Sequential acid/reduction response of triblock copolymeric nanomicelles to release camptothecin and toll-like receptor 7/8 agonist for orchestrated chemoimmunotherapy

Background Immunosuppressive tumor immune microenvironment (TIME) lowers immunotherapy effectiveness. Additionally, low penetration efficiency and unpredictable drug release in tumor areas restrict tumor therapy. Methods A triblock copolymeric micelle (NanoPCPT+PIMDQ) was developed to carry the chemotherapeutic drug camptothecin (CPT) and the TLR7/8 agonist 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c] quinoline-4-amine (IMDQ) to achieve deep tumor penetration and on-demand drug release by responding to acid and reduction stimuli sequentially. The synergistic antitumour efficacy of NanoPCPT+PIMDQ was assessed both in vitro and in vivo. Results NanoPCPT+PIMDQ is composed of a hydrophilic PEG(polyethylene glycol) outer layer, an acid-sensitive EPEMA middle layer, and a drug inner core. Upon intratumoral injection, (i) NanoPCPT+PIMDQ first responds to the acidic tumor microenvironment and disintegrates to PIMDQ and PCPT, penetrating deep regions of the tumor; (ii) tumor cells are killed by the released CPT; (iii) DCs are activated by PIMDQ to increase the infiltration of cytotoxic T lymphocyte (CTL); and (iv) both downregulated Foxp3+ Tregs by CPT and repolarized M2 macrophages by PIMDQ can relieve the TIME. Conclusion This pH/GSH-responsive triblock polymer-drug conjugate reduces immunosuppression and enhances the infiltration of CTLs by codelivering CPT and IMDQ in a controllable manner, providing a promising platform for synergistic tumor chemoimmunotherapy. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-022-01577-5.

"nonimmunogenic phenotypes" into "immunogenic phenotypes" tumors could be a viable solution to solve this issue. To improve the response rate of nonimmunogenic tumors (i.e., colorectal cancer) [6], it is urgent to simultaneously reverse the immunosuppressive state and enhance the infiltration of CTLs [7,8]. CPT, a commonly used chemotherapeutic agent, is reported to inhibit Treg cells by reducing the expression of Forkhead box P3 (Foxp3) [9]. Emerging evidence indicates that IMDQ, as a variant of IMQ (imiquimod), promotes macrophage repolarization and stimulates dendritic cell (DC) maturation in tumors and tumor-draining lymph nodes (TDLNs), enhancing CTL infiltration and therapeutic efficacy [10,11]. Therefore, combining CPT and IMDQ could reverse the immunosuppressive state (decreasing Treg cells and repolarizing M2 macrophages) and increase CTL infiltration, providing a platform to sensitize nonimmunogenic tumors.
Although promising, their precise delivery requires overcoming multiple barriers to penetrate deeply into tumors and achieve on-demand release, which remains challenging. Focusing on these obstacles, previous studies have reported the strategies of charge inversion [12] and size variation [13], demonstrating favorable deep tumor penetration activity. Additionally, a design with responsiveness to tumor microenvironment stimuli, such as pH and GSH, can be used to control drug release [14][15][16]. Notably, the GSH concentration inside cancer cells (2 − 10 × 10 −3 M) is remarkably higher than that in normal tissue (2 − 10 × 10 −6 M) [17,18]. Taking advantage of these properties, a GSH-responsive strategy was adopted here to release drugs in tumor cells and optimize the therapeutic efficacy.
