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• Tumor immunotherapy has promising application prospects in tumor prevention and treatment [1-5]. The tumor vaccine is an active tumor immunotherapy that triggers the patient’s autoimmunity through tumor antigens, inducing a specific immune response to eradicate tumor cells [6-8]. Tumor antigens come from various sources and can be divided into predefined (known) antigens and anonymous (unknown) antigens [9,10]. After vaccination, antigens are captured and processed by antigen-presenting cells (APCs), particularly dendritic cells (DCs). Then, the antigenic peptides are presented to the naïve T cells. Afterwards, the activated T cells are differentiated into helper CD4⁺ T cells, cytotoxic CD8⁺ T cells, and memory T cells. Tumor vaccines can directly kill tumor cells and prevent tumor metastasis and recurrence. However, the practical application of tumor vaccines is often limited by poor immunogenicity of tumor antigen, low delivery efficiency, weak antigen cross-presentation, and immunosuppressive tumor microenvironment (ITME).

  Adjuvants can enhance the specific response ability of the body to the antigen, reduce the amount of antigen used, or change the type of immune response [11-13]. Among them, Toll-like receptor (TLR) agonists can recognize pathogen-associated molecular patterns and have the ability to activate the immune system by increasing the expression of surface markers and the secretion of cytokines [14-16]. Cytosine-phosphate-guanine (CpG) is a TLR9 agonist composed of the primary CpG sequence that mimics the deoxyribonucleic acid (DNA) of bacteria and viruses. It can stimulate the activation of DCs and natural killer (NK) cells and then activate effector T cells to initiate a series of innate and adaptive immune responses [17,18]. In addition, it can transform tumor-associated macrophages (TAMs) from the pro-tumor type to the anti-tumor type, thus reprogramming ITME to effectively inhibit tumor growth and recurrence [19]. Studies have shown that polyethylenimide (PEI) can bind to the TLR4 receptor on the cell membrane to activate the TLR4 pathway and facilitate the internalization of nanoparticles by immune cells [20,21]. After phagocytosis, PEI can help antigens escape from the lysosome to the cytoplasm via the proton sponge effect to enhance antigen cross-presentation by DCs and induce the production of cytotoxic T lymphocytes. As an effective adjuvant, PEI can also precisely reverse TAMs from type M2 to type M1 for reprogramming and reconstructing anti-tumor immune responses [22,23]. To break the limitations of single adjuvant, combining two or more adjuvants can further regulate or enhance the immune response and improve the immune effect. In our previous work, we combined the TLR4 agonist monophosphoryl lipid A (MPLA) and TLR7 agonist imiquimod (IMQ) to synergistically activate DCs by both extra- and intra-cellular TLRs for enhancing adaptive immune responses [14]. Nevertheless, free antigens and adjuvants have poor molecular stability and are easily inactivated. To meet therapeutic needs, high-dose or repeated administrations cannot be avoided, which may lead to drug resistance and further increase systematic toxicity.

  The nano-delivery system can improve the biostability and bioavailability of antigens and adjuvants, deliver them to the same APCs, enhance cellular endocytosis efficiency, and mitigate the potential for off-target effects, thus significantly improving the effectiveness of tumor vaccines [5,24,25]. Various nanostructures-based tumor vaccines have been developed using inorganic or organic materials. However, most nano-carriers exhibit relatively poor drug loading ability, complex preparation process, and defective metabolism ability. In recent years, carrier-free nanoparticles entirely or mostly composed of pure active pharmaceutical ingredients (APIs) have been rapidly developed for disease theranostics. They are considered one of the most promising delivery vehicles for tumor vaccines with the advantages of extraordinary API loading, simple preparation process, avoidable carrier-induced toxicity, and strong clinical translational potential.

