Efficient chemo-immunotherapy leveraging minimalist electrostatic complex nanoparticle as “in situ” vaccine integrated tumor ICD and immunoagonist

Graphical abstract

An efficient chemo-immunotherapy leveraging minimalist electrostatic complex nanoparticle (NP) integrated tumor immunogenic cell death (ICD) and immunostimulatory agonist was developed as a watertight “in situ” tumor vaccine for cancer therapy through convenient intratumoral administration with minimized systemic toxicity.

Highlights

• •Ingenious chemo-immunotherapy leveraging minimalist electrostatic complex nanoparticle.
• •Convenient integration of tumor immunogenic cell death and immunostimulatory agonist.
• •Intratumoral injection ensured minimized systemic toxicity and promoted therapy outcome.
• •This ‘‘in situ” tumor vaccine effectively boosted a watertight potent antitumor immunity.
• •The idea provided a feasible and promising paradigm for cancer chemo-immunotherapy.

Abstract

Introduction

Immunotherapy has unprecedentedly opened up a series of neoteric tactics for cancer treatment. As a burgeoning approach, chemo-immunotherapy has innovatively expanded the accomplishments of conventional chemotherapeutic agents for cancer governing.

Objectives

An efficacious chemo-immunotherapy leveraging minimalist electrostatic complex nanoparticle (NP) integrated tumor immunogenic cell death (ICD) and immunoagonist was developed as a watertight “in situ” vaccine for cancer therapy through convenient intratumoral administration with minimized systemic toxicity.

Methods

Chemical-modified pH-sensitive cis-aconityl-doxorubicin (CAD) and immunoadjuvant unmethylated cytosine-phosphate-guanine (CpG) were co-packaged by polycationic polyethylenimine (PEI) though electrostatic-interaction to construct PEI/CpG/CAD NP. By intratumoral injection, this positively charged NP could be detained at tumor site and endocytosed by tumor cells effortlessly. Then, doxorubicin was released through cis-aconityl cleavage induced by endosomal-acidity and further triggered tumor ICD, the moribund tumor cells could release damage-associated molecular patterns (DAMPs) to recruit dendritic cells (DCs). Meanwhile, the entire tumor debris derived into diversified antigens and cooperated with immunostimulatory CpG to excite DC maturation and activated comprehensive antitumor immunity.

Results

Prominent tumor suppression was achieved in aggressive mouse melanoma tumor model, which verified the feasibility and effectiveness of this minimalist CAD/CpG-codelivered NP.

Conclusion

This study has provided a convenient and promising paradigm for potent cancer chemo-immunotherapy.

Introduction

Immunotherapy has set off a huge heat wave in oncotherapy over the past decade, and it also has boosted bright interpretations for traditional antitumor therapies in the aspect of immunology [1], [2], [3]. Chemo-immunotherapy has won blowout attention in recent years, some of the conventional chemotherapeutants, such as doxorubicin (DOX), paclitaxel and oxaliplatin, were proved to possess the capability of inducing tumor immunogenic cell death (ICD) [4], [5], [6], [7]. These chemotherapeutic agents can drub tumor cells to release damage-associated molecular patterns (DAMPs) for recruiting antigen-presenting cells (APCs) to the drug-treated tumor region [8]. The deciduous cell debris from the moribund tumor cells will be recognized and captured as tumor-associated antigens (TAAs) by APCs [9]. Then, these educated APCs will process and present TAAs to T cells for further activating tumor-specific immune responses to fight against malignancies [10], [11], [12], [13].