Herein, a cascade-responsive trilayer polymeric micelle (Nano PCPT+PIMDQ ) was developed to codeliver CPT and TLR7/8 agonist in a controllable manner by sequentially responding to pH/GSH to trigger deep tumor penetration and on-demand release. The design of Nano PCPT+PIMDQ is based on a triblock copolymer consisting of a hydrophilic poly(ethylene glycol) (PEG) segment, acid-sensitive poly(2-(N-ethyl-N-propyl amino) ethyl methacrylate) (PEPEMA) chain and hydrophobic drug segment (reduction-responsive CPT) prodrug (PCPT) chain or poly(IMDQ) (PIMDQ) chain), which are obtained by RAFT polymerization. Upon intratumoral injection (i.t.), the protonated PEPEMA becomes a hydrophilic fragment with positive charges and subsequently promotes the decomposition of Nano PCPT+PIMDQ into PCPT and PIMDQ, which is beneficial for increasing cellular uptake and tumor penetration. TLR 7/8 is stimulated by PIMDQ to activate DCs, and protumoral M2 macrophages are repolarized to antitumoral M1 macrophages. Subsequently, the high GSH concentration in tumor cells triggers CPT release from PCPT, which not only provides anticancer cytotoxic effects but also decreases the expression of Foxp3 in Treg cells. Furthermore, combination with TLR7/8 agonists elicits a robust antitumor CTL response in primary and distal tumors (Scheme 1).

Preparation and characterization of Nano PCPT+PIMDQ
The preparation process, pH responsiveness and drug release mechanism of Nano PCPT+PIMDQ are illustrated in Fig. 2a. The structure of EPEMA changed to a hydrophilic quaternary amine in the acidic tumor milieu, allowing for charge reversal and dispersion of Nano PCPT+PIMDQ . These changes not only enhanced the cellular uptake of the conjugate but also increased drug penetration into tumors. To verify our hypothesis, we studied the effect of pH on the size and morphology of Nano PCPT+PIMDQ . As illustrated in Fig. 2b-d, the hydrodynamic particle size of Nano PCPT+PIMDQ at pH 7.4, 6.5, and 6.0 determined by dynamic light scattering (DLS) was 172.43 ± 9.57 nm, 112.83 ± 6.41 nm, and 52.78 ± 5.41 nm, respectively, with a polydispersity index (PDI) of 0.22 ± 0.00, 0.21 ± 0.01, and 0.76 ± 0.01. Transmission electron microscopy (TEM) images obtained under different pH conditions indicated the pH-induced dissociation of Nano PCPT+PIMDQ . Specifically, the morphology of Nano PCPT+PIMDQ was spherical at pH 7.4 with a size of 100-130 nm, tended to be irregular at pH 6.5, and completely collapsed at pH 6.0, further revealing the pH dependence of its morphology. The count rate, representing the average scattering intensity of samples [19], decreased as the pH decreased, especially at pH 6.0, indicating the disassembly of Nano PCPT+PIMDQ (Fig. 2e). To confirm the charge reversal induced by pH, the zeta potential of Nano PCPT+PIMDQ was investigated at different pH values. The zeta potentials of Nano PCPT+PIMDQ reversed from − 5.95 ± 0.87 mV at pH 7.4 to 10.87 ± 0.40 mV at pH 6.5 and 24.53 ± 1.17 mV at pH 6.0 (Fig. 2f ).
The release kinetics of free CPT in vitro verified the reduction-sensitive release characteristics of nanomicelles designed to control drug release and avoid the toxicity of chemotherapy to normal cells [20]. The samples were dialyzed against various buffered medium in the absence or presence of dithiothreitol (DTT, 10 mM) to study whether the reduction circumstances influenced the CPT release characteristics of Nano PCPT . Negligible CPT was released from Nano PCPT without DTT, no matter under which pH conditions the samples are. However, free drug was constantly released from Nano PCPT if they were immersed into 10 mM DTT solution under different pH (7.4, 6.5, and 5.5), indicating that the release of Scheme 1. A schematic diagram of the cascade-responsive trilayer polymeric micelles (Nano PCPT+PIMDQ ): a Preparation of Nano PCPT+PIMDQ ; b Nano PCPT+PIMDQ responds to the acidic tumor microenvironment, triggering deep tumor penetration and releasing PCPT and PIMDQ on demand. PCPT releases CPT in tumor cells in response to GSH, and the remaining CPT is released from dying tumor cells into the tumor microenvironment. Subsequently, Tregs are downregulated by CPT. d PIMDQ not only elicits an immune response by activating DCs but also alleviates the immunosuppressive environment by repolarizing M2 TAMs. The combination of IMDQ and CPT relieved the immunosuppressive milieu and boosted the infiltration of CTLs, thus improving colorectal cancer therapy. Figure created using BioRender.com drug is dependent on the reduction condition, not or a little bit dependent on the pH conditions in the tumor microenvironment ( Fig. 2g and Additional file 1: Fig.  S13). Altogether, the combined results revealed that Nano PCPT+PIMDQ exhibited sequential pH/GSH responsiveness, laying the groundwork for our subsequent research.