  In this study, we synthetized fluoroalkyl substituted PEI material (F-PEI) to reduce toxicity from excessive positive charges of PEI. Then, double adjuvant carrier-free nanoparticles (FPC-NPs) were formulated by electrostatic adsorption of positively charged F-PEI and negatively charged CpG, in which F-PEI was approved for clinical trials while CpG was approved for clinical use [23,26]. The particle size, zeta potential, encapsulation efficiency, and in vitro antigen-trapping ability of FPC-NPs were evaluated. Tumor-associated antigens (TAAs) produced by tumor cells treated with doxorubicin (DOX) were extracted and successfully adsorbed onto the nanoadjuvant to obtain personalized tumor nanovaccine FPC-NPs@TAAs. As shown in Scheme 1, the personalized tumor nanovaccine enhanced cellular uptake by DCs, activated DCs through both TLR4 and TLR9 signaling pathway, and promoted cross-presentation of antigens. In addition, our results demonstrated that FPC-NPs@TAAs could induce macrophage repolarization, showing potential in remodeling tumor immune microenvironment and enhancing tumor immunotherapy.

  Scheme 1

  

  Scheme 1.  Schematic mechanism of personalized double-adjuvant tumor nanovaccine for enhancing immune response.

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  As PEI is known to be cytotoxic, the fluoroalkane-grafted PEI was obtained in our study via an amine-epoxide reaction between fluoride and PEI [27]. The synthesized F-PEI was then mixed with CpG at a 1:1 mass ratio to form F-PEI/CpG nanoparticles (FPC-NPs) through electrostatic adsorption between the cationic F-PEI and negatively charged CpG (Fig. 1A). The details of preparation and characterization were provided in Supporting information. Atomic force microscopy (AFM) photographs showed that the synthetic FPC-NPs had a uniform spherical structure (Fig. 1B). The average hydrodynamic size of FPC-NPs was 177.27 ± 1.23 nm with polydispersity index (PDI) of 0.215 (Figs. 1C and D), and the zeta potential was ~25.7 ± 0.2 mV (Fig. 1E). Additionally, FPC-NPs exhibited characteristic ultraviolet (UV) absorption peaks similar to those of free CpG, confirming the successful encapsulation of CpG within FPC-NPs (Fig. 1H). The encapsulation efficiency of CpG was 98.4%, which is much higher than other conjugated loading methods [28]. It is worth noting that the prepared nanoadjuvants consisted solely of two types of TLR agonists without additional carrier materials, which addresses the issues associated with low drug loading, complex preparation processes, and potential carrier toxicity commonly encountered in traditional nanovaccine development.

  Figure 1

  

  Figure 1.  (A) Schematic illustration to show the preparation of FPC-NPs. (B) AFM image of FPC-NPs. (C) The hydrodynamic size distribution of FPC-NPs. (D) The hydrodynamic size and (E) zeta potential of FPC-NPs and FPC-NPs@TAAs. (F) AFM image of FPC-NPs@TAAs. (G) The hydrodynamic size distribution of FPC-NPs@TAAs. (H) UV–vis absorption spectra of F-PEI, CpG, and FPC-NPs. Data were presented as mean ± SEM (n = 3).

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  Then, we evaluated the capability of our nanoadjuvants to bind TAAs by the outer positively charged F-PEI. As we all know, chemotherapy is a widely utilized strategy to induce immunogenic cell death (ICD), which can not only release danger-associated molecular patterns (DAMPs) but also promote the release of TAAs [29]. The TAAs released from DOX-treated tumor cells were collected and mixed with FPC-NPs by mild vibration to simulate the antigen capture process in vivo. After incubation with TAAs, FPC-NPs@TAAs showed spherical structure (Fig. 1F) and exhibited a significant increase in the particle size (397.60 ± 13.93 nm, PDI 0.234) and a decrease in the zeta potential (−13.7 ± 0.2 mV) compared with FPC-NPs, confirming that TAAs were successfully adsorbed on the surface of the nanoadjuvants (Figs. 1B–G). The quantitative analysis revealed that 298.27 ± 15.73 µg of proteins were bound onto the FPC-NPs, corresponding to an antigen loading efficiency of 52.05%. These results underscored the potential of the nanoadjuvants to generate personalized tumor vaccines through self-assembly with tumor antigens released in vivo after DOX killed tumor cells.