For chemotherapeutic drugs, traditional administration pattern is systemic injection. However, decades of clinical implementation had witnessed the terrible toxic side-effects of these drugs with nonselective carnage between tumors and normal tissues after systemic biodistribution. For instance, DOX has a wide antitumor spectrum and it has been widely employed in treating various cancers [14]. Apart from its antitumor property, DOX can arouse severe cardiotoxicity and liver damage. In addition, the toxic side-effect of DOX also involves thrombocytopenia, leukopenia, nausea and vomiting, inappetence and hair loss [15]. To diminish the side-effects, topical administration, such as intratumoral injection, is an emerging programme for solid tumor therapy [16], [17]. Compared with systemic administration, local management has preferable safety to circumvent systemic toxicity and adverse side-effects [18], [19]. Meanwhile, intratumoral administration can directly deliver high dose of drugs to the intended tumor site to better undertake tumor accumulation for maintaining effective drug therapeutic concentration [20], [21]. Moreover, local injection usually requires lower dose than that of systemic injection, which is beneficial to lessen drug dosage and save treatment cost for patients [22]. Therefore, intratumoral administration is a noteworthy approach for future oncotherapy [23], [20].

Toll-like receptor (TLR) agonists have exhibited potentials in stimulating immune responses [24]. Unmethylated cytosine-phosphate-guanine (CpG) is a TLR9 agonist, as a promising immune adjuvant, CpG can stimulate many immune cells to instigate innate and adaptive immune responses [25]. The potent immunostimulatory capacity is closely involved with its characteristic structure [26]. CpG sequence is universally existed in bacterium and prokaryote DNA, but less in mammals (in a methylated dormant status and unable to stimulate lymphocytes) [27]. When unmethylated CpG situates in mammals, it will be recognized as pathogen-related molecular patterns (PAMPs) and phagocytized by lymphocytes, and further bonded with TLR9 to trigger immune responses [28]. In the reported studies, the administration of immunostimulatory CpG to tumors was inclined to reverse the immunosuppressive tumor microenvironment (TME) from “cold” to “hot” for igniting antitumor immune responses [29], [30], [31]. However, systemic administration of immunostimulatory agents inevitably distributed to normal tissues, the off-target effect would cause immune-related adverse events (irAEs) by the overstimulation of systemic immunotoxicity [32], [33]. Therefore, intratumoral injection of CpG is more dependable for performing effective treatment by confining the immunomodulatory agent within tumor area [34], [35].

Here, a convenient minimalist electrostatic complex nanoparticle (NP) integrated tumor ICD and immunostimulatory agonist was developed as “in situ” tumor vaccine for efficient chemo-immunotherapy via intratumoral administration (Fig. 1A). Chemical-modified pH-sensitive cis-aconityl-doxorubicin (CAD) and immunoadjuvant CpG were co-packaged by polycationic polyethylenimine (PEI) though electrostatic-interaction to form PEI/CpG/CAD NPs. By intratumoral administration, the positively charged PEI/CpG/CAD NPs could be accumulated at tumor region and readily endocytosed by tumor cells. In tumor endosome, the cis-aconityl linkage of CAD was sensitive to acidic pH and cleaved to release DOX for triggering tumor ICD. These moribund tumor cells further released DAMPs to recruit dendritic cells (DCs). Meanwhile, the heterogeneous antigens derived from tumor cell debris cooperated with the immunostimulatory CpG further excited DC maturation and activated specific antitumor-immunity. A series of experiments were implemented to verify our hypothesis on the effectiveness of the designed CAD and CpG codelivery NPs. By inducing robust ICD, the minimalist PEI/CpG/CAD NPs could convert whole tumor cells into ‘‘in situ” tumor vaccines to effectively boost a watertight antitumor immunity by the adequate recognition of entire TAAs and broadly promote the activation of comprehensive types of tumor-specific T cells for immunotherapy. As expected, prominent tumor suppression was accomplished in aggressive mouse melanoma tumor model by intratumoral injection of PEI/CpG/CAD NPs with a low drug dosage. The study has provided a convenient and promising paradigm for potent cancer chemo-immunotherapy.

Fig. 1.

(A) Schematic of the efficacious chemo-immunotherapy leveraging minimalist electrostatic complex PEI/CpG/CAD NP as “in situ” vaccine integrated DOX-induced tumor ICD and CpG-assisted immunostimulation, along with the guaranteed biosafety via intratumoral injection. (B) Zeta potential and (C) particle size of the PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs. (D) The PDI of PEI/CpG/CAD NPs. (E) In vitro drug release of PEI/CpG/CAD NPs under diverse pH.

