Tumor antigens are produced by genetic mutations or abnormal or faulty protein translation modifications (42). They are either tumor-specific expressed, which means they are not present in normal tissue cells and are called tumor-specific antigens(TSAs) (43), or present in the normal body but are overexpressed in cancer cells, called tumor-associated antigens(TAAs) (44). According to the different sources of tumor antigens, nanovaccines are mainly categorized into four types: peptide vaccines, DNA vaccines, mRNA vaccines, and cell vaccines.

The abundance of mutations observed in tumor genomes can be leveraged as a valuable resource for the development of tumor vaccines. Neoantigens are mutant peptides resulting from gene mutation transcription and translation that can specifically induce T lymphocytes (45). Neoantigens are highly specific and immunogenic, have no thymus-negative screening, and are expressed only in tumor tissues (43). Therefore, tumor neoantigens are considered ideal targets for immunotherapy, especially for individualized tumor vaccines. To identify neoantigens, whole genome or exonic sequencing of somatic and tumor cells from the same patient is required to recognize mutations in individual tumor protein-coding genes (46). Not all mutant peptides are neoantigens; only those that can be recognized and presented by MHC and elicit a T-cell immune response can be called neoantigens. Many clinical trials of personalized therapeutic cancer vaccines based on neoantigens are ongoing. In an ongoing phase II trial in patients with advanced non-small cell lung cancer, four aqueous peptide mixtures were synthesized, each containing six neoantigen peptides. Extensive stability studies at −25°C or −80°C showed that the formulation was stable for up to 32 weeks, which provided guidance for the development of new personalized therapeutic vaccines (47). However, screening neoantigens is a complex process, different databases or algorithms may produce different results, and it is necessary to comprehensively consider the time, tool, labor costs, and treatment benefits.

Nanocarriers are promising tools to carry multiple therapeutic agents, increase the solubility of cargo, stabilize therapeutic activity in vivo, enhance the targeted delivery of drugs, and reduce drug-related side effects (65). Some carriers possess adjuvant properties, which activate the immune system and enhance immune responses, thereby improving the efficacy of vaccines (66). Liposomes, polymer nanocarriers, inorganic nanocarriers, and biomimetic nanocarriers are among the commonly used nanocarriers today.

Immune adjuvants, also known as immune boosters, are key components of vaccines that significantly enhance the magnitude, breadth, and persistence of the immune response (88). The immunostimulatory properties of adjuvants are essential for enhancing the immunogenicity of subunit antigens (89). Adjuvants can be classified into two categories according to their mechanism of action, vaccine delivery systems and immunostimulants. Mineral salts, emulsion adjuvants, liposomes, and biogenic nanomaterials, and are commonly used as vaccine delivery systems to more effectively present antigens to effector cells, achieve controlled antigen release, and induce long-lasting immunity. Immune adjuvants can activate innate immunity either by acting directly as cytokines or indirectly through the recognition of specific patterns by pattern recognition receptors(PRRs) (90, 91). The main types and functions of immune adjuvants are summarized in Table 2 . Although adjuvants are extremely critical for the efficacy of most vaccines, they also have the potential to induce undesirable immune reactions. Therefore, how to achieve an efficient combination of antigenic adjuvants while producing minimal side effects is a factor to be considered before vaccine design.

The effectiveness of tumor vaccines is strongly associated with their immunogenicity (114). However, endogenous antigens produced by immunogenic death are often inadequate to elicit robust anti-tumor immunity. The low immunogenicity of tumors is one of the major bottlenecks of tumor immunotherapy. Enhancing tumor immunogenicity can improve the efficacy of tumor immunotherapy by increasing tumor antigen exposure and DCs targeting, and supplementing with adjuvant (115).

The delivery of exogenous immunogenic antigens and the release of endogenous immunogenic antigens by inducing immunogenic cell death (ICD) are the two main ways to increase antigen exposure. In the former case, these immunogenic antigens can be peptides (116), TCLs (32), nucleic acids (117), and neoantigens (112). In the latter case, inducing ICDs in tumor cells, resulting in the release of various danger-associated molecular patterns (DAMPs) and TAAs, can effectively stimulate anti-tumor immune responses, promote DC maturation, and facilitate the infiltration of CTLs (104).