  Although CpG has been approved by the Food and Drug Administration (FDA), the poor stability and low cellular uptake greatly limit further clinical utility [30,31]. Encapsulation by nanoparticles can effectively improve the cell internalization. We then investigated the influence of FPC-NPs on the cellular internalization of CpG. In this study, fluorescent labeled CpG-Alexa 488 was employed to formulate FPC-NPs, and then free CpG and FPC-NPs were cultured with DC2.4 cells for further determination. Confocal laser scanning microscope (CLSM) imaging confirmed the progressive accumulation of green fluorescence-labeled CpG facilitated by the cellular delivery of FPC-NPs over time (Fig. 2A). Flow cytometry results also showed that free CpG was hardly internalized by DC2.4 cells, whereas FPC-NPs effectively facilitated the internalization of adjuvants (Figs. 2B and C). Furthermore, a time-dependent adjuvant cellular delivery was observed with an increase of incubation time from 1 h (6.5%) to 12 h (57.0%). Nanoparticles with suitable size were readily phagocytized by cells directly [32], while F-PEI also mediated an assisted endocytosis through TLR4 receptors located on the cell membrane of DCs. Notably, the fluoroalkyl chains with hydrophobic and lipophobic characteristics would be easily fused across the cell membrane, which may also play a unique role in augmenting the cellular uptake of FPC-NPs [27,33].

  Figure 2

  

  Figure 2.  (A) Confocal images of DC2.4 cells treated with free CpG or FPC-NPs for 1 or 12 h. Scale bar: 10 µm. CpG labeled by Alexa 488 (green) and the nuclei were stained with Hoechst (blue). (B) Representative flow cytometry histograms of adjuvants internalization by DC2.4 cells after treatment for 1 and 12 h. (C) Proportion of adjuvants internalization by DC2.4 cells after treatment for 1 and 12 h. (D) DC2.4 cells survival ratio after incubating with different concentrations (5, 10, 20 µg/mL) of PEI, F-PEI, free CpG, and FPC-NPs. (E) Representative flow cytometry scatter plots of in vitro cytotoxicity of BMDCs after cultivating with different preparations. Data were presented as mean ± SEM (n = 3). ^***P < 0.001.

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  Then, the ability of FPC-NPs@TAAs and several control formulations (phosphate buffer saline (PBS), CpG, F-PEI, and FPC-NPs) to promote the maturation of DCs and facilitate antigen cross-presentation in vitro was investigated. First, the toxicities of these adjuvants and NPs were evaluated. The MTS assay showed that unmodified PEI exhibited pronounced cytotoxicity against the DC2.4 within the tested concentration range. When 10 µg/mL of PEI was applied, <40% of the cells survived (Fig. 2D). In comparison, F-PEI exhibited much reduced cytotoxicity. We also evaluated their cytotoxicity towards bone marrow-derived dendritic cells (BMDCs) by examining cellular apoptosis using Annexin V/7-amino-actinomycin D (7-AAD) staining. After incubating BMDCs with unmodified PEI, we observed that >5% of the cells were in the advanced apoptotic stage or were already dead (Annexin V⁺, 7-AAD⁺), and simultaneously, over 10% of the cells were in the early apoptotic stage (Annexin V⁺, 7-AAD⁻), indicating the cytotoxic nature of unmodified PEI. The percentages of early or advanced apoptotic cells incubated with other formulations were <5% (Fig. 2E). The results confirmed that fluoroalkane-grafted PEI significantly reduced the cytotoxicity of unmodified PEI.