In vitro cellular uptake and cytotoxicity of nanomicelles
Although CPT has an excellent curative effect, its clinical application is limited due to its low water solubility and high toxicity [21]. Traditional nano-formulations can effectively improve the in vivo pharmacokinetics of CPT, but the physical encapsulation of CPT will still cause damage to the body due to quick leakage in systemic circulation [22]. Covalent ligation to polymeric carriers might offer chemically better-defined alternatives to physical encapsulation technology. Studies have shown that the positive charge of nanomedicine is beneficial to cellular uptake [23]. To evaluate the effects of various pH levels on the cellular uptake of nanomicelles, 5(6)-TAMRA cadaverine (Rho) was used to label nanomicelles, yielding Nano Rho . When incubated with CT26 cells at pH = 6.5, Nano Rho showed a higher mean fluorescence intensity (MFI) than that at pH = 7.4 (*p < 0.05). This result was attributed to the charge reversal from negative to positive of Nano Rho at pH 6.5 (Fig. 3a), which was beneficial to enhance the uptake by tumor cells. To evaluate the tumor penetration ability of Nano PCPT+PIMDQ by 3D multicellular spheroids (MCSs) of CT26 cells, cy5.5 was chosen instead of CPT or IMDQ to label triblock polymeric micelles, yielding Nano cy5.5 . Confocal laser scanning microscopy (CLSM) showed that more Nano cy5.5 penetrated the interior zone of MCSs at pH 6.5 after 6 h of incubation than at pH 7.4 (Fig. 3b), which was related to the charge The achievement of charge reversal from negative to positive in the nanomicelles was merely one of the requirements for their efficacy. Subsequently, the cytotoxicity of Nano blank polymer against CT26 cells was assessed by a CCK-8 assay. According to studies, zwitterionic polymers are neutral and thus have low cytotoxicity [24]. Negligible cytotoxicity was observed when the concentration of Nano blank polymer reached 500 μg·mL −1 (Additional file 1: Fig. S14), indicating that it was cytocompatible at a given concentration range. It is worth noting that the concentration of GSH in tumors (2-10 × 10 -3 M) is reported to be 1000 times higher than that in normal cells (2-10 × 10 -6 M) [25]. The in vitro release of CPT demonstrated the reduction-triggered ability of Nano PCPT . To further confirm the reduction-triggered specific drug release in tumor cells, we investigated the toxicity of free CPT and Nano PCPT to tumor cells (CT26 cells) and normal cells (RAW 264.7 cells) (equal to 3 μM CPT), respectively. As expected, free CPT had equal toxicity to RAW 264.7 cells and CT26 cells, while the viability of macrophages incubated with Nano PCPT was lower than that of CT26 cells (***p < 0.001) (Fig. 3f ). Additionally, we explored the toxicity of various concentrations of Nano PCPT against CT26 cells. The cell viability exhibited a dosedependent effect, and the half-maximal inhibitory concentration of the Nano PCPT group determined to be the CPT-equivalent concentration was 8 μM (Fig. 3c). The results indicated that the carrier, triblock polymer, had high biocompatibility, and Nano PCPT had excellent reduction-induced drug release performance against tumor cells rather than normal cells. Therefore, Fig. 2 Physicochemical characterization of Nano PCPT+PIMDQ . a Schematic diagram of the structure, self-assembly and disassembly of Nano PCPT+PIMDQ . b, d DLS results showing the hydrodynamic diameter of Nano PCPT+PIMDQ at pH b 7.4, c 6.5, and d 6.0. e Count rate of Nano PCPT + PIMDQ accompanied by TEM images at pH 7.4 (bar: 500 nm), pH 6.5 (bar: 100 nm), and pH 6.0 (bar: 100 nm). f Zeta potentials of Nano PCPT+PIMDQ under various pH conditions. g DTT-triggered CPT release from Nano PCPT in vitro. Data are shown as the mean ± standard deviation (SD) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with other CPT nanocarriers, our carrier could be taken up by cells at tumor sites and had a powerful specific killing effect on tumor cells.