Materials and methods

Materials

PEI (25 kDa) and cis-aconitic anhydride (CA) were from Sigma-Aldrich. CpG 1826 and Cy5-modified CpG (CpG-Cy5) were from Sangon (Shanghai, China). DOX·HCl was from Macklin (Shanghai, China). CAD was prepared by decorating DOX with CA as our reported methods [36], [37]. ELISA kits for mouse HMGB1, ATP, IL-6, TNF-α and IFN-γ were from R&D Systems (MA, USA). Antibody CD11c-PE, CD86-APC, CD80-FITC, CD3e-PE and CD8a-FITC for flow cytometry (FCM) were from eBioscience (CA, USA). CD8a antibody for immunofluorescence was from Invitrogen (CA, USA). CRT and FITC-labeled secondary antibody were from ABclonal (Wuhan, China).

Preparation and characterization of PEI/CpG/CAD NPs

PEI/CpG/CAD NPs were formulated conveniently by simple electrostatic-adsorption. Briefly, PEI (2 mg/mL), CpG (1 mg/mL) and CAD (1 mg/mL) were separately dissolved in PBS (pH 7.4, 0.01 M) and mixed together in equal volume, the mixture was instantly vortexed 20 s and incubated 20 min to form PEI/CpG/CAD NPs (mass ratio = 2:1:1). Zeta potential and particle size were measured by Zetasizer Nano ZS90 analyzer (Malvern Instruments Ltd., UK).

In vitro drug release

In vitro drug release of CAD from PEI/CpG/CAD NPs was investigated in PBS release medium under different pH (7.4, 6.8, 6.0 and 5.0). Specifically, 2 mL prepared PEI/CpG/CAD NPs (containing 50 μg CAD) were sealed in dialysis bag (3.5 kDa), and incubated in 48 mL corresponding release medium under gentle shaking at 100 rpm in 37 °C. At predetermined time intervals, 2 mL release medium was pipetted out for testing, and 2 mL fresh PBS was replenished. The released drug from PEI/CpG/CAD NPs was measured by UV spectrophotometry at 480 nm.

Cellular uptake

Cellular uptake of PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs in B16 cells was observed by FCM. Cells were seeded in 6-well plate (2 × 10⁵ cells/well) and severally incubated with PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs (FITC-labeled PEI was used), the working concentration of CAD was 5 μg/mL. After 4 h incubation, B16 cells were detected by FCM (BD FACS AriaIII, USA).

Intracellular localization

B16 cells were seeded on sterile coverslips in 6-well plate (2 × 10⁵ cells/well) and PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs (FITC-PEI and Cy5-CpG were used) were added, the working concentration of CAD was 5 μg/mL. After 4 h incubation, B16 cells were gently fixed with 4 % paraformaldehyde solution and nuclei were tagged by DAPI, further observed by CLSM (Leica TCS SP8, Germany).

Cytotoxicity assay

MTT assay and live/dead cell staining were performed to evaluate in vitro cytotoxicity. B16 cells were seeded in 96-well plate (8000 cells/well) and PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs were added, the working concentration of CAD was 5 μg/mL. After 24 h, cytotoxicity was measured by MTT at 492 nm with Bio-Rad 680 microplate reader. For live/dead cell staining, B16 cells were seeded in 6-well plate (2 × 10⁵ cells/well), after incubated with PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs for 24 h, cells were stained with calcein-AM and PI, subsequently photographed by inverted fluorescent microscope (DMI4000B, Leica, Germany).

