A2, cat 100 228), PE anti‐mouse CD4 (Clone GK1.5, cat 116 006), PerCP/Cyanine5.5 anti‐mouse CD8a (Clone 53–6.7, cat 100 734), PE anti‐mouse CD284 (TLR4) Antibody (Clone SA15‐21, cat 145 404), Ultra‐LEAF Purified anti‐mouse CD279 (PD‐1) Antibody (Clone 29F.1A12, cat135250) and FITC anti‐mouse IFN‐γ (Clone XMG1.2, cat 505 806) were procured from BioLegend. BD Horizon BV421 Rat Anti‐Mouse CD282 (TLR2) (Clone CB225, cat 565 908) and BD OptiBuild BV605 Mouse Anti‐Mouse CD286 (TLR6) (Clone C1N2, cat 743 268) were procured from Becton Dickinson. ELISA Kits for cytokine testing, including IFN‐γ (ab100689), IL‐12 (ab119531), and TNF‐α (ab285327), and Rabbit anti‐mouse antibodies targeting MAPK p38 (Clone HL1006, cat ab308333), MAPK p‐p38 (Clone EPR18120, cat ab195049), NF‐κB p‐65 (Clone E379, cat ab32536), NF‐κB p‐p‐65 (cat ab264271), Myd88 (Clone EPR21824, cat ab219413), TRAF6 (Clone EP591Y, cat ab33915), anti‐mouse IgG2c(ab97255) and Beta‐actin (cat ab8227), were sourced from Abcam. Additional antibodies included Anti‐mouse IgG (cat 31 430), IgG1(cat A10551), Goat anti‐Rabbit IgG(H+L) Cross‐Adsorbed Secondary Antibody DyLight 800 (cat SA5‐10036), FluoZin‐3, AM, cell permeant (cat F24195), LDH (cat C20301), Propidium Iodide (cat P1304MP) and Calcein AM (cat C3099) which were obtained from Thermo Fisher Scientific.

Synthesis of Zinc Peroxide Nanoparticles and Passivated Zinc Peroxide Nanoparticles

The synthesis of Zinc Peroxide Nanoparticles (ZPNs) and passivated Zinc Peroxide Nanoparticles (p‐ZPNs) was performed using the following procedure. First, 0.12 g of zinc acetate dihydrate (Zn(OAc)₂·2H₂O) and 0.1 g of polyvinylpyrrolidone (PVP) were dissolved in 5.0 mL of water under stirring. Subsequently, 0.5 mL of hydrogen peroxide (H₂O₂) was rapidly added to the solution under vigorous stirring, and the reaction mixture was allowed to react for 24 h at room temperature. The resulting ZPNs were collected, washed thoroughly, and re‐dispersed in water.To prepare passivated ZPNs (p‐ZPNs), 3 mg of the purified ZPNs were suspended in 10 mL of a pH 2.53 solution and stirred vigorously at 90 °C for 1 h. After the passivation process, the p‐ZPNs were washed three times with ethanol to remove residual by‐products and unreacted materials.

Material Characterization

Material characterization was conducted using the following methods: Transmission electron microscopy (TEM) images were obtained using a JEOL‐1010 microscope‐ray diffraction (XRD) patterns were recorded using a Rigaku Mini Flex X‐ray diffractometer with Ni‐filtered Cu Kα radiation (λ = 1.5406 Å).X‐ray photoelectron spectroscopy (XPS) profiles were acquired using a Kratos Axis ULTRA.

Release Profiles of Zn Ions, H₂O₂ and OVA

The release of Zn²⁺ and H₂O₂ from ZPNs and p‐ZPNs was evaluated under different pH conditions (pH 5.0 and 7.4) in phosphate‐buffered saline (PBS). Specifically, 1 mg of ZPNs or p‐ZPNs was dispersed in 1 mL of PBS at the designated pH. At specified time points, the supernatant was collected for analysis. Zn²⁺ release was quantified using inductively coupled plasma optical emission spectrometry (ICP‐OES), with calibration performed using Zn²⁺ standard solutions. Concurrently, H₂O₂ release was measured using a hydrogen peroxide assay kit following the manufacturer's instructions. The results provided insight into the pH‐responsive release behavior of Zn²⁺ and H₂O₂ from the nanoplatforms.