  Given that DCs represent a pivotal class of APCs, their maturation and activation are indispensable for triggering subsequent immune responses [23]. Thus, we subsequently assessed the expression of co-stimulatory molecules on BMDCs after a 24-h incubation with various formulations using flow cytometry. In comparison to the soluble form of CpG or F-PEI alone, the treatment of BMDCs with FPC-NPs led to a significant up-regulation in the expression of CD40, CD80, and CD86, which could be due to the notably increased cellular uptake and the activation of a synergistic immune response. It is worth noting that FPC-NPs@TAAs, the nanovaccine prepared by co-incubating FPC-NPs with TAAs, exhibited a much more potent immune stimulation effect for BMDCs maturation than FPC-NPs, which may be caused by the delivery of both antigens and adjuvants to the same BMDCs (Figs. 3A–F). In addition, major histocompatibility complex (MHC) molecules, which can form complexes with processed antigenic peptides and be expressed on the surface of DCs to activate T cells, were evaluated to demonstrate the effect of FPC-NPs on antigen presentation by DCs. Compared to the PBS control, free CpG only slightly increased the presentation of MHC Ⅰ and MHC Ⅱ on the surface of BMDCs, and F-PEI demonstrated a stronger ability to induce MHC presentation by BMDCs (Figs. 3G–J). This enhanced presentation can be attributed to F-PEI’s nature as a TLR4 agonist and its cationic structure, which facilitates binding to cell membranes and subsequent uptake by cells. Remarkably, both the FPC-NPs and FPC-NPs@TAAs groups achieved a significantly improved antigen presentation efficiency, with FPC-NPs@TAAs inducing the highest levels of MHC molecules on the surface of BMDCs. This result emphasized the potential of FPC-NPs@TAAs for promoting antigen cross-presentation in vitro.

  Figure 3

  

  Figure 3.  The maturity level of BMDCs. Representative flow cytometry histograms and quantitative analysis of (A, B) CD40⁺, (C, D) CD80⁺, (E, F) CD86⁺, (G, H) MHC Ⅰ⁺, and (I, J) MHC Ⅱ⁺ expression on the surface of DCs after various treatments. Data were presented as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. FPC-NPs@TAAs group.

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  In addition to DCs, as an integral component bridging innate and adaptive immunity, macrophages exhibit the ability to non-specifically phagocytize pathogens and serve as APCs that process antigens while regulate immunity through secretions. Studies have shown that reprogramming TAM can effectively improve ITME [7]. The cellular uptake study was conducted to ascertain the internalization of nanovaccines by RAW264.7, a macrophage cell line. Confocal images revealed that green fluorescent intensity of CpG in FPC-NPs group was significantly increased compared to free CpG after incubation with RAW264.7 for 12 h (Fig. 4A). The quantitative analysis of cellular uptake was further confirmed through flow cytometry, revealing the similar trends (Figs. 4B and C). Except as the APCs, the phenotypic characteristics of macrophages play a pivotal role in initiating subsequent immune responses and shaping the composition of the tumor microenvironment. Among them, M2 macrophages, a subset to exhibit a tumor-promoting function, can potentially promote the proliferation and metastasis of primary tumors by inhibiting the immune activity of effector T cells and increasing intratumoral infiltration of regulatory T cells (Tregs). We then analyzed the proportion of M2 macrophages. The results demonstrated that treatment with FPC-NPs or FPC-NPs@TAAs significantly reduced the percentage of M2 macrophages (F4/80⁺, CD206⁺) compared to both the PBS control group and the groups receiving single adjuvants alone (Fig. 4D). Repolarizing TAMs from the M2-subtype to M1-subtype state could be a promising strategy to remove the immunosuppressive obstacles [34]. Herein, our results showed that after treatment of F-PEI, FPC-NPs, and FPC-NPs@TAAs, the ratio of M1/M2 bone-marrow-derived macrophage cells (BMDMs) was increased (Fig. 4E). This observation suggested that the NPs incorporating both F-PEI and CpG can efficiently increase the proportion of M1 macrophages and reduce the proportion of M2 macrophages. Previous research indicated that F-PEI can reduce M2 macrophages through TLR4 signaling [23] and CpG can “awaken” macrophages to eliminate tumor cells [35]. Our result demonstrated that FPC-NPs had synergistic effect on reprogramming macrophages, showing great promise in tumor immunotherapy.