In vitro induction of ICD

To investigate in vitro ICD induction, cell surface CRT, extracellular release of HMGB1 and ATP were examined. CRT was assessed by FCM and CLSM. For FCM, 2 × 10⁵ B16 cells were seeded into 6-well plate and incubated with PBS, PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs for 24 h, the working concentration of CAD was 5 μg/mL. Then cells were incubated with CRT antibody and analyzed via FCM. For CRT visualizing, after 24 h of NP-coincubation, B16 cells were fixed and nuclei were marked by DAPI, further incubated with CRT antibody and visualized by CLSM. The extracellular release of HMGB1 and ATP was examined by ELISA kits. After 24 h NP-treatment, culture supernatant was collected for ELISA.

In vitro DC maturation

Bone marrow-derived dendritic cells (BMDCs) were cultivated to evaluate DC maturation in vitro through detecting costimulatory molecules (CD80 and CD86) and cytokines (TNF-α and IL-6). Firstly, B16 cells were severally treated with PBS, PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs (CAD working concentration: 5 μg/mL). After B16 cells were treated by the NPs for 24 h, BMDCs were added into the plate and co-cultured with these NP-treated B16 cells. After 24 h, the cells were collected and stained with CD11c-PE, CD80-APC and CD86-FITC antibodies to measure the costimulatory molecules on BMDCs by FCM. Besides, cytokine levels of TNF-α and IL-6 in culture medium were measured by ELISA.

In vivo antitumor therapy

Female C57BL/6 mice (6-week-old) were purchased from Vital River (Beijing, China). To generate subcutaneous tumor model, mice were inoculated with 5 × 10⁵ B16 cells. When tumors reached certain volume, mice were randomly divided into 6 groups (5 mice/group) and intratumorally injected with PBS, PEI, CpG/CAD, PEI/CAD, PEI/CpG and PEI/CpG/CAD NPs (the dosage was 2 mg/kg on PEI basis, CpG and CAD was 1 mg/kg) every 3 days, and 5 times in total. During therapy, tumor size and body weight were monitored every other day. After therapy, tumors and main organs were collected for analysis. Tumor suppression rate (TSR) = [(V_c - V_x)/V_c] × 100 %, V_c and V_x respectively represented the mean tumor volume of PBS control group and individual treatment group.

Analysis of immune cells

Next, immune cells were analyzed through FCM, including intratumoral DCs, mature DCs in tumor-draining lymph nodes (TDLNs) and CD8⁺ T cells in tumors. Fresh tumors and TDLNs were harvested and processed into single-cell suspension. Intratumoral DCs were detected by CD11c-PE antibody, mature DCs in TDLNs were identified by CD11c-PE and CD86-FITC antibodies. For CD8⁺ T cells in tumors, the cells were marked with CD3e-PE and CD8a-FITC antibodies further sorted by FCM.

Inflammatory cytokines

The inflammatory cytokines (IFN-γ and TNF-α) in tumors were evaluated by ELISA kits. Fresh tumors were cut into pieces and homogenized in ice bath to obtain supernatant. The tests were carried out following the ELISA instructions, and the concentrations of IFN-γ and TNF-α were quantified via standard curves which established according to standard samples.

Histology and immunofluorescence

To check biocompatibility, mouse main organs and tumors were carefully collected for histopathological analysis by H&E staining. For immunofluorescence, CRT exposure and CD8⁺ T cells in tumors were performed separately. For CRT exposure, tumor sections was incubated with CRT antibody and further stained with DAPI. To visualize tumor-infiltrating CD8⁺ T cells, tumor slices were successively incubated with CD8a antibody and DAPI, the obtained tumor slices were observed by CLSM.

Statistical analysis

All tests were carried out independently for 3 times, and data were presented as mean ± SD. ANOVA with Bonferroni's post hoc test was utilized for statistical significance. *p < 0.05 meant statistically significant, **p < 0.01 and ***p < 0.001 represented highly significant.

Ethics statement

All animal procedures were conducted in accord with the guidelines established by the Animal Care and Use Committee of Shandong Second Medical University (Approval no. 2021SDL075).