In vitro immune stimulation evaluation
The ability of Nano PIMDQ to activate bone marrowderived dendritic cells (BMDCs), which are the most effective antigen-presenting cells, was determined by activation assays. For this purpose, either IMDQ or Nano PIMDQ was pulsed with BMDCs, and the extent of DC activation was quantified by flow cytometric measurement of the surface expression of MHC-II and the costimulatory molecules CD80, CD86, and CD40. The intracellular uptake results ( Fig. 3d and Additional file 1: Fig. S15) indicated that Rho had a stronger fluorescent signal than Nano Rho after a short period of incubation. The fluorescence of Nano Rho was stronger after 24 h of incubation, whereas that of free Rho was comparatively weaker. That might be due to the fact that free Rho, as a small molecule, was quickly taken up but also quickly pumped out by cells. However, Nano Rho , with a certain size, was gradually more taken up by BMDCs over time, which was conducive to exerting efficacy. Furthermore, BMDCs activation experiments (Fig. 3e) showed that the upregulation of MHC-II, CD80, CD40 and CD86 was dose-dependent on both the IMDQ and Nano PIMDQ . BMDCs expressed more MHC-II, CD40, and CD86 after treatment with Nano PIMDQ than that treatment of equal IMDQ (5000 nM), mainly due to the difference in cellular uptake levels of IMDQ. Therefore, the results indicated that Nano PIMDQ was still capable of activating DCs, even to a greater extent than the equivalent amount of IMDQ under the experimental conditions.
Tumor-associated macrophages (TAMs) are the major innate immune cells, accounting for approximately 50% of human tumor cells, and thus play vital . c CCK-8 was used to detect the vitality of CT26 cells incubated with Nano PCPT (n = 6). d In vitro BMDC uptake using Rho as a trace at 24 h (n = 3). e In vitro BMDC maturation induced by Nano PIMDQ and the FlowJo quantification of CD40, CD80, CD86, and MHC-II gated on CD11c + cells (n = 3). f A CCK-8 assay was used to detect/compare the viability of CT26 cells and macrophages incubated with Nano PCPT (n = 5). g, h Flow cytometric analysis results of macrophage repolarization remodeling experiment by RAW264.7 cells in vitro (n = 3). Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 roles in antitumor immunotherapy [26]. TAMs assume two opposing phenotypes: antitumoral M1 macrophages and protumoral M2 macrophages. It was reported that TAMs predominantly exhibit M2-type functions promoting intratumoral infiltration of Treg cells, thus contributing to T-cell dysfunction [27]. Therefore, reprogramming M2 macrophages into M1 macrophages could be a promising therapeutic strategy [28,29]. Here, the effect of free IMDQ and Nano PIMDQ on macrophage repolarization was investigated with IL4-pretreated Raw264.7 cells as a model of protumoral M2 macrophages. CD200R was used to mark M2 macrophages, and iNOS was used to mark M1 macrophages. As shown in Fig. 3g, h, flow cytometric analysis first indicated that Nano blank polymer (without IMDQ conjugation) did not affect the inflammatory responses, suggesting that these nanomicelles were inherently nonimmunogenic under the experimental conditions. Second, both free IMDQ and Nano PIMDQ treatment induced a dramatic increase in the percentage of M1 macrophages. Briefly, Nano PIMDQ notably reprogrammed M2 macrophages to M1 macrophages to alleviate the immunosuppressive tumor microenvironment. Simultaneously, the percentage of M2 macrophages also slightly increased, which could result from a compensatory immune response. Thus, in our work, IMDQ-ligated nanomicelles are sufficiently capable of activating innate immune responses as well as remodeling macrophages to antitumor M1 phenotypes.