  Figure 4

  

  Figure 4.  (A) Confocal images of RAW264.7 cells treated with free CpG or FPC-NPs for 1 or 12 h. Scale bar: 10 µm. CpG was labeled by Alexa 488 (green) and the nuclei were stained with Hoechst (blue). (B) Representative flow cytometry histograms and (C) proportion of adjuvants internalization by RAW264.7 cells after treatment for 1 and 12 h. (D) Representative flow cytometry plots of BMDMs. (E) Quantitative analysis of M1/M2 BMDMs after various treatments. (F) The representative in vivo fluorescence images detected at 1, 6, 12, and 24 h after subcutaneous injection of free CpG, FPC-NPs, and FPC-NPs@TAAs. (G) The fluorescence images of lymph nodes isolated at 24 h postinjection. Data were presented as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ^##P < 0.01 vs. FPC-NPs@TAAs group.

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  The success of immune responses largely relies on the motility of immune cells that constantly circulate between peripheral tissues and lymphoid organs [36]. Therein, mature DCs migration to lymph nodes to encounter with naïve T cells and facilitate T cell priming is a crucial process for triggering immune response [11]. For the in vivo lymph node migration experiments, we injected Cy7-labeled free CpG or nanoparticles subcutaneously into the tail root of mice. All animal experiments were approved and conducted in accordance with the guidelines for the Animal Ethical and Welfare Committee of the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences. The fluorescence signal of Cy7 was imaged at different time points. It was found that the Cy7 signal in the lymph nodes of mice treated with FPC-NPs and FPC-NPs@TAAs began to appear at 12 h, and gradually became obvious at 24 h (Fig. 4F). After 24 h, the mice bilateral inguinal lymph nodes were removed for ex vivo imaging. The results showed that FPC-NPs and FPC-NPs@TAAs had better lymph node migration ability than free CpG (Fig. 4G). We will further explore the in vivo therapeutic effect and immune improvement of this nanoadjuvant combined with other tumor therapies that can generate TAAs in vivo and form in situ personalized nanovaccine in later works.

  In summary, carrier-free double-adjuvant nanoparticles (FPC-NPs) with the ability to trigger both TLR4 and TLR9 signaling pathway were self-assembled by electronic adsorption of F-PEI and CpG. In vitro experiments showed that the nanostructure can promote the uptake of adjuvants by DCs and macrophages. In addition, FPC-NPs can bind TAAs to form personalized tumor nanovaccines (FPC-NPs@TAAs), showing the capability to effectively activate DCs and promote antigen cross-presentation. Furthermore, the immune nanoadjuvants significantly increased the proportion of anti-tumor macrophages and reduced the proportion of inhibitory macrophages, thus constructing a highly immunogenic tumor microenvironment to improve the immunotherapeutic effect of tumor vaccines. In vivo experiments showed that nanoadjuvants with or without TAAs can successfully migrate to lymph nodes, thus having the capability to further activate the systemic immune response. Our work has demonstrated a simple yet effective strategy to construct immune adjuvants for enhancing the efficacy of personalized tumor vaccines.

  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.

  CRediT authorship contribution statement

  Hanyong Wang: Writing – original draft, Methodology, Investigation, Data curation. Weijia Zhang: Methodology, Investigation. Chenlu Huang: Validation, Supervision, Project administration. Xinyu Yang: Investigation, Data curation. Qingyu Yu: Methodology, Investigation. Hai Wang: Validation, Software, Resources. Wen Li: Writing – review & editing, Supervision, Conceptualization. Linhua Zhang: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Dunwan Zhu: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

  Acknowledgments

  This work was supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (No. 2023ZD0500800), National Natural Science Foundation of China (Nos. 82302390, 82172090 and 82072059); CAMS Innovation Fund for Medical Sciences (Nos. 2021-I2M-1-058, 2022-I2M-2-003 and 2023-I2M-2-008); China Postdoctoral Science Foundation (No. 2022M720502); Tianjin Municipal Natural Science Foundation (Nos. 22JCQNJC00070 and 24ZXZSSS00200); CAMS Union Young Scholars Support Program (No. 2022051); and Fundamental Research Funds for the Central Universities (No. 2019PT320028).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.111092.