Receptors exist on the surface of DCs that mediate the uptake of extracellular antigens and their import into the phagosomal compartment of DCs. Therefore, direct targeting of antigens via nanocarriers to specific extracellular receptors expressed by DCs, such as DEC-205, CLEC9a, XCR1, and mannose receptors (118), is an effective strategy to enhance immunogenicity. Feng et al. utilized nanoliposomes to encapsulate OVA and dulcimer, which were targeted to DCs using anti-DEC-205 receptor antibodies (30). The study found that the nanoliposomes enhanced the proliferation and maturation of DCs, improved their phagocytic efficiency, and stimulated both cellular and humoral immunity. In some cases, adjuvants such as cytokines, chemokines, TLR agonists, and STING agonists are co-administered with tumor antigens to further enhance the immunogenicity of tumors. CLEC9a is a C-type-lectin-like receptor that is specifically expressed in DCs. CLEC9a antibodies induce humoral, CD4^+, and CD8⁺ Tcell responses through antigen-specific delivery to DCs. Researchers constructed a 12-peptide vector CBP-12 with high affinity to Clec9a to deliver exogenous or endogenous antigens (119). Following this work, a nanovaccine drug delivery system coated with an engineered peptide expressing bionic cancer cell membrane (EPBM) was designed (PLGA/STING@EPBM) to deliver STING agonists and tumor antigens to Clec9a(+)DC, which significantly enhanced IFN-stimulated gene expression and antigen-cross-presentation of Clec9a(+)DC, resulting in a strong anti-tumor effect in both anti-PD-1 responsive and drug-resistant tumor models (120). Wylie and colleagues constructed a novel vaccine containing XCL1 ligands to target the uptake of XCR1(+) cross-presenting DCs and a cell-penetrating peptide (CPP) with endosome escape properties to enhance antigen entry into the cross-presenting pathway, which showed promising tumor suppression in a B16 murine melanoma model (13). Similarly, DC-targeting mannose and immune adjuvant CpG-ODN were assembled on the surface of liposomes, loaded with melanoma-specific TRP2 peptide as liposome vaccine, which could be effectively taken up by DCs and led to increased DCs activation, enhancing the efficacy of tumor vaccine (95).

Lymph nodes (LNs) are small, bean-shaped organs located throughout the body that are an essential part of the immune system. Within the lymph nodes, immune cells such as DCs, macrophages, B cells, and T cells interact and collaborate to initiate and coordinate adaptive immune responses against foreign invaders like viruses, bacteria, and cancer cells (121). DCs residing in peripheral tissues capture antigens and then migrate through afferent lymphatic vessels toward draining lymph nodes under the action of the chemokine receptor CCR7 (122). Antigen-presenting DCs cells that reach the lymph nodes induce the activation of homologous naive T lymphocytes, stimulating and amplifying the immune response of tumor antigen-reactive T lymphocytes (123). After activation in the lymph nodes, the antigen-specific T lymphocytes travel throughout the body via the efferent lymphatic vessels and circulatory system. These T cells can directly or indirectly recognize and respond to the antigen and migrate to the tumor site to eliminate tumor cells containing the same antigen (124).

The immune microenvironment of peritumor lymph nodes, such as tumor-draining lymph nodes (TDLNs), is thought to be immunosuppressive, which can create favorable conditions for cancer cells to migrate to their proliferating lymph nodes and even spread to other organs (125). To achieve a full anti-tumor effect, effective delivery of the vaccine to lymph nodes is required (124). Nanoparticle sizes between 10-50 nm are more likely to be primed to the lymph nodes. A polymeric micelle containing a platinum-based anticancer drug with a diameter of 30 nm was reported to be more effective in preventing tumor lymph node metastasis by better tumor penetration and accumulation compared to nanomicelles with a diameter of 70 nm under similar conditions of stability, zeta potential, drug release rate, and plasma clearance (126). Neutral or negatively charged NPs are more likely to flow freely into lymphatic vessels than positively charged NPs. Researchers constructed phenylboronic ester modified trp2 tumor antigen-containing peptide nanovaccine and demonstrated that TRP2 nanovaccine could be effectively trapped into lymph nodes, taken up by DCs, and induced DCs maturation as one of the results of increasing negative charge (18). Different shapes of nanomaterials lead to different targeting and accumulation effects. Mueller and his colleagues reported that cylindrical (80 × 180 nm) hydrogel platforms, which are more amenable to LNS targeting and aggregation than conventional spherical NPs, can elicit stronger immune responses (127). Improving the ability of peripheral DCs to homing to lymph nodes by surface modification of NPs is also a target for cancer immunotherapy. In addition, since the targeted migration of DCs to lymph nodes requires CCR7 for initiation, achieving high expression of CCR7 via nanovectors is also one of the strategies to enhance lymph node targeting. Encapsulation of OVA and plasmid DNA encoding CCR7 in nanomicelles achieves active DCs targeting and cytoplasmic release of antigen, which not only promotes DCs activation and maturation but also facilitates DCs migration to lymph nodes by increasing CCR7 expression, thereby enhancing CD8⁺ T cell immune responses (109).

