Nanodrug Delivery Systems in Antitumor Immunotherapy

Application of nanodrug delivery systems in ICB therapy

To overcome the limitations of the immune checkpoint inhibitors mentioned above, nanodrug delivery systems have been applied in immunotherapy and have achieved excellent therapeutic results. Firstly, the superior targeting ability of nanodrug delivery systems can help ICB therapeutic drugs target tumors precisely, and they can target tumor tissues through the enhanced permeation and retention effect or modification on the surface of NPs to improve the drug delivery efficiency and therapeutic effect [52]. In addition, nanodrug delivery systems can also carry drugs across some physiological tissues (such as the blood–brain barrier) to achieve ICB therapeutic drug delivery. For example, Wang et al. [53] employed the choline analog methyl N-phneyl carbamate (MPC) as a nanocarrier loaded with anti-PD-1 to cross the blood–brain barrier for immunotherapy against glioma. In addition to targeting tumor cells, nanodrug delivery systems can also target immune cells. Schmid et al. [54] achieved targeted delivery of transforming growth factor β (TGFβ) signaling inhibitors and Toll-like receptor 7/8 (TLR7/8) agonists to PD-1-expressing T cells by modifying antibodies on the surface of poly(lactic-co-glycolic acid) (PLGA)/PEG NPs. This strategy greatly decreased the concentration of drug or antibody and reduced the toxicity while ensuring the efficacy of treatment. Secondly, drug delivery through nanodrug delivery systems can prevent the degradation of drugs by enzymes in the human body, and it can also prolong the residence time of drugs in the circulatory system [55]. Reducing antibody disclosure by modifying PEG on the surface of nanodrug delivery systems is a common approach used by researchers. Wang et al. [56] wrapped the PD-L1 antagonist in the NPs by self-assembly and prevented the drug degradation in vivo through a PEG protective layer on the surface. When the NPs reached the melanoma, the PEG protective layer on the surface was destroyed by MMP-2 overexpressed in the melanoma and released the PD-L1 antagonist, which significantly improved the immunotherapeutic effect in combination with the inhibitor of apoptosis proteins (IAP) antagonist AZD5582 without severe toxic side effects. Huang et al., Guo et al., and Wang et al. [57–59] have also employed this strategy to reduce anti-PD-L1 (aPD-L1) leakage and significantly reduce aPD-L1-induced immune-related adverse effects (Fig. ​(Fig.3A3A to C). Thirdly, the combined application of nanodrug delivery systems and immunotherapy can amplify the antitumor effect of ICB and enhance the systemic antitumor immune effect [16,55]. For example, Ma et al. [60] prepared a kind of NP, Vc-ICG@LP NPs (VIP) by using PEG-PLGA loaded with vitamin C and indocyanine green (ICG), which could improve the immune response of the TME. After the combination of VIP and anti-PD-L1 therapy, the expression of CD206, a marker of tumorigenic M2-type macrophages, was significantly down-regulated in TME, and CD80, a marker of antitumor M1-type macrophages, was significantly up-regulated (Fig. ​(Fig.3D),3D), suggesting that the combination therapy promoted the transformation of macrophages from the M2-type to the M1-type, which contributed to enhancing the immune infiltration in TME. When vitamin C was combined with anti-PD-L1, the percentage of CD4⁺ T cells in tumors increased from 7.67% to 12.95% and that of CD8⁺ T cells from 8.19% to 13.33%. When the combination was mediated by nanodrug delivery systems, the percentages of CD4⁺ T cells and CD8⁺ T cells increased to 21.58% and 19.36%, respectively, and the infiltration percentage was significantly increased (Fig. ​(Fig.3E).3E). In addition, TLRs are important targets for stimulating the immune system and supporting T-cell activation, and nanodrug delivery system-mediated codelivery of TLR agonists and ICB therapeutic drugs can significantly amplify the ICB-induced systemic immune response. Li et al. [61] prepared TME-targeted NPs by self-assembly of Zn²⁺ with pIC (polycytidylic aid), which was able to activate the TRL3 pathway and significantly enhance the tumor therapeutic effect of αPD-L1: the percentage of CD8⁺ T cells, DCs, and macrophages in tumors was significantly increased. Yu et al. [62] developed a PD-L1/TLR7 dual-targeted nanoantibody-conjugated drug that showed stronger antitumor activity by codelivery of anti-PD-L1 with a TLR7 agonist: The infiltration of CD4⁺ T cells, CD8⁺ T cells, and NK cells in tumors was significantly increased, enhancing the antitumor immune response mediated by anti-PD-L1. Finally, nanodrug delivery systems can be used as a platform to combine immunotherapy with other therapeutic approaches such as photodynamic therapy (PDT), photothermal therapy (PTT), CT, and radiation therapy (RT) to improve tumor treatment efficacy and amplify systemic antitumor immune responses, and we will describe these approaches below.

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Fig. 3.

Application of nanodrug delivery systems in immune checkpoint blockade therapy. (A) Schematic illustration of the sensitivity and in vivo performance of the pH and MMP- 2 dual-sensitive PEG-coated nanodrug PEG-CDM-aPD-L1/Ce6 abbreviated as P-aPD-L1/C for tumor-targeting immuno-sonodynamic combination therapy of cancer. Adapted with permission from [57], copyright 2021 Biomaterials. (B) PEG-sheddable nanodrug PEG-LP@αPD-L1/ DMXAAst (P-LPD) with pH and MMP-2 dual sensitivity for codelivery of αPD-L1 and STING activator DMXAAst. Adapted with permission from [58], copyright 2022 Nano Today. (C) Tumor acidity-triggered shedding of both long-chain PEG and aPD-L1 from nanodrug. Adapted with permission from [59], copyright 2023 Acta Biomaterialia. (D) Multiplex immunohistochemical images of CD80⁺ and CD206⁺ in tumor tissues after different treatments. (E) Flow cytometry analysis of CD3⁺, CD4⁺, and CD8⁺ T cell infiltration in tumor tissues for each mouse group: APC antimouse CD3, PE (phycoerythrin) antimouse CD8, and FITC antimouse CD4. FITC, fluorescein isothiocyanate. Adapted with permission from [60], copyright 2022 ACS Nano.