  
  
• 1. [1]

     L. Drew, Nature 627 (2024) S33. doi: 10.1038/d41586-024-00840-z

  2. [2]

     M. Gupta, A. Wahi, P. Sharma, et al., Vaccines 10 (2022) 2011. doi: 10.3390/vaccines10122011

  3. [3]

     Y. Xie, X. Li, J. Wu, et al., Chin. Chem. Lett. 34 (2023) 108202. doi: 10.1016/j.cclet.2023.108202

  4. [4]

     S. Liang, J. Yao, D. Liu, et al., Chin. Chem. Lett. 36 (2025) 109856. doi: 10.1016/j.cclet.2024.109856

  5. [5]

     H. Guo, Y. Hou, C. Wang, et al., Oncologie 26 (2024) 343–348. doi: 10.1515/oncologie-2024-0037

  6. [6]

     C. Huang, H. Wang, X. Yang, et al., Adv. Funct. Mater. 34 (2024) 2401489.

  7. [7]

     W. Xu, Y. Su, Y. Ma, et al., Sci. China Chem. 66 (2023) 1150–1160. doi: 10.1007/s11426-022-1441-7

  8. [8]

     H. Wang, X. Yang, C. Hu, et al., Chin. Chem. Lett. 33 (2022) 4179–4184. doi: 10.1016/j.cclet.2022.02.017

  9. [9]

     M.J. Lin, J. Svensson-Arvelund, et al., Nat. Cancer 3 (2022) 911–926. doi: 10.1038/s43018-022-00418-6

  10. [10]

      L. Diao, M. Liu, Adv. Sci. 10 (2023) e2300121.

  11. [11]

      X. Zhang, B. Yang, Q. Ni, et al., Chem. Soc. Rev. 52 (2023) 2886–2910. doi: 10.1039/d2cs00647b

  12. [12]

      K. Liu, J. Peng, Y. Guo, et al., ACS Nano 18 (2024) 11910–11920. doi: 10.1021/acsnano.4c01691

  13. [13]

      Y. Su, W. Xu, Q. Wei, et al., Sci. Bull. 68 (2023) 284–294.

  14. [14]

      N. Wang, Y. Zuo, S. Wu, et al., Acta Pharm. Sin. B 12 (2022) 4486–4500.

  15. [15]

      R.G. Everson, W. Hugo, L. Sun, et al., Nat. Commun. 15 (2024) 3882.

  16. [16]

      A. Kaur, J. Baldwin, D. Brar, et al., Curr. Opin. Chem. Biol. 70 (2022) 102172.

  17. [17]

      Y. Wang, S.L. Qiao, J. et al., Adv. Mater. 36 (2024) e2306248.

  18. [18]

      J. Wei, D. Wu, S. Zhao, et al., Adv. Sci. 9 (2022) e2103689.

  19. [19]

      Z. Liu, S. Li, Y. Xiao, et al., Adv. Sci. 11 (2024) e2402678.

  20. [20]

      J. Xu, J. Lv, Q. Zhuang, et al., Nat. Nanotechnol. 15 (2020) 1043–1052. doi: 10.1038/s41565-020-00781-4

  21. [21]

      V. Mulens-Arias, J.M. Rojas, S. Pérez-Yagüe, et al., Biomaterials 52 (2015) 494–506.

  22. [22]

      J. Wang, F. Meng, B.K. Kim, et al., Biomaterials 217 (2019) 119296.

  23. [23]

      Z. Huang, Y. Yang, Y. Jiang, J. et al., Biomaterials 34 (2013) 746–755.

  24. [24]

      S. Meng, H. Du, X. Li, et al., ACS Nano 18 (2024) 3134–3150. doi: 10.1021/acsnano.3c08792

  25. [25]

      X. Duan, H. Zou, J. Yang, et al., J. Control. Release 375 (2024) 285–299.

  26. [26]

      J.D. Campbell, Methods Mol. Biol. 1494 (2017) 15–27. doi: 10.1007/978-1-4939-6445-1_2

  27. [27]

      Z. Zhang, W. Shen, J. Ling, et al., Nat. Commun. 9 (2018) 1377.

  28. [28]