Notably, the route of administration of nanovaccine can also affect their organ targeting. Several studies have shown that transdermal administration via microneedles promotes vaccine accumulation in TDLNs, enhances cellular uptake, activates DCs maturation, and enhances Th1 immune response (128–130).

DCs are specialized APCs that are crucial in initiating and modulating immune responses (131). They play a key role in the recognition, capture, and presentation of antigens to T cells, which in turn activate effector cells that can eliminate pathogens, infected cells, or cancer cells (132) ( Box 1 ). A widely accepted view is that immature DCs promote immune tolerance, while mature DCs stimulate immune responses to foreign antigens (133). More and more nanovaccines are developed with the inclusion of immunostimulatory components to promote DCs maturation, in addition to effective antigen loading. PRRs play a crucial role in DCs maturation and are essential components of innate immunity required to activate DCs, serving as a bridge between innate and adaptive immunity (134). Toll-like receptors(TLRs) are a widely known family of PRRs, and their agonists have been extensively studied as adjuvant components of nanovaccines (135). Zhang et al. used graphene oxide (GO) nanosheets as a carrier and used electrostatic interactions to couple OVA and adjuvant CpG ODN, aTLR 9 agonists, to GO-PEI nanosheets to effectively induce DCs maturation. It significantly inhibited tumor growth and prolonged survival in a mouse model of cholera (136). In recent years, researchers have gained a better understanding of DCs antigen cross-presentation mechanisms, and endosome-targeted nanovaccines are playing an increasingly important role in promoting antigen cross-presentation and DCs maturation. For instance, a TLR7/8 agonist-conjugated nanovaccine (TNV) was synthesized with an ultrasensitive pH-responsive component, iPDPA polymer. This polymer can detect a slight decrease in endosomal pH upon disassembly into monomers. Upon treatment with histone B, the free Toll-like receptor agonist imidazoquinoline (IMDQ) compound is released, which stimulates TLR7/8 localized on the endosomal membrane. This stimulation leads to DCs maturation and elicits specific cellular immunity (105).

In the context of vaccine development, exogenous antigens are first taken up by DCs via endocytosis and can be further processed and trimmed by resident cysteine protease histone proteases, such as histone S. Alternatively, these antigens can be directly digested by active proteasomes, and the resulting antigenic peptides are loaded onto MHC-I molecules derived from circulating endosomes, leading to activation of CD8⁺ T cells. To enhance the antigen cross-presentation process, one study utilized nanocarriers to attach ovalbumin to substrates of histone S in phagocytosed endosomes, effectively targeting histone S. The results of this study demonstrated that the use of nanocarriers significantly enhanced the efficiency of resident histone-mediated cross-presentation in DCs (143). In addition to targeting specific extracellular receptors on DCs, endosomal escape is another strategy to facilitate antigen cross-presentation. Large intracytoplasmic delivery of antigens can be induced by endosomal escape, which enhances antigen cross-presentation. For instance, bacterial-derived outer membrane vesicles (OMVs) have been used as an mRNA delivery platform to promote endosomal escape. In one study, RNA-binding protein L7Ae and lysosomal escape protein listeriolysin O were surface-modified onto OMVs to deliver mRNA to DCs. The results showed that the vaccine significantly inhibited tumor progression, promoted tumor regression, and established long-term immunity for a certain time (101). Cell-penetrating peptides, which are composed of 4-40 amino acids, are effective tools for delivering mRNA and have been shown to facilitate endosomal escape (144). Wu et al. used cytoplasmic localized internalizing peptide 6 (CLIP6), a specific CCP, to couple to the OVA for direct translocation across the cell membrane (145). The uptake of this nanoparticle by DCs is significantly increased compared to bare OVA and, more importantly, significantly enhances antigen cross-presentation and induces a stronger CTL-mediated immune response with the help of adjuvants. Similarly, researchers synthesized a DC-targeted pH-sensitive liposome nanovaccine, which consisted of a pH-sensitive polymer that became hydrophobic under weakly acidic conditions in endosomes. This property promoted the fusion of liposomes and endosomal membranes, enhancing antigen release to the cytoplasm and improving cross-presentation efficiency by DCs (106).