Nano-PDT drug delivery systems combined with ICB therapy to amplify antitumor immune response

PDT plays an antitumor role mainly through the generation of reactive oxygen species (ROS) by photosensitizers during the antitumor process. Photosensitizers react with oxygen to produce ROS and singlet oxygen species (¹O₂) by absorbing specific wavelengths of light (near-infrared [NIR] light). Both ROS and ¹O₂ can selectively induce tumor cell apoptosis without damaging other normal cells [63,64]. PDT also provides a large amount of tumor-associated antigens (TAAs), which triggers immunogenic cell death (ICD) effects and induces antitumor immune responses in vivo [64]. However, when used in clinical practice, PDT has limitations such as insufficient tumor targeting ability, a hypoxic TME in tumor tissue that limits the production of ROS, difficulty in light reaching deep tissues, and poor stability of photosensitizers [65].

At present, many studies have been carried out to overcome these shortcomings of PDT therapy using nanodrug delivery systems with positive results. Nanodrug delivery system-mediated PDT therapy can not only solve the shortcomings of PDT therapy, but more importantly, it can enhance the immune response generated by the combination of PDT therapy and ICB therapy. Combined treatment can further improve the therapeutic effect on tumors and expand the scope of the clinical treatment of tumors. Wang et al. [66] reported an antitumor immunotherapy through serum albumin-loaded photosensitizer PcM combined with PD-1/PD-L1 blockade therapy, which solved the deficiency of poor PDT tumor targeting ability by nanodrug delivery systems (Fig. ​(Fig.4A).4A). The nanodrug delivery system-mediated combination of PDT with αPD-1 antibody had the highest primary tumor suppression rate (Fig. ​(Fig.4B),4B), and the lowest tumor volume and weight (Fig. ​(Fig.4C4C and D). More importantly, in the 4T1 delayed distant primary tumor model, the abscopal effect of combination therapy was more obvious, as the highest inhibition rate of the delayed distant primary tumor (up to 59.21%) was more evident in the combination therapy group (Fig. ​(Fig.4E).4E). Using flow cytometry to analyze CD8⁺ T cells, PD-1 and myeloid-derived suppressor cells (MDSCs) in TME after distal tumor treatment, the results showed that the combination therapy group had the highest percentage of CD8⁺ T cells, the highest PD-1 expression, and the lowest ratio of MDSCs, suggesting that nanodrug delivery systems enhanced the systemic antitumor adaptive immunity generated by the combined application of PDT and ICB therapy (Fig. ​(Fig.4F).4F). When evaluating the systemic antitumor immune effects, in addition to the delayed distant primary tumor model described above, Huang et al. [67] found that nanodrug delivery systems combined with PDT and ICB therapy significantly increased the ratio of effector memory T cells (Tem) and central memory T cells (Tcm) in the spleen of mice, achieving long-term protection by activating the immune system (Fig. ​(Fig.4G4G and H).

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Fig. 4.

Nano-PDT drug delivery systems combined with ICB therapy to amplify antitumor immune response. (A) Schematic illustration of the structure of NanoPcM, the mechanism of its tumor targeted fluorescence turn-on imaging, and the synergistic antitumor effect caused by NanoPcM based PDT and αPD-1-based immunotherapy. (B) Tumor growth curves in BALB/c mice after NanoPcM injection (n = 5 for each group). NanoPcM was injected intravenously when the tumor size reached ∼100 mm³, and 24 h later, the tumors were irradiated with a 655-nm laser for 10 min at a power density of 0.2 W/cm2. The αPD-1 antibody was intravenously injected a total of 4 times every 2 d, starting from 24 h after laser irradiation. (C) The tumor weight was measured after resection at the end of the study. (D) Representative photographs of tumor tissue from each group at the end of the study. (E) Tumor growth curves of 4T1primary tumors and 4T1 delayed primary tumors (on the opposite side). (F) Flow cytometric analysis of infiltrating leukocytes from delayed primary tumor tissues excised at the end of the study. Relative proportions of CD8⁺ T cells (CD3⁺CD4⁻ CD8⁺) versus CD4⁺ T cells (CD3⁺CD4⁺ CD8⁻) among CD3⁺ T cells (top panel); exhausted/activated T cells (PD-1⁺ TIM3⁻ PD-1⁻ TIM3⁺, andPD-1⁺ TIM3⁺) among CD8⁺ T cells (middle panel); and MDSCs (CD11b⁺ Gr-1⁺) among total leukocytes (bottom panel). Adapted with permission from [66], copyright 2022 ACS Nano. (G and H) The determination of effector memory cell (CD45⁺CD3⁺CD4⁺CD44⁺CD62-and CD45⁺CD3⁺CD8⁺CD44⁺CD62-) and Tcms (CD45⁺CD3⁺CD4⁺CD44⁺CD62⁺and CD45⁺CD3⁺CD8⁺CD44⁺CD62⁺) in CD8⁺ T cells and CD4⁺ T cells in spleens. Data are presented as mean ± SD. Statistical significance was calculated via 1-way ANOVA with Tukey’s posttest. *indicates P < 0.05; **indicates P < 0.01; ***indicates P < 0.001; ****indicates P < 0.0001. Adapted with permission from [67], copyright 2022 Advanced Materials.

Nano-PTT drug delivery systems combined with ICB therapy to amplify the antitumor immune response

PTT produces heat therapy through a photothermal agent under the irradiation of NIR light, which causes tumor cell apoptosis and tumor ablation. PTT works well in treating local tumors but cannot effectively prevent tumor recurrence and metastasis [68]. One strategy to overcome the shortcomings of PTT is to combine it with immunotherapy by activating the body’s own antitumor immune system against metastatic tumors and residual tumor cells [69]. PTT can release many TAAs after tumor elimination, thus activating the body’s immune system. However, a single PTT cannot induce an effective antitumor immune response due to the influence of immune checkpoints and other factors [70]. After PTT ablates tumors, ICB therapy can solve the problem of tumor recurrence and metastasis, while nanodrug delivery systems can serve as carriers for the combination of the 2 therapies, and simultaneously promote the antitumor immune response generated by the combined therapy.