For the study involving ovalbumin (OVA), ZPNs and p‐ZPNs were loaded with OVA for 24 h with 1 mg nanoparticles and 1 mg OVA in 1 mL PBS. The release and loading of OVA from ZPNs (OVA) and p‐ZPNs (OVA) were then assessed under the same pH conditions. For the release study, 1 mg of OVA‐loaded nanoparticles was suspended in 1 mL PBS at pH 5 or pH 7.4, and the release behavior was monitored over 24 h. The amount of OVA released was quantified using a bicinchoninic acid (BCA) protein assay kit.

Cell Viability Test

Cell viability was assessed using the tetrazolium dye (MTT) colorimetric assay.^{[ 39 ]} Dendritic cells (DC 2.4) were seeded in 96‐well plates at a density of 8 × 10³ cells per well and allowed to adhere and grow overnight, and treated with PBS (control), p‐ZPNs, ZPNs at various concentrations. After 24 h of incubation, 10 µL of MTT reagent (5 mg mL⁻¹ in PBS) was added to each well and incubated for 4 h at 37 °C. The culture medium was then removed, and the resulting formazan crystals were solubilized in 100 µL of DMSO. Absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage relative to the untreated control using the following formula:

$$\mathrm{Cell}\mathrm{viability}\left(\%\right)=\frac{\mathrm{O}{\mathrm{D}}_{570}\mathrm{sample}}{\mathrm{O}{\mathrm{D}}_{570}\mathrm{control}}\times 100$$ (1)

To assess cytotoxicity using LDH activity, cells were seeded at the same density as used in the MTT assay and incubated with ZPNs or p‐ZPNs at a concentration of 2 µg mL⁻¹. Cytotoxicity was measured using the CyQUANT LDH Cytotoxicity Assay Kit (Cat. No. C20301, Thermo Fisher Scientific), following the manufacturer's instructions. To determine maximum LDH activity, 10 µL of 10X lysis buffer was added to the designated wells. After 45 min of incubation, 50 µL of each sample (spontaneous, maximum, and treatment group) was transferred to a new 96‐well plate. The LDH reaction mixture was then added, and the plate was incubated for 30 min at room temperature, protected from light. Following incubation, stop solution was added, and the absorbance was measured at 490 and 680 nm. LDH activity was calculated by subtracting the background absorbance at 680 nm from the absorbance at 490 nm, and the percentage of cytotoxicity was normalized using the formula below.

$$\begin{array}{ccc}\hfill \%\mathrm{Cytotoxicity}& =& \frac{\mathrm{Compound}\mathrm{treated}\mathrm{LDH}\mathrm{activity}-\mathrm{Spontaneous}\mathrm{LDH}\mathrm{activity}}{\mathrm{Maximum}\mathrm{LDH}\mathrm{activity}-\mathrm{Spontaneous}\mathrm{LDH}\mathrm{activity}}\hfill \\ & & \times 100\hfill \end{array}$$ (2)

PI/Calcein AM Dual Staining

A total of 1.5 × 10⁵ DC 2.4 cells were seeded into a culture plate and incubated for 24 h to allow cell attachment and stabilization. ZPNs and p‐ZPNs were then added to the cells at a final concentration of 2 µg mL⁻¹, followed by an additional 24‐h incubation. After incubation, the cells were gently washed with PBS to remove non‐adherent particles. The cells were then stained with a prepared mixture of Calcein AM and propidium iodide (PI) staining solution and incubated for 30 min at 37 °C in the dark. Fluorescence images were captured using a fluorescence microscope. Calcein AM stains live cells, emitting green fluorescence, while PI stains dead or membrane‐compromised cells, emitting red fluorescence. All experiments were performed in triplicate to ensure reproducibility.

Intracellular ROS and Zn2+ Detection by Flow Cytometry

To investigate intracellular levels of reactive oxygen species (ROS) and Zn²⁺ using flow cytometry, the following procedure was employed: DC 2.4 were initially seeded in 12‐well plates at a density of 1.5 × 10⁵ cells per well. Following the overnight incubation, the culture medium was carefully removed, and the cells were washed twice with phosphate‐buffered saline (PBS). Next, the cells were treated with nanoparticles at a concentration of 2 µg mL⁻¹ for 6 h. After that, the cells were stained with ROS detection probe, 2′,7′‐dichlorofluorescein diacetate (DCF‐DA). DCF‐DA was a compound that was converted to a fluorescent form when it comes into contact with ROS. To perform this measurement, a solution containing DCF‐DA at a concentration of 100 µM in PBS was added to the cells for 15 min incubation. Following the incubation with the ROS probe, the cells were washed with PBS to remove any excess probe. Finally, the fluorescence was analyzed using a Beckman Coulter CytoFLEX flow cytometer system. All experiments were performed in triplicate.