The suppressive effects of the TME often result in reduced infiltration or dysfunction of immune cells such as DCs, and their defective function is one of the key factors responsible for tumor evasion of immune surveillance (146). In the TME, nanovaccines are an attractive tumor treatment that initiates the cancer immune cycle by inducing the immunogenic death of tumor cells and generating a rich pool of tumor antigens (147, 148). Enhancing the function of DCs in response to the characteristics of the TME is crucial in tumor immunotherapy to transform “cold” tumors and metastasis into “hot” tumors (149). In a recent study, a novel charge-reverting polymeric nanomodulator, SPDMCN, was designed to effectively target the TME. In the blood circulation, SPDMCN maintained a negative charge to ensure high stability and aggregation toward the tumor site. Once SPDMCN reached the acidic TME, the surface charge reversed to a positive charge, releasing immunomodulators and enhancing tumor site penetration. Under NIR laser irradiation, SPDMCN generated single linear state oxygen, which induced immunogenic death and released TAAs. This process effectively promoted DCs maturation and antigen-specific CTL infiltration, ultimately leading to a robust anti-tumor immune response (150). Hypoxia is a major feature of most solid tumors and one of the main reasons for the failure of many tumor immunotherapies in clinical practice. Hypoxia can affect the maturation, migration, and Th-cell polarization of functional anti-tumor DCs (151). To overcome this challenge, a potential solution is the use of manganese porphyrin-based multifunctional (Mn-MOF) nanoplatforms with peroxidase-like activity. These nanoplatforms can catalyze the production of O2 from tumor-overexpressed H₂O₂ to alleviate tumor hypoxia. The results show that Mn-MOF significantly increased the number of activated CD8⁺ T lymphocytes and mature DCs in tumor tissues, effectively remodeling the tumor immune microenvironment (152). These studies suggest that reversing the immunosuppressive TME can effectively restore the function of DCs and initiate the body’s anti-tumor immune response, which is a promising strategy for tumor immunotherapy.

Most immunotherapies aim to restore the immune system or more precisely the ability of immune cells to sense and eliminate cancer (153). As an important component of the immune system, CTLs are the key effectors of anticancer immunity, specifically recognizing and killing neoplastic cells (154). Activation of tumor-specific T lymphocytes is dependent on the antigen-presenting function of DCs (155). Zhang et al. developed a thiolated nanovaccine that allows direct cytoplasmic delivery of neoantigens and the Toll-like receptor 9 agonist CpG-ODN. This approach was able to bypass endocytosis/lysosomal degradation, increase the uptake and local concentration of neoantigen and CpG-ODN, activate APCs, and significantly enhance anti-cancer T cell immunity (107). One of the major barriers to DCs initiation and activation of T lymphocytes needs to be overcome is the suppressive immune checkpoint molecules expressed on tumor-infiltrating T lymphocytes, such as CTLA-4 molecule and PD-1. By blocking their expression, it helps to improve the efficiency of T cell activation by DCs vaccines (156, 157). CTLs need to reach tumor tissue to perform effector functions effectively. Studies have shown that local application of STING can promote vaccine-initiated tumor infiltration of CD8⁺ T lymphocytes, overcoming to some extent the dilemma of poor efficacy of tumor vaccination (158).