Based on the strategy of combined PTT and ICB therapy, He et al. [71] prepared the nanocluster FerH by self-assembling Fe³⁺ with HMT (a natural polyphenol), which significantly improved the retention time and infiltration depth of the photosensitizer in the tumor region through nanodrug delivery systems, and had a significant effect on tumor killing. FerH could produce powerful ICD effects by NIR irradiation and promote DCs maturation after killing tumor cells in vivo. After synergistic effects with anti-PD-L1, the combined therapy had a more obvious inhibition effect on the distal tumor, and the tumor inhibition rate reached 59.8% (Fig. ​(Fig.5A).5A). Immunofluorescence staining was used to detected the infiltration of T cells, and the results showed that CD3⁺, CD4⁺ and CD8⁺ T cells had stronger fluorescent signals in the combined treatment group, indicating that the infiltration of T cells in tumors was significantly increased (Fig. ​(Fig.5B).5B). Combined treatment also greatly increased the maturation of DCs, with notably higher expression of CD80 and CD86 (Fig. ​(Fig.5C5C and D). In addition, the expression of IL-6 and TNF-α also increased significantly (Fig. ​(Fig.5D),5D), suggesting that the combined therapy based on nanodrug delivery systems amplified the antitumor immune response. In another article, Zhou et al. [72] developed an albumin-phenformin-based NP to block the PD-1/PD-L1 pathway by loading PM (a metformin analog) to induce adenosine 5′-monophosphate-activated protein kinase phosphorylation to reduce PD-L1 expression in tumor cells, achieving PD-1/PD-L1 blockade and solving the problem of tumor recurrence and metastasis after PTT treatment (Fig. ​(Fig.5E)5E) [72]. Cano-Mejia et al. [73] triggered PTT by cytosine-phosphate-guanine motifs (CpG) oligodeoxynucleotide-coated Prussian blue nanoparticles (CpG-PBNPs), which showed significantly improved antitumor effects and were able to provide long-term effective antitumor immune protection to the organism when combined with CTLA-4 blocking antibodies. Huang et al. [74] suggested an all-in-one macrophage strategy and they constructed a nanodrug delivery system loaded with oxaliplatin prodrug and photosensitizer, which was transported to TME through macrophages. NIR light triggered photosensitizer-induced PTT combined with oxaliplatin prodrug-induced CT to kill tumor cells, while activating the ICD effect to enhance the antitumor response (Fig. ​(Fig.5F5F to H), which significantly increased the ratio of CD8⁺ T cells and CD4⁺ T cells in TME (Fig. ​(Fig.5I5I and J). After combination with anti-PD-L1, this strategy can significantly eliminate primary and bone metastatic tumors.

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Fig. 5.

Nano-PTT drug delivery systems combined with ICB therapy to amplify the antitumor immune response. (A) The volume of distant tumors from the 4T1 tumor-bearing mice. Data are presented as mean ± SD. (n = 5). (B) Immunofluorescence investigation of distant tumor tissues after treatment with the following markers: CD3, CD4, CD8, FOXP3 (green: CD4, yellow: CD3, orange: CD8, pink: Treg, blue: nuclei). Scale bar, 50 μm. (C) Flow cytometry analysis of CD11c and CD80 (gated on CD11c⁺) in tumor-draining lymph nodes after different treatments and quantitative analysis of in vivo DC maturation level, experiments performed in triplicate with similar results (n = 3). (D) Relative mRNA expression in DCs and serum, including CD80, CD86, TNF-α, and IL-6 (n = 3). For (C) and (D), statistical significance was calculated via 1-way ANOVA with Tukey’s multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001). Adapted with permission from [71], copyright 2022 Theranostics. (E) The number of 4T1 metastatic foci in the lungs of Balb/C mice on day 26 (n = 5). Statistical analysis was performed via the 2-tailed Student t test. *P < 0.05; **P < 0.01; ***P < 0.001. Adapted with permission from [72], copyright 2021 Journal of Nanobiotechnology. Quantitative determination of (F) adenosine triphosphate secretion (***P < 0.0001, ***P < 0.0001) and (G) HMGB-1 release of 4T1 cells after various treatments (n = 3, ***P < 0.0001, **P = 0.0017). (H) Flow cytometric examination of CRT exposure on the surface of 4T1 cells (n = 3). (I) CD4⁺ T cells (**P = 0.0060, ***P = 0.0002, *P = 0.0102) and (J) CD8⁺ T cell in tumors analyzed on day 6 after various treatments (**P = 0.0047, **P = 0.0038, **P = 0.0098). Adapted with permission from [74], copyright 2021 Nature Communications.

Nano-CT drug delivery systems combined with ICB therapy to amplify antitumor immune response

CT is a treatment that uses chemically synthesized drugs systemically or locally to inhibit DNA synthesis, nucleic acid synthesis, protein synthesis, and other pathways of cancer cells to control tumor growth or kill tumor cells effectively. At present, CT is a common method for tumor treatment, but it has many disadvantages. Firstly, most chemotherapeutic drugs cannot target specific cells, which leads to the indiscriminate killing of normal cells and tissues and causes severe side effects. Secondly, CT produces definite immunosuppressive effects, and tumors are more resistant to antitumor immunity after CT. For example, CT promotes the secretion of IL-18 by macrophages, increases the expression of CD47 (the immune checkpoint of osteosarcoma), and promotes the immune escape of tumor cells [75]. Finally, CT has limitations in controlling the metastasis and escape of tumors, and single CT often cannot effectively prevent tumor metastasis and recurrence.

There are many limitations of single CT to cure tumors, and it is difficult to achieve the desired therapeutic outcome. Notably, nanodrug delivery systems have great potential to address these issues. Firstly, nanodrug delivery systems can solve the problem of poor tumor targeting of CT drugs by actively or passively targeting tumors. Qiao et al. [76] reported a type of bionic metal-organic skeletal nano-microspheres with erythrocyte membranes encapsulating TGFBR1 and the chemotherapeutic drug adriamycin, which achieved massive accumulation of chemotherapeutic drugs in tumor tissues through nanodrug delivery systems. Secondly, nanodrug delivery systems can reduce the toxicity of chemotherapeutic drugs and do not affect the efficacy of chemotherapeutic drugs. Weng et al. [77] utilized water-soluble pH-dependent antioxidant nanomaterials to deliver chemotherapeutic drugs, which reduced the damage of chemotherapeutic drugs to the kidney without affecting the antitumor efficacy. Tian et al. [78] reported a nanoantidote composed of dendrimer molecular wrapped by reduced cysteine, which significantly reduced the toxicity of chemotherapeutic drugs. In the cure of glioblastoma, this nano-antidote was only distributed in normal tissues and did not cross the blood–brain barrier, thus not affecting the tumor-killing effect of the chemotherapeutic drug temozolomide (TMZ, the glioblastoma standard CT), while capturing TMZ and significantly reducing the toxic side effects of TMZ on other normal tissues. Wu et al. [79] constructed a nanocomposite hydrogel to deliver chemotherapeutic and immunotherapeutic drugs. This nanocomposite hydrogel degraded in TME over time after injection, greatly reducing the toxicity of chemotherapeutic drugs to other normal tissues.