Results and discussion

Zeta potential and particle size

PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs were assembled by facile electrostatic-interaction between polycationic PEI and negative CAD and CpG. Firstly, zeta potential and particle size were characterized and presented in Fig. 1B and C. All of the PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs were positively charged, and compared with the other two fomulations, PEI/CpG/CAD NPs had lowered zeta potential (Fig. 1B), which implied the successful co-hitchhike of the CpG and CAD. The particle size of PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs were respectively 441 nm, 282 nm and 247 nm (Fig. 1C), which indicated that the formed PEI/CpG/CAD NPs had more compact nanostructure. Further polydispersity index (PDI) result displayed that the PEI/CpG/CAD NPs had a uniform narrow size-distribution, the PDI was 0.149 (Fig. 1D). The positive zeta potential of PEI/CpG/CAD NPs could significantly facilitate the interaction with negative cell membrane for promoted cellular uptake, besides, the nano-size would also be advantageous for being endocytosed by the cells.

In vitro drug release

Drug release of PEI/CpG/CAD NPs was investigated in vitro under different pH (7.4, 6.8, 6.0 and 5.0) for mimicking counterpart pH gradient from blood circulation to TME and intracellular endosome/lysosome. The result (Fig. 1E) showed that CAD release from PEI/CpG/CAD NPs was pH-dependent, and the cumulative CAD release rate increased accompanying with pH decrease. This pH-sensitive release of CAD from PEI/CpG/CAD NPs could be attributed to the responsive breakage of cis-aconityl linkage in acidic condition, which had been clearly verified in our previous studies [36], [37].

Cellular uptake and intracellular localization

Cellular uptake of PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs was evaluated in B16 melanoma cells by FCM (Fig. 2A, B and C). Compared with PBS, all groups had excellent cellular uptake, and the uptake percentages were close to 100 %. This magnificent endocytosis was attributed to PEI, as a widely studied polycationic carrier, PEI had been broadly reported to possess prominent cellular uptake efficiency [38], [39], this was also the motivation why we selected PEI to co-hitchhike CpG and CAD. To further determine the intracellular localization of the PEI/CpG/CAD NPs in B16 cells, PEI-FITC (green) and CpG-Cy5 (yellow) were utilized for intracellular tracking, and CAD was visualized by the fluorescence of DOX (red). As shown in Fig. 2D, conspicuous internalization was accomplished for all the groups, which was in accord with the FCM result. For PEI/CpG/CAD NPs, these three kinds of fluorescence exhibited robust intensity and co-localization in B16 tumor cells, which illustrated that the PEI/CpG/CAD NPs could efficiently co-deliver CpG and CAD into the B16 cells.

Fig. 2.

(A, B, C) Cellular uptake, mean fluorescence intensity and representative FCM plot of PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs in B16 cells for 4 h detected by FCM. (D) Intracellular localization of these NPs in B16 cells for 4 h presented by CLSM. PEI-FITC and CpG-Cy5 were displayed in green and yellow, CAD was tracked by DOX fluorescence in red (scale bar = 10 μm). (E) Cytotoxicity of PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs for 24 h. (F) Live/dead staining of B16 cells treated with different NPs for 24 h (scale bar = 200 μm).

Cytotoxicity assay

The cytotoxicity of PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs against B16 cells was evaluated by MTT assay. As displayed in Fig. 2E, about 20 % death cells were detected when incubated with PEI alone, the cytotoxicity of PEI was widely known as its unfavorable influence on cells induced by its abundant amino groups with positive charges [40], [41]. After complexing negative CpG, the cell viability of PEI/CpG was slightly increased. When CAD was hitchhiked, the cytotoxicity of the PEI/CAD and PEI/CpG/CAD NPs was enhanced, the CpG and CAD co-loaded NPs produced significantly intensified tumor-killing effect. For further visualized the cytotoxicity, live/dead cell staining was conducted and shown in Fig. 2F. It was noticeable that the cytotoxic tendency of live/dead cell staining was consistent with MTT result, and these data expressly indicated that the PEI/CpG/CAD NPs had excellent cytotoxicity against B16 tumor cells.