      J. Meng, P. Zhang, Q. Chen, Z. et al., Adv. Mater. 34 (2022) e2202168.

  29. [29]

      B. Wiernicki, S. Maschalidi, J. Pinney, et al., Nat. Commun. 13 (2022) 3676.

  30. [30]

      Q. Ni, F. Zhang, Y. Liu, et al., Sci. Adv. 6 (2020) eaaw6071.

  31. [31]

      J.L. Perry, S. Tian, N. Sengottuvel, et al., ACS Nano 14 (2020) 7200–7215. doi: 10.1021/acsnano.0c02207

  32. [32]

      C. Foged, B. Brodin, S. Frokjaer, et al., Int. J. Pharm. 298 (2005) 315–322.

  33. [33]

      M. Wang, H. Liu, L. Li, et al., Nat. Commun. 5 (2014) 3053.

  34. [34]

      S. Liang, Y. Liu, T. Gao, et al., Adv. Funct. Mater. 31 (2021) 2401489.

  35. [35]

      M. Liu, R.S. O’Connor, S. Trefely, et al., Nat. Immunol. 20 (2019) 265–275. doi: 10.1038/s41590-018-0292-y

  36. [36]

      Z. Alraies, C.A. Rivera, M.G. Delgado, et al., Nat. Immunol. 25 (2024) 1193–1206. doi: 10.1038/s41590-024-01856-3

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  Scheme 1  Schematic mechanism of personalized double-adjuvant tumor nanovaccine for enhancing immune response.

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  Figure 1  (A) Schematic illustration to show the preparation of FPC-NPs. (B) AFM image of FPC-NPs. (C) The hydrodynamic size distribution of FPC-NPs. (D) The hydrodynamic size and (E) zeta potential of FPC-NPs and FPC-NPs@TAAs. (F) AFM image of FPC-NPs@TAAs. (G) The hydrodynamic size distribution of FPC-NPs@TAAs. (H) UV–vis absorption spectra of F-PEI, CpG, and FPC-NPs. Data were presented as mean ± SEM (n = 3).

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  Figure 2  (A) Confocal images of DC2.4 cells treated with free CpG or FPC-NPs for 1 or 12 h. Scale bar: 10 µm. CpG labeled by Alexa 488 (green) and the nuclei were stained with Hoechst (blue). (B) Representative flow cytometry histograms of adjuvants internalization by DC2.4 cells after treatment for 1 and 12 h. (C) Proportion of adjuvants internalization by DC2.4 cells after treatment for 1 and 12 h. (D) DC2.4 cells survival ratio after incubating with different concentrations (5, 10, 20 µg/mL) of PEI, F-PEI, free CpG, and FPC-NPs. (E) Representative flow cytometry scatter plots of in vitro cytotoxicity of BMDCs after cultivating with different preparations. Data were presented as mean ± SEM (n = 3). ^***P < 0.001.

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  Figure 3  The maturity level of BMDCs. Representative flow cytometry histograms and quantitative analysis of (A, B) CD40⁺, (C, D) CD80⁺, (E, F) CD86⁺, (G, H) MHC Ⅰ⁺, and (I, J) MHC Ⅱ⁺ expression on the surface of DCs after various treatments. Data were presented as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. FPC-NPs@TAAs group.

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  Figure 4  (A) Confocal images of RAW264.7 cells treated with free CpG or FPC-NPs for 1 or 12 h. Scale bar: 10 µm. CpG was labeled by Alexa 488 (green) and the nuclei were stained with Hoechst (blue). (B) Representative flow cytometry histograms and (C) proportion of adjuvants internalization by RAW264.7 cells after treatment for 1 and 12 h. (D) Representative flow cytometry plots of BMDMs. (E) Quantitative analysis of M1/M2 BMDMs after various treatments. (F) The representative in vivo fluorescence images detected at 1, 6, 12, and 24 h after subcutaneous injection of free CpG, FPC-NPs, and FPC-NPs@TAAs. (G) The fluorescence images of lymph nodes isolated at 24 h postinjection. Data were presented as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ^##P < 0.01 vs. FPC-NPs@TAAs group.

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