Despite the immunosuppressive effects of CT, it has also been shown that CT also has certain immunostimulatory effects, such as cyclophosphamide (which inhibits the transcription of tumor cell DNA into RNA) which induces immunogenic death of cancer cells, triggers the immune system, and activates DCs and T cells when treating tumors [80]. Single CT is not sufficient to induce a strong immune response to resolve tumor metastasis and recurrence. However, by combination with ICB therapy through nanodrug delivery systems, it can induce a strong systemic antitumor immune response and effectively prevent tumor cells from escaping [81]. Jiang et al. [82] improved the delivery of the chemotherapeutic drug OxPt/SN38 through nanodrug delivery systems. Although it could significantly inhibit tumor growth and trigger strong ICD effects, it also up-regulated the expression of PD-L1 in tumor cells. The combination of this nanodrug delivery system with αPD-L1 produced a stronger antitumor immune response to eliminate subcutaneous MC38, CT26, and KPC tumors (Fig. ​(Fig.6A6A to C). In addition to being used in combination with immune checkpoint inhibitors, some nanomaterials themselves can also play a part in blocking immune checkpoint pathway. For example, Wang et al. [83] reported a kind of lipid bilayer NPs self-assembled by podophyllotoxin and poly (butyl acrylate) (PBA), which enhanced the delivery of the CT drug podophyllotoxin and significantly reduced its systemic toxicity [83]. Most importantly, the LNPs can significantly reduce the expression of PD-L1 while inducing tumor cell apoptosis and successfully combine CT and ICB therapy (Fig. ​(Fig.6D6D to G). Cai et al. [84] developed a nanodrug delivery platform (MS NPs) by assembling metformin (Met) and 7-ethyl-10-hydroxycamptothecin (SN38) through hydrogen bonding and electrostatic interactions, which effectively improved chemotherapeutic drug delivery [84]. Met can induce PD-L1 degradation in tumor cells and act as a PD-1/PD-L1 pathway blocker in the treatment system. MS NPs significantly reduced in the expression levels of PD-L1 in tumor cells through Met and inhibited tumor cell metastasis, especially lung metastasis, in vivo (Fig. ​(Fig.6H6H and I). The immunofluorescence staining results showed that the number and activity of CD8⁺ T cells in the tumors of tumor-bearing mice mediated by MS NPs were greatly increased, and the effect of immunotherapy was obviously enhanced (Fig. ​(Fig.6J).6J). Tong et al. [85] reported a polymeric NP—SPN@Pro-Gem with a particle size of 120 nm at neutral pH and transformed to small particles of approximately 10 nm in the slightly acidic TME (Fig. ​(Fig.6K).6K). SPN@Pro-Gem could pass through the tumor mesenchyme and deliver the chemotherapeutic drug gemcitabine (Gem) to the tumor parenchyma, enhancing the effect of CT in treating tumors. The combination of SPN@Pro-Gem with PD-1 antibody resulted in further improvements in tumor suppression and, more importantly, in increased levels of T cells during immunotherapy: a decrease in the number of MDSCs in the blood (Fig. ​(Fig.6L);6L); and an effective increase in the number of CD4⁺ T cells and CD8⁺ T cells in the tumor (Fig. ​(Fig.6M6M and N). Zhu et al. [86] developed a lung-targeting nanodrug delivery system, Lip-CExo@PTX, based on a bispecific CRA-T cell-derived exosome that targeted mesothelin and programmed death ligand-1, loaded with chemotherapeutic agent paclitaxel (PTX). Lip-CExo@PTX enhanced the chemotherapeutic effects of PTX by delivering it to the tumor cells at lung tissues. At the same time, the anti-PD-1 single-chain antibody on the surface of exosomes prevented T-cell depletion by blocking the PD-1/PD-L1 pathway. Lip-CExo@PTX significantly prolonged the survival time of CT26 metastatic lung cancer model mice, which provided a prime example for the combination of CT and ICB therapy.

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Fig. 6.

Nano-CT drug delivery systems combined with ICB therapy to amplify antitumor immune response. (A) Tumor growth curves of MC38 tumor-bearing C57BL/6 mice after indicated treatments, n = 6. (B) Tumor growth curves of CT26 tumor-bearing BALB/c mice after indicated treatments, n = 6. (C) Tumor growth curves of KPC tumor-bearing C57BL/6 mice after indicated treatments, n = 5. PBS, OxPt plus IRI, OxPt NCP, ZnP/SN38, or OxPt/SN38 was intravenously dosed once every 3 d with 3.5 mg/kg OxPt or and 6.2 mg/kg SN38 equiv to the MC38 model (8 doses) and CT26 and KPC models (6 doses). Adapted with permission from [82], copyright 2022 ACS Nano. (D and E) Western blot analysis of PD-L1 production in PPT- and PPCN-treated H460 cells. (F) Quantification of PD-L1 production in PPCN-treated H460 cells by flow cytometry. (G) Quantification of PD-L1 mRNA by reverse-transcription quantitative real-time polymerase chain reaction analysis. Data in (E) to (G) are means ± SDs, n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant. Adapted with permission from [83], copyright 2022 ACS Nano. (H) Western blot image of changes in PD-L1 level from MS NPs-treated MB231 cells. (Lane 1: MB231 cells, lanes 2 to 4: MB231 cells treated with different concentrations of MS NPs (20, 40, and 80 μg/ml respectively). (I) Densitometric analysis of PD-L1 bands. (J) Immunofluorescence staining of PD-L1 (green) (upper row), the magnified images (bottom row) and CD8⁺CTLs (red) and GzyB (green). Adapted with permission from [84], copyright 2021 Theranostics. (K) pH-Dependent size change of mPEG-G4-DnPEA44, mPEG-G4-DnPEA37, and mPEG-G4-DnPEA30 analyzed by dynamic light scattering (DLS). (L) Numbers of MDSCs in blood. (M) Numbers of CD8⁺ T cells per gram of the tumor upon various treatments. (N) Numbers of CD4⁺ T cells per gram of the tumor upon various treatments. Adapted with permission from [85], copyright 2021 Small.