In vitro induction of ICD

To verify ICD induction, representative DAMPs including CRT, HMGB1 and ATP were detected in vitro. CRT exposure was analyzed by FCM and CLSM, as shown in Fig. 3A and B, the increasing of CRT⁺ B16 cells confirmed that PEI/CpG/CAD NPs dramatically induced much more CRT exposure on B16 cells than other groups (Fig. 3A). And CLSM result also exhibited that significant CRT exposure was incurred by PEI/CpG/CAD NPs (Fig. 3B). Next, HMGB1 release and ATP secretion were examined by ELISA (Fig. 3C and D), compared with other groups, PEI/CpG/CAD NPs also significantly enhanced the release of HMGB1 and ATP from B16 cells. The above results collectively suggested that PEI/CpG/CAD NPs could remarkablely induce ICD of B16 tumor cells.

Fig. 3.

(A) FCM quantification of CRT⁺ B16 cells treated by PEI, PEI/CpG, PEI/CAD and PEI/CpG/CAD NPs for 24 h. (B) CLSM image of CRT exposure (scale bar = 10 μm). (C, D) ELISA detection for HMGB1 and ATP release from B16 cells treated by NPs for 24 h. (E-H) In vitro DC maturation analyzed through FCM of costimulatory molecules (CD80 and CD86). (I, J) Cytokine TNF-α and IL-6 secretion for DC maturation.

In vitro DC maturation

After confirming the ICD-inducing effect of PEI/CpG/CAD NPs, we further evaluated the stimulation capacity on DC maturation in vitro. Beforehand, B16 cells were treated with NPs for 24 h, then BMDCs were added and co-cultured with the NP-treated B16 cells for another 24 h. Costimulatory molecules (CD80 and CD86) and cytokines (TNF-α and IL-6) were detected for assuring DC maturation. For costimulatory molecules (Fig. 3E–H), the CD80⁺ and CD86⁺ DCs were significantly higher for the PEI/CpG/CAD NPs than other groups. The maturation-related cytokine TNF-α and IL-6 secreted by DCs were analyzed through ELISA, PEI/CpG/CAD NPs also exhibited the topmost secretion of these cytokines (Fig. 3I and J). These results collectively demonstrated that efficacious ICD of B16 tumor cells was induced by treating with PEI/CpG/CAD NPs, the generated tumor cell debris (can act as comprehensive diversified TAAs) and the released DAMPs could effectively promote DC maturation, which would be a forceful prerequisite for further activate multifarious tumor-specific T cells for a watertight antitumor immunity.

In vivo antitumor therapy

In vivo antitumor therapy was evaluated in B16 tumor-bearing mice, and different NPs were intratumorally injected every 3 days for 5 times in total (Fig. 4A). Tumor size and body weight were monitored and the results were displayed in Fig. 4B and C. Compared with PBS group, free PEI and PEI/CpG NPs showed modest tumor inhibition, while CpG/CAD and PEI/CAD groups revealed enhanced tumor suppression correlated with the participation of CAD. Encouragingly, the PEI/CpG/CAD NPs presented the most prominent antitumor efficiency, with TSR% up to 87.71 %. Besides, from the photo of tumor tissues (Fig. 4D) and the recorded tumor weight (Fig. 4E), remarkable antitumor outcome could be visually observed for the PEI/CpG/CAD NPs. In addition, the body weight (Fig. 4C) of the treated mice for all the groups had steadily risen, which implied that the intratumoral administration of these positive NPs could effectively guarantee an admissible systemic tolerance and biosecurity.

Fig. 4.

(A) Therapeutic schedule of in vivo antitumor therapy. (B) Tumor volume change. TSR%: tumor suppression rate. (C) Body weight of the treated mice. (D) Photo of the tumors after therapy. (E) Tumor weight of different groups.