In Vitro Maturation of APC, Cytokine Secretion Measurement and MHC‐I/II Presentation

The in vitro maturation of antigen‐presenting cells (APCs), specifically DC 2.4 cells, was investigated. Cells were initially seeded at a density of 1.5 × 10⁵ cells per well in 12‐well plates and incubated overnight to allow for adherence and stabilization. The cells were then treated with either PBS (control), ZPNs, or p‐ZPNs at final concentrations of 2 and 4 µg mL⁻¹, followed by incubation for 24 h. After treatment, the cells were harvested and subjected to Fc‐blocking to prevent non‐specific antibody binding. Subsequently, the cells were incubated with antibodies specific to CD80 and CD86, two key surface markers indicative of APC maturation. The expression levels of CD80 and CD86 were analyzed by flow cytometry using a Beckman Coulter CytoFLEX flow cytometer.DC 2.4 cells (seeded in 12 well plates at a density of 1.5 × 10⁵ cells/well overnight) were treated with PBS, lipopolysaccharide (LPS) 50 ng mL⁻¹, P‐ZPNs (2 µg mL⁻¹) and ZPNs (2 µg mL⁻¹) for 24 h. The supernatant was collected and analysed using ELISA kits for IFN‐γ, IL‐12, IL‐4, and TNF‐α.

The cross‐presentation of major histocompatibility complex class I (MHC‐I) and class II (MHC‐II) molecules were studied in DC2.4 cells. These cells were initially seeded at a density of 1.5 × 10⁵ cells per well in 12‐well plates and allowed to incubate overnight for optimal adherence. Four distinct treatment groups were added, comprising PBS, Ovalbumin (OVA), ZPNs (OVA), and p‐ZPNs (OVA). Following a 24‐h incubation, DCs were harvested and stained with antibodies specific to either MHC class I –OVA257–264 complexes or total MHC class II molecules. Flow cytometry was performed using a Beckman Coulter CytoFLEX flow cytometer to evaluate surface expression levels of MHC‐I–OVA complexes and overall MHC‐II molecules.

All experiments were conducted in triplicate.4.9 TLR 2, 4, 6 Staining: DC 2.4 cells were seeded at a density of 1.5 × 10⁵ cells per well in a 6‐well plate and incubated for 24 h to allow cell attachment and stabilization. After 24 h, cells were treated with samples (ZPNs, p‐ZPNs, or control) at a final concentration of 2 µg mL⁻¹ and incubated for an additional 24 h. Following incubation, the culture medium was removed, and the cells were gently washed with cold PBS. Cell permeabilization was performed using BioLegend's permeabilization reagent (0.1%–0.2% Triton X‐100) for 10–15 min at room temperature, following the manufacturer's instructions. After permeabilization, cells were stained with fluorophore‐conjugated antibodies specific to TLR2, TLR4, and TLR6, and incubated for 30 min at 4 °C in the dark. The cells were then washed twice with cold PBS containing 2% FBS to remove unbound antibodies and subsequently fixed with 4% paraformaldehyde prior to flow cytometry analysis.

Western Blot

DC 2.4 cells were treated with various groups, including ZPNs, p‐ZPNs (both at a final concentration of 2 µg mL⁻¹), LPS (50 ng mL⁻¹), and a control group treated with PBS. After 24 h of incubation, the cells were harvested and lysed using Radio‐Immunoprecipitation Assay (RIPA) buffer supplemented with a protease inhibitor cocktail. The total protein content in the cell lysates was quantified using the Bicinchoninic Acid (BCA) assay.