Nanodrug delivery systems improve the presentation of tumor vaccines in vivo and enhance targeting capability

TAAs applied in tumor vaccines are autoantigens, which exist not only in tumor cells but also in normal human cells, so the human body will develop certain tolerance to these antigens, making it difficult to induce a vigorous immune response. In addition, traditional tumor vaccines are ineffective in clinical treatment due to their low efficiency of antigen encapsulation capacity, poor lymph node targeting performance, and weak lysosomal escape ability [103]. Therefore, the precise targeting property of nanodrug delivery system-mediated tumor vaccines is important for successful tumor vaccine design. Tumor vaccines based on nanodrug delivery systems can not only efficiently encapsulate antigens and adjuvants through electrostatic interactions, hydrophobic interactions, and covalent binding and protect antigens and adjuvants during vaccine delivery but also precisely target lymph nodes or immune cells and efficiently release them in APCs based on their specific physical or chemical properties [103–105].

DCs are specialized APCs in the human body that can capture tumor cells after apoptosis and cross-present TAAs to CD8⁺ T cells [106,107]. DC vaccination is an immunological approach that targets DCs and induces DCs maturation, followed by activation of CD8⁺ T cells through cross-presentation of DCs to induce a strong antitumor immune response in vivo. The effective delivery and subsequent activation of antigens to DCs are major challenges in the development of DC vaccines, and nanodrug delivery systems not only enable the targeting ability of tumor vaccines but also enhance lysosomal escape and thus improve the efficiency of antigen cross-presentation [102]. Xu et al. [108] reported a polymeric pathogen-like nanovaccine (MPVax) with mannan as the shell and poly (lactic acid)-poly(ethyleneimine) (PLA-PEI) assembled NPs as the core that adsorbed the tumor antigen protein ovalbumin (OVA) and Toll-like receptor 9 (TLR9) agonist CpG by electrostatic interactions. MPVax significantly improved the recognition ability of DCs to vaccines by modifying mannans on the surface, which in turn improved the presentation efficiency of nanovaccines to DCs. Compared to NPs without mannans, the presentation efficiency of DCs was increased 2-fold and compared to free CpG/OVA, the presentation efficiency was increased 6-fold (Fig. ​(Fig.8A).8A). Su et al. [109] reported a nanovaccine (NVs^cp) made by growing calcium peroxide in situ based on OVA self-assembly, which significantly improved the cross-presentation efficiency of exogenous antigens. NVs^cp activated the NADPH oxidase 2 complex by forming lipid peroxidation products on the surface, promoting vaccine escape via endo-/lysosomal lipid peroxidation and drainage into the lymph nodes, thereby enhancing cross-presentation to lymph node DCs (Fig. ​(Fig.8B).8B). In addition to DCs, NVs^cp also significantly increased the uptake and presentation of vaccines by macrophages. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to detect T cells in the spleen of mice injected with NVs^cp and their IL-2 secretion after in vitro activation. The results showed that compared with mice injected with NVs, the percentages of IFN-γ⁺CD4⁺ T cells and IFN-γ⁺CD8⁺ T cells in the spleen of mice injected with NVs^cp were much higher, and IL-2 secretion was also significantly higher, and the results were best in the NVs^cp-high group (Fig. ​(Fig.8C8C to E), suggesting that NVs^cp induced an effective antitumor immune response in vivo. Chen et al. [110] constructed an AlNEs-OVA-CpG nanovaccine by adsorbing aluminum hydroxide on the surface of the oil-in-water emulsion MF59 and wrapping antigen (OVA) and immune enhancer (CpG). The in vitro results showed that the uptake efficiency of DC2.4 cells and bone marrow-derived cells (BMDCs) on AlNEs-OVA-CpG and cross-presentation efficiency to APCs were much higher than those of free OVA/CpG, and the uptake efficiency of DC2.4 cells on AlNEs-OVA-CpG was 45 times higher than that of free OVA/CpG (Fig. ​(Fig.8F8F and G). Notably, other normal cells (human umbilical vein endothelial cell and murine L929 cells) showed low uptake efficiency of AlNEs-OVA-CpG, which suggested a strong targeting ability of AlNEs-OVA-CpG. The nanovaccine also significantly improved the targeted delivery efficiency to lymph nodes, with colocalization experiments showing that nanodrug delivery systems improved the presentation efficiency of the vaccine to lymph node DCs in vivo by 17.37-fold and macrophages by 29.29-fold (Fig. ​(Fig.9H9H to K).

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Fig. 8.

Nanodrug delivery systems improve the presentation of tumor vaccines in vivo and enhance targeting capability. (A) Antigen cross-presentation analysis of BMDCs after coculture with free CpG/OVA/, PVax-CpG/OVA and MPVax-CpG/OVA for 24 h and staining with antimouse H-2Kb bound to SIINEFKL antibody. (ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Adapted with permission from [108], copyright 2022 Biomaterials. (B) NVs and NVscp distribution in DCs and 826 macrophages in ILN. (C and D) Flow cytometry was used to determine the percentages of OVA specific (C) IFN-γ-producing CD4⁺ cells and (D) IFN-γ-producing CD8⁺ T cells. (E) The release of IL-2 from splenocytes of immunized C57BL/6 mice after ex vivo restimulation with OVA 257-264 peptide (SIINFEKL) was determined using ELISA. Adapted with permission from [109], copyright 2021 Biomaterials. (F) Uptake efficiency of AlNEs-OVA-CpG (containing FITC-labeled OVA) by BMDCs and DC2.4. (G) AlNEs-OVA-CpG significantly promoted cross-presentation of OVA on BMDCs. (H) Percentages of FITC-OVA⁺ cells in total lymph nodes cells. (I and J) Percentages of OVA- and CpG-positive cells in DCs (I) or macrophages (J) of the popliteal lymph nodes cells. (K) Percentage of CD40⁺CD11c⁺DCs in total lymph node cells at day 3 after administration. Adapted with permission from [110], copyright 2022 Journal of Controlled Release. (L) Representative confocal images of DC2.4 and RAW264.7 cells incubated with Cy5.5 labeled mOVA and mRLNPs for 6 h, showing cellular uptake and endo/lysosome escaping of mOVA. The endo/lysosomes were stained with Lyso Tracker Green (green). Scale bar: 20 μm. (M) Cellular uptake of DC2.4 and RAW264.7 cells incubated with saline, mOVA, LNPs, mLNPs, and mRLNPs for 6 h determined with flow cytometry. Adapted with permission from [116], copyright 2022 Advanced Functional Materials.