Analysis of immune cells and inflammatory cytokines

Next, immune cells were analyzed through FCM, including intratumoral DCs, mature DCs in tumor-draining lymph nodes (TDLNs) and CD8⁺ T cells in tumors. Generally, after intratumor injection of the PEI/CpG/CAD NPs, it was speculated that the induced ICD effect would release DAMPs and TAAs to recruit DCs to tumor area, so intratumoral DCs were detected. As shown in Fig. 5A and B, the treatments of PEI/CAD and PEI/CpG/CAD NPs could recruit more DCs into tumor tissues. Afterwards, mature DCs in TDLNs were tested, and PEI/CpG/CAD NPs also gained the highest quantity of mature DCs in TDLNs (Fig. 5C and D). Finally, CD8⁺ T cells in tumors were investigated, and the FCM result (Fig. 5E and F) verified that the tumor-infiltrating CD8⁺ T cells for PEI/CpG/CAD NPs were 21 times compared with PBS group and also marvellously higher than other treatment groups, which indicated that the PEI/CpG/CAD treatment could markedly promote the infiltration of CD8⁺ T cells into tumors. Meanwhile, the inflammatory cytokine IFN-γ and TNF-α were evaluated by ELISA, and the concentrations of the two cytokines (Fig. 5G and H) were significantly increased for PEI/CpG/CAD NPs. These results suggested that PEI/CpG/CAD NPs could induce potent ICD for efficiently recruiting DCs and activating CD8⁺ T cells to boost effective antitumor immune responses.

Fig. 5.

(A, B) DCs in tumors analyzed by FCM. (C, D) Mature DCs in TDLNs. (E, F) CD8⁺ T cells in tumors. (G, H) Cytokine levels of IFN-γ and TNF-α in tumors.

Histology and immunofluorescence

The biosafety was evaluated by H&E staining, and histological analysis of major organs presented no tissue damage and pathological abnormality for all groups (Fig. 6A), suggesting a satisfactory biosafety of PEI/CpG/CAD NPs for intratumor administration. Furthermore, severe tumor cell death and nucleus dissolution were observed for PEI/CpG/CAD NPs, which exhibited conspicuous antitumor effect. To further confirm the ICD-induction capacity of PEI/CpG/CAD NPs in vivo, CRT exposure in tumors was analyzed by immunofluorescence staining (Fig. 6B), and PEI/CpG/CAD NPs exhibited significantly enhanced CRT exposure. Finally, CD8⁺ T cells in tumors were inspected, as visualized in Fig. 6C, PEI/CpG/CAD NPs boosted higher level of CD8⁺ T cells infiltrated in tumors, which firmly indicated that this CpG and CAD co-hitchhiked nanoplatform could trigger potent antitumor immune responses and accomplish feasible cancer chemo-immunotherapy.

Fig. 6.

(A) H&E staining of major organs and tumors (scale bar = 50 μm). (B, C) Immunofluorescence labeling of CRT and CD8⁺ T cells in tumors (scale bar = 100 μm).

Conclusion

In summary, a convenient minimalist electrostatic complex PEI/CpG/CAD NP which integrated tumor ICD and immunostimulatory agonist was developed as “in situ” tumor vaccine for efficient chemo-immunotherapy. By intratumoral administration, the positive PEI/CpG/CAD NPs could be maximally accumulated at tumor site and readily endocytosed by tumor cells for triggering robust ICD and convert whole tumor cells into ‘‘in situ” tumor vaccines to effectively boost a comprehensive antitumor immunity. As expected, preeminent tumor suppression was accomplished in aggressive mouse melanoma tumor model by intratumoral injection with maximum limitation of systemic toxicity and adverse effects. This research has provided an efficacious and feasible nanoplatform paradigm for potent cancer chemo-immunotherapy.

CRediT authorship contribution statement

Yunfei Han: Methodology, Investigation. Mingxia Jiang: Investigation, Data curation, Writing – original draft preparation. Yanju Sun: Validation, Data curation. Wenqiang Chen: Visualization, Software. Yanli Zhao: Formal analysis. Xiuwen Guan: Conceptualization, Funding acquisition, Project administration, Writing – review & editing. Weifen Zhang: Supervision.

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.