For each treatment group, 30 µg of total protein was separated by SDS‐PAGE and transferred onto a PVDF membrane. The membranes were incubated with primary antibodies (rabbit anti‐mouse) targeting key proteins, including MAPK p38, phosphorylated MAPK p‐p38, NF‐κB p65, phosphorylated NF‐κB p‐p65, MyD88, TRAF6, and Beta‐actin, which served as a loading control. After primary antibody incubation, membranes were washed and incubated with IRDye 800CW‐labeled goat anti‐rabbit secondary antibodies. Protein bands were visualized using the Odyssey CLx Infrared Imaging System. All experiments were performed in triplicate to ensure the reliability and reproducibility of the results.

Animal Studies

All animal work was conducted in accordance with approved Animal Ethics protocols (AIBN/2022/AE000646) at the University of Queensland. Female C57BL/6 mice, 6–8 week, ordered from Ozgene, were housed in individually ventilated cages with standard rodent chow and water provided ad libitum under a 12‐h light/dark cycle. All experimental procedures were performed in compliance with institutional animal welfare guidelines. Mice were euthanized when tumor volumes reached 1000 mm³ to minimize suffering. All injections were carried out under anesthesia to ensure animal welfare.

Evaluation of DC Maturation

Female C57BL/6 mice were divided into six groups and subjected to different immunization regimens, including PBS, OVA alone, MPLA (OVA), ZnO (OVA), ZPNs (OVA), and p‐ZPNs (OVA). Immunizations were administered via injection into the left flank over the course of 3 weeks, with one injection per week. For each immunization, the OVA dose was kept at 300 µg per mouse across all groups. In the MPLA (OVA) group, MPLA was administered at a dose of 10 µg per injection. For the nanoparticle groups (ZnO (OVA), ZPNs (OVA), and p‐ZPNs (OVA), the nanoparticle dose was 1 mg per injection. Mice were sacrificed 48 h after the final (third) immunization, and lymph nodes and spleens were collected to isolate single‐cell suspensions for further analysis. To assess DC maturation, isolated cells were first incubated with Fc Block antibodies to minimize non‐specific binding, followed by surface staining with antibodies targeting CD11c, CD80, and CD86. Flow cytometry was performed to analyze the expression of these maturation markers, and data analysis was conducted using FlowJo 10.0 software. Doublets were excluded from analysis based on forward and side scatter profiles.

Evaluation of T Cells

To assess T cell responses in this study, female C57BL/6 mice aged 5 to 6 weeks were randomly divided into six groups, with six mice per group. Mice were immunized via subcutaneous injection into the left flank following different regimens, including PBS, OVA alone, MPLA (OVA), ZnO (OVA), ZPNs (OVA), and p‐ZPNs (OVA). Immunizations were administered three times at one‐week intervals. For each immunization, the OVA dose was maintained at 300 µg per mouse, MPLA was administered at 10 µg per injection in the MPLA (OVA) group, and for the nanoparticle groups (ZnO (OVA), ZPNs (OVA), and p‐ZPNs (OVA), the nanoparticle dose was 1 mg per injection. Five days after the final immunization, lymph nodes and spleens were harvested, and single‐cell suspensions were prepared for flow cytometry analysis. The isolated cells were first incubated with Fc Block (TruStain fcX, anti‐mouse CD16/32 Antibody, BioLegend) for 10 min to minimize non‐specific binding. Following Fc blocking, the cells were surface stained with Brilliant Violet 421 anti‐mouse CD3, PE anti‐mouse CD4, and PerCP/Cyanine5.5 anti‐mouse CD8a recombinant antibodies to characterize T cell subsets. To evaluate intracellular IFN‐γ production, the cells were fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BioLegend), followed by intracellular staining with fluorophore‐conjugated anti‐IFN‐γ antibodies. The cells were then washed and fixed with 4% paraformaldehyde prior to analysis. Flow cytometry was performed using an LSR II flow cytometer, and data were analyzed using FlowJo 10.0 software.

Immunization of Mice and Detection of OVA‐Specific Antibody and Cytokine Production

In the process of immunizing mice and assessing the production of OVA‐specific antibodies and cytokines, several steps were undertaken. Mice were first weighed and randomly divided into six groups, with each group containing six mice. Prior to immunization, pre‐immunization blood samples were collected through retro‐orbital bleeds to establish baseline measurements. The immunization doses consisted of OVA (300 µg) combined with either 1000 µg of ZnO, ZPNs, or p‐ZPNs, or 10 µg of MPLA, all prepared in saline. These immunization formulations were administered via subcutaneous (s.c) injection at the tail base, utilizing a sterile needle (Terumo, Tokyo, Japan). A total of three injections were given at three‐week intervals, and mice were euthanized five days after the final immunization.