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Fig. 9.

The adjuvant properties of nanodrug delivery systems can amplify the antitumor immune response. (A to E) BMDCs were incubated with PBS, OVAT, VLP-WT, or VLP-OVAT for 24 h and then stained with antibodies to assess surface markers including CD40 (A), CD80 (B), CD86 (C), MHC-I (D), and MHC-II (E) by FACS. (F to H) Supernatants were collected for analysis of the expression of IL-6 (F), IL-12 (G), and IFN-γ (H) by ELISA. Scale bars, 20 μm. Data are expressed as the mean ± SEM (n = 3) (*P < 0.05; **P < 0.01; ***P < 0.001). Adapted with permission from [120], copyright 2021 Biomaterials. (I) Representative fluorescence microscopy images of ROS expressed by DCs after incubation with different nanoformulations. Intracellular ROS was stained by 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Adapted with permission from [121], copyright 2022 Advanced Materials. (J) Confocal fluorescence images of DC2.4 cells incubated with IC8@Cy5 and IC8/Mn@Cy5 in 10% fetal bovine serum (FBS) medium for 5 h. DAPI (4′,6- diamidino-2-phenylindole; blue) was used to track the cell nuclei, and Lyso Tracker Green DND-26 (green) was used to track lysosomes; red fluorescence was from Cy5 mRNA. (K) Flow cytometry examination of BMDCs maturation (CD11c⁺CD80⁺CD86⁺) 24 h after incubation with IC8@OVA and IC8/Mn@OVA. FITC, fluorescein isothiocyanate. Adapted with permission from [122] , copyright 2022 Science Advances.

In addition to improving the delivery of tumor antigen vaccines, nanodrug delivery systems can also serve as carriers for nucleic acid vaccines, improving their delivery behavior in vivo. Nucleic acid vaccines are mainly divided into DNA vaccines and mRNA vaccines. Although DNA vaccines can stimulate the body’s immune response much better, there is a risk of genetic mutation. In contrast, mRNA vaccines do not carry the risk of gene mutation, as they do not integrate into the genome, and they also have the advantages of high efficiency and are economical, so they have been widely researched [111,112]. mRNA vaccines stimulate an immune response by delivering messenger RNA sequences to the body that produce proteins similar to tumor antigens. Nanodrug delivery system-mediated delivery of mRNA vaccines can effectively protect nucleic acids and improve mRNA stability. LNPs are currently FDA-approved mRNA carrier materials with several advantages, such as simple formulation, high mRNA payload capacity, good biocompatibility, and they have important advantages in both cell-mediated and humoral immunity activation in vivo [113]. Furthermore, LNPs can protect mRNA from degradation, facilitate intracellular delivery, and promote the endosomal escape of mRNA cargo [114]. For example, Hashiba et al. [115] developed a novel branched ionizable lipid that significantly improved mRNA stability and delivery efficiency when contained in LNPs. LNP-mediated delivery of mRNA vaccines could greatly improve the delivery efficiency of mRNA and without toxicity or unnecessary immunogenicity, making LNPs ideal carriers for developing mRNA tumor vaccines currently. LNPs have made good progress as nonviral delivery carriers to deliver mRNA to the cytoplasm, but the storage and transport of mRNA-LNP vaccines have limited their clinical applications. To solve the problem of storage and transportation, Jia et al. [116] constructed an mRNA nanovaccine using LNPs as a carrier for mRNA that achieved nanovaccine preservation by cross-linking with a hyaluronan dynamic hydrogel. This nanodrug delivery system significantly improved the stability of the mRNA vaccine. Western-blot data showed that compared to free mOVA-treated DC2.4 and RAW264.7 cells, the nanodrug delivery system-mediated mOVA-treated cells showed high expression of OVA, indicating that this nanodrug delivery system-mediated mOVA was not degraded (Fig. ​(Fig.8L8L and M). All of the above examples showed that nanodrug delivery systems could not only improve the delivery efficiency of tumor vaccines but also help preserve tumor vaccines, which have excellent clinical application prospects. In addition to vaccination applications, mRNA can be used to target immune cells in the vivo to achieve antitumor effects.

The adjuvant properties of nanodrug delivery systems can amplify the antitumor immune response

In addition to improving the delivery and targeting of tumor vaccines, some biomaterials can be used as immune adjuvants to activate the immune system. Virus-like particles (VLPs) are natural nanomaterials derived from various plant viruses, phages and mammalian viruses that can bind to tumor-specific antigens by chemical/peptide chain binding and constitute nanovaccines. The nanovaccine can effectively enter the lymph nodes and be phagocytosed by DCs after local injection, activating the major histocompatibility complex-I (MHC-I) signaling pathway and achieving effective antigen presentation [117,118]. Apart from delivery and targeting, VLPs can also act as immune adjuvants, binding to CD209 on DCs and inducing the release of cytokines IFN-γ and TNF-α to produce robust cellular immune responses [119,120]. Alam et al. [119] constructed a series of mannoside ligands by modifying VLPs (derived from phage Qβ), among which VLPs modified by aryl mannoside induced maturation of DCs and expression of proinflammatory cytokines characteristic of the Th1-like immune response. When constructing nanovaccines, these aryl mannoside-modified VLPs could not only serve the function of targeting DCs but also participate in inducing an antitumor cell immune response and play the role of an immune adjuvant, which has good application prospects. Li et al. [120] combined the B and T epitopes (OVA_B and OVA_T) of the model antigen OVA with the C segment of VLPs (derived from phage P22), respectively, and self-assembled them with scaffolding protein to form 2 nanovaccines, VLP-OVA_B and VLP-OVA_T. VLP-OVA_B induced strong antibody titers (>10⁵) and a Th1-type immune response, while VLP-OVA_T significantly increased the percentages of CD4⁺, CD8⁺ and effector T cells and decreased the percentage of MDSCs in tumors or spleens in vivo. In this antitumor research, in addition to performing the role of targeting antigen delivery, VLPs also showed that the expression of CD40, CD80, CD86, MHC-I, and MHC-II on the cell membranes of BMDCs induced by blank VLPs and the concentrations of IL-6, IL-12, and IFN-γ in the supernatant were not significantly different from those of VLP-OVA_T, suggesting that VLPs themselves could be used as immune adjuvants to induce the activation of BMDCs (Fig. ​(Fig.9A9A to H).