To evaluate the immune response, the production of anti‐OVA antibodies, specifically IgG1, IgG2c, and total IgG, was assessed in plasma on day 21, which corresponded to five days after the third injection. This assessment was conducted using enzyme‐linked immunosorbent assay (ELISA) techniques. In addition, titers were calculated using a threshold set at twice the optical density (OD) of the PBS control group. The IgG2c/IgG1 ratio was determined by comparing the IgG2c levels of each sample to the average titer IgG1 levels. Furthermore, mouse serum samples were collected to measure the levels of cytokines, including IFN‐γ, IL‐12, and TNF‐α, using ELISA kits. These analyses provided valuable insights into the immune response induced by the different immunization strategies, shedding light on the potential immunomodulatory effects of the tested formulations and adjuvants.

Antigen Collection

B16‐F10 cell membrane was used as antigen loading to nanoparticles. The protocol involves culturing cells, detaching them with 2 mM EDTA PBS solution on ice for 20–30 min, and collecting the cells using a scraper. The cells were then washed with PBS three times via centrifugation at 500 g for 10 min. They were re‐suspended in hypotonic lysing buffer, frozen in liquid nitrogen or dry ice, thawed in a 37 °C‐water bath, and sonicated on ice, repeating this cycle for 6–10 times. The cells were centrifuged at 3200 g for 5 min, and the supernatants were collected and further centrifuged at 20 000 g for 30 min. The resulting supernatants were saved. For purification, the supernatant was centrifuged again at 80 000 g for 1.5 h. The pellets containing plasma membranes were washed with Tris‐HCl and EDTA, resulting in purified cell membranes ready for further experiments. The antigen was collected and loaded in nanoparticle in at 4 °C for 24 h.

In Vivo Preventive and Antitumor Efficacy

For the preventive study, female C57BL/6 mice were weighed and randomly divided into five groups. The groups received subcutaneous (s.c.) injections of PBS, MPLA‐M (10 µg MPLA), ZnO‐M, ZPNs‐M, or p‐ZPNs‐M (each containing 1 mg of nanoparticles) on days −21, −14, and −7. Each injection contained 0.1 mg of B16‐F10 cell membrane (M) as the antigen. On Day 0, all mice were challenged with 2 × 10⁵ B16‐F10 cells via subcutaneous injection. Tumor growth and body weight were monitored for 30 days to evaluate the preventive efficacy of the different formulations. For the therapeutic (antitumor) study, mice were similarly divided into six groups and subcutaneously injected with 2 × 10⁵ B16‐F10 cells on Day 0. Mice then received s.c. injections of PBS, MPLA‐M (10 µg MPLA), ZnO‐M, ZPNs‐M, p‐ZPNs‐M, or p‐ZPNs‐M combined with anti‐PD‐1 (p‐ZPNs‐M‐anti‐PD‐1), on days 3, 7, and 14. Each injection contained 1 mg of nanoparticles and 0.1 mg of B16‐F10 cell membrane (M) as the antigen. In the p‐ZPNs‐M‐anti‐PD‐1 group, mice also received intraperitoneal injections of anti‐PD‐1 antibody (60 µg per dose) on days 4, 8, and 15. Tumor growth and body weight were monitored over a 30‐day period to assess the therapeutic efficacy of the different treatment regimens.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, CA, USA). For comparisons between two groups, unpaired two‐tailed Student's t‐tests were used. No data transformation or outlier exclusion was applied prior to statistical testing. Data were presented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM), as indicated in figure legends. A minimum of three biological replicates (n ≥ 3) were used for each experiment unless otherwise specified. P values less than 0.05 were considered statistically significant and denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

Conflict of Interest

The authors declare no conflict of interest

Supporting information

Supporting Information

Luo J., Wang Y., Dai Z., et al. “Passivation‐Engineered Zinc Peroxide Nanoparticles as Th1‐Biased Adjuvant for Antitumor Immunity.” Small 21, no. 42 (2025): e05250. 10.1002/smll.202505250

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.