In addition to the above VLPs, other nanomaterials can also serve as immune adjuvants in the construction of nanovaccines and participate in the stimulation of antitumor immune responses in vivo. Chen et al. used MF59 and aluminum hydroxide to construct a kind of nanodrug delivery system in which aluminum hydroxide was involved in coating antigens and adjuvants and acted as a Th2-type immune adjuvant to enhance the immune response [110]. Meng et al. [121] constructed a water-soluble nanomaterial IONP (iron oxide nanoparticle) by replacing the oleic acid ligand of Fe₃O₄ particles with DIP-PEG-MAL. Then, IONPs were attached to CpG and OVA and wrapped with a lipid membrane of the DC-targeted cyclic peptide P30 to generate the nanovaccine IONPC/O@LP. IONPC/O@LP could deliver OVA and CpG to the cytoplasm and lysosomes of immature DCs and enhance the activation of DCs. Notably, the nanomaterial IONPs not only imparted nanovaccine detectability by magnetic resonance imaging but also generated ROS in cells with good adjuvant effects (Fig. ​(Fig.9I).9I). In addition to tumor antigen vaccines, adjuvant properties of nanomaterials can also enhance the immune response of mRNA tumor vaccines in vivo. Fan et al. [122] constructed an mRNA nanodrug delivery system (IC8/Mn LNPs) with high immunogenicity by adding Mn to the novel ionizable lipid IC8. The addition of Mn endowed this nanodrug delivery system with adjuvant properties, which enhanced mRNA expression by promoting lysosomal escape and stimulated the maturation of APCs by activating the STimulator of Interferon Genes (STING) pathway (Fig. ​(Fig.9J9J and K). The results showed that the best antigen presentation of APCs was achieved when Mn/mRNA = 1:1.

Nanodrug delivery systems in CAR-T therapy

CAR-T therapy focuses on isolating T cells from patients’ blood, genetically modifying them in vitro to enhance their ability to target and kill cancer cells, and then infusing them back into their bodies to multiply and kill cancer cells [129]. Using T cells to target tumors usually solves problems such as weak signals released from dead tumor cells, lack of T-cell recognition due to the absence of MHC class I molecules in tumor cells, deficiency of T-cell infiltration in tumors, and T-cell dysfunction, so CAR-T therapy aims to activate or enhance the antitumor immune system in the body [130]. After artificial construction, CAR-T cells can recognize various cell surface structures. For example, the surface of CAR-T cells is composed of outer parts used for antigen recognition cells and has strong tumor cell recognition ability, which makes CAR-T cells independent of the presentation of MHC class I molecules, greatly enhancing their targeting and killing ability to tumor cells [131]. In addition to targeting tumor cells, CAR-T cells can also be used for other cells. Tisagenlecleucel and axicabtagene ciloleucel are 2 CAR-T products that have been approved to target CD19 cells for the treatment of relapsed diffuse large B-cell lymphoma, both of which have achieved excellent therapeutic results [132–134]. Moreover, idecabtagene vicleucel (Abecma), which targets B-cell maturation antigens for the treatment of myeloma, has been approved by the FDA [135].

CAR-T therapy has achieved remarkable results in antitumor therapy, but the serious toxicity and side effects that are associated with the application of CAR-T therapy should also be considered. The 2 most common toxic and side effects are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome. CAR-T cells release large amounts of perforin and cytokines after recognizing antigens, leading to tumor scorching (inflammatory cell death), and the released cytokines, such as IL-2, also activate macrophages, releasing inflammatory factors. All of these processes ultimately lead to the overactivation of immune effector cells and proinflammatory cytokines over normal physiological levels, which triggers CRS [132]. Inflammation caused by CAR-T cells can also lead to injury or even death of endothelial cells in the blood–brain barrier, which in turn destroys the blood–brain barrier, allowing immune cells and cytokines to penetrate, leading to neuronal damage and dysfunction and triggering immune effector cell-associated neurotoxicity syndrome [135]. Therefore, when treating with CAR-T therapy, the impacts of the therapy’s toxic side effects need to be considered. Another limitation of the application of CAR-T-cell therapy is its high production cost. The isolated T cells require specialized facilities such as viral vectors to deliver CAR genes into the T cells so that the T cells can express CAR proteins. In addition, the production rate of CAR-T cells limits their use in some rapidly developing tumors.

When cooperating with CAR-T therapy in the treatment of tumors, nanodrug delivery systems have the following 4 main advantages: (a) improving the transport of CAR-T cells in vivo, enhancing cytotoxicity and targeting; (b) reducing the toxicity and side effects of CAR-T in vivo; (c) shortening the cycle of CAR-T therapy for tumors; and (d) reducing treatment costs [136]. Hu et al. [137] used NPs to encapsulate IL-15 and delivered by nanofiber hydrogel to maintain the proliferation and activity for CAR-T cells, which could improve the therapeutic efficacy of CAR-T therapy. In 2017, Smith et al. developed DNA NPs to import CAR genes into the nucleus of T cells, enabling the production of CAR-T cells in vivo and achieving excellent antitumor effects [138]. Parayath et al. [139] reported an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA. Nanoparticles targeted CD8⁺ T cells by electrostatic adsorption of CD8 antibody on their surface and delivered mRNA to T cells, which induced T cells to express tumor-specific CARs. In mouse models of human leukemia, prostate cancer, and hepatitis B-induced hepatocellular carcinoma, NPs all showed excellent therapeutic results. In these methods of targeting the delivery of NPs loaded with specific genes to T cells in vivo, nanodrug delivery systems and CAR-T therapy have demonstrated good synergy, successfully performing in situ programming of T cells in the body and solving the problems of production cost and action cycle of CAR-T therapy. To address the weaknesses of CAR-T therapy in terms of killing and toxic side effects, Luo et al. [140] developed a human serum albumin NP loaded with IL-12 and conjugated this NP to CAR-T cells, which significantly strengthened the effect of CAR-T cells in treating solid tumors and reduced the toxic side effects. IL-12 could enhance the killing ability of T cells against tumor cells and significantly improve antitumor immune response. However, IL-12 has side effects such as proinflammatory toxicity, so it is necessary to accurately target tumor tissue. Based on human serum albumin NPs, Luo et al. designed an IL-12 nanostimulator (INS) and successfully prepared INS-CAR-T cells by biological hybridization with CAR-T cells. Incorporation of INS significantly enhanced the tumor infiltration ability of CAR-T cells (Fig. ​(Fig.11A).11A). The high recognition ability of CAR-T cells to tumors solved the problem of targeted delivery of IL-12 to tumor tissue, greatly reducing the tumor load of tumor-bearing mice (Fig. ​(Fig.11B11B to D). After reaching tumor tissue, IL-12 improved the cytotoxicity of CAR-T cells, showing good synergistic effects. After injection of INS-CAR-T cells into the body and stimulation by Raji tumor cells, INS was isolated, and IL-12 was gradually released to promote the tumor-killing effect mediated by CAR-T cells, significantly reducing the tumor volume and weight (Fig. ​(Fig.11E11E to G). As another typical example, Zhou et al. constructed a LNP system loaded with IL-6 silencing RNA (shRNA) and CD19-CAR binding gene with surface-modified CD3 antibody, which could produce the same antitumor effect as conventional CAR-T in vivo while simplifying the CAR-T preparation process and reducing the CRS caused by CAR-T [141]. LNPs modified with CD3 antibody can significantly target lymph nodes and induce the highest number of specific CAR-T cells in vivo on the 21st day, with the same antitumor effect as CAR-T cells prepared in vitro, but with a significantly reduced preparation cycle (Fig. ​(Fig.11H11H and I). This nanodrug delivery system-based CAR-T therapy produced CD8⁺ CAR-T cells dramatically on days 14 to 21, and the number of CD8⁺ CAR-T cells was significantly decreased on days 35 to 90 after killing the tumor (Fig. ​(Fig.11I).11I). In addition, under the influence of IL-6 shRNA, the toxicity of CRS was significantly reduced, improving the safety of CAR-T therapy in vivo. In the above example, nanodrug delivery systems down-regulated cytokine release by delivering silenced genes to improve the safety of CAR-T therapy. In addition to this approach, nanodrug delivery systems can also improve the safety of CAR-T therapy by accurately targeting tumor sites. Nguyen et al. [142] optimized the design of CAR-T cells and obtained LiCAR-T cells that respond to blue or NIR light. After binding to tumor antigens, LiCAR-T cells can kill tumor cells under the activation of blue or NIR light. Researchers further combined LiCAR-T cells with highly efficient upconversion nanoparticles, using nanoparticles as light sensors to convert NIR light with strong tissue-penetrating ability into blue light, thereby achieving activation of LiCAR-T cells in situ and precise targeting of cellular immune responses, avoiding side effects related to immunotherapy, and greatly improving the safety of CAR-T cells.

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Fig. 11.

Nanodrug delivery systems in adoptive cell therapy. (A) Infiltration of INS-CAR T and CAR T cells. Raji cell spheroids (anti-CD19-PE, green) were incubated with CAR T cells or INS-CAR T (APC-conjugated anti-CD3, red) for 48 h, and images were recorded with the confocal quantitative imaging cytometer CQ1. The tumor spheroid is marked with a white dotted circle. (B) Experimental scheme. Luci-Raji tumor-bearing mice were intravenously injected with CAR T cells (1 × 10⁷ cells), IL-12⁺CAR T cells (0.25 μg of IL-12, 1 × 10⁷ cells) or INS-CAR T (0.25 μg of IL-12, 1 × 10⁷ cells) on day 8 post tumor inoculation. (C and D) Tumor-bearing mice were monitored by bioluminescence imaging (C) and quantified by detection of the radiance in the region of interest over a course of 31 d (D). Bioluminescence imaging units: Avg Rad ×10⁶ (p/s/cm2/sr). (E) Individual tumor growth curves were constructed by measuring tumor volume using calipers at the indicated time points. (F and G) Photographs and weights of excised tumors at the end of experiments. Data are presented as the means and standard errors. Significance was calculated using 1-way ANOVA with Tukey’s posttest, n = 5. *P < 0.05, **P < 0.01. Adapted with permission from [140], copyright 2022 Biomaterials. (H) Biodistribution of fluorescent CD3 antibody targeting or nontargeted LNPs 24 h after tail-vein injection. Data are from 5 mice per treatment condition. The mice on the left were administered LNP/ CAR19 + shIL6, and the mice on the right were administered AntiCD3-LNP/CAR19 + shIL6. The CD3 antibody modified the LNPs targeting the lymphoid tissue in which the T cells were located. (I) The number of CAR-T cells produced by LNPs in vivo. The number of CAR-T cells in T + AntiCD3-LNP/CAR19 + shIL6 group was significantly higher than that in other groups on the 21st day. Data are represented as mean ± SD of 5 mice in each group. Adapted with permission from [141], copyright 2022 Journal of Controlled Release. (J) Quantitation of the purity and recovery of FACS/MACS/MATIC for isolating human NSCLC samples. *P < 0.05, **P < 0.01, ***P < 0.001, log-rank test for survival curve, unpaired t test for bar plot, mean ± SD. Each dot represents a biological replicate. Adapted with permission from [150], copyright 2022 Journal of Controlled Release.

Funding: This work was supported by National Natural Science Foundation of China (Grant number 82274111).

Author contributions: ZS.G. organized data and literature, made graphs, and was a major contributor in writing the manuscript. JH.Y. wrote and revised the manuscript. XH.C. wrote and edited review. TS.W. wrote and edited review and investigation. Y.Z. wrote and checked the manuscript. KL.Y. wrote and checked the manuscript. SY.D. participated in project administration and provided funding. PY.L. supervised the writing of the manuscript and provided funding. All authors read and approved the final manuscript.

Competing interests: The authors have declared that no competing interest exists.