Spatiotemporal biodistribution of Nano cy5.5 in vivo
The tissue distribution and tumor retention of triblock copolymeric nanomicelles were evaluated in CT26 tumor-bearing BALB/c mice. Nanomicelles were labeled with cy5.5-amine, injected intratumourally and subsequently imaged using an in vivo imaging system (IVIS) at different time intervals post-injection. I.t. injection of Nano cy5.5 yielded a strong and long-term persisting fluorescent signal in the tumor sites compared with that of free Cy5.5. This finding was further confirmed by the increased cy5.5 signal in isolated tumor tissues treated with Nano cy5.5 (Fig. 4a-c). To observe the cellular uptake in detail, cancer cells and hepatocytes were isolated separately. Notably, the uptake of Nano cy5.5 in tumor cells was higher (****p < 0.0001) than that of free cy5.5, while it was lower (**p < 0.01) in hepatocytes, according to flow cytometry analysis (Fig. 4d, e). We speculate that the nanomicelles will disintegrate at the tumor site to achieve better tumor penetration, and the disintegration of the drug with a positive charge increased the adsorption of the cells, which induced our nanomicelles to have a longer retention effect after i.t. injection than the free Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 drug. In summary, our nanomicelles have a longer retention effect in tumors, which was conducive to minimizing systemic toxicity.

In vivo antitumor efficacy
Encouraged by the positive antitumor activity and enhanced immune responses of nanomicelles in vitro, we assessed the therapeutic potential of nanomicelles in vivo. The antitumor efficacy of nanomicelles was evaluated using bilateral CT26 cell-bearing BALB/c mice, where the tumor growing on the right side of the back of BALB/c mice was the primary tumor and that on the left was the distal tumor. When the primary tumor volume reached 50-60 mm 3 , mice were randomly grouped (n = 6) and then treated with PBS, CPT, Nano PCPT , Nano PIMDQ , or Nano PCPT+PIMDQ intratumorally at equivalent doses of CPT (10 mg·kg −1 ) and IMDQ (0.5 mg·kg −1 ) according to the design schedule shown in Fig. 5a. The tumor volume was monitored using calipers, and the tumors from each treatment group were collected and weighed at the end of the survival study. Nano PCPT+PIMDQ generated the most substantial inhibition in both primary and distant tumors, as shown in Fig. 5b-g, which demonstrated that the combination of CPT-mediated chemotoxicity and Treg cells inhibition, together with TLR agonist-induced immune responses, might inhibit tumor growth synergistically. Images of primary excised tumors indicated that one out of six tumors were completely eradicated after treatment with the combined CPT and IMDQ therapies. In addition, no significant changes in body weight were observed, and the Kaplan-Meier curve displayed improved survival of the mice treated with Nano PCPT+PIMDQ .
To further evaluate the antitumor efficacy of Nano PCPT+PIMDQ in vivo, Ki67, H&E, and TUNEL stains were applied (Fig. 5h). The Ki67 staining images indicated less proliferation in the Nano PCPT+PIMDQ group. The H&E staining images demonstrated that the Nano PCPT+PIMDQ group had extensive cell necrosis. The TUNEL staining images showed the presence of multiple apoptotic cells in the Nano PCPT+PIMDQ group. In summary, compared with the control groups, Nano PCPT+PIMDQ exhibited outstanding therapeutic efficacy and prolonged survival in CT26bearing mice. There was no obvious tissue damage in the Nano PCPT+PIMDQ -treated group (Additional file 1: Fig.  S17).

In vivo exploration of the antitumor immune mechanism
TLR agonists can promote TLR recognition of patternassociated molecular patterns to initiate innate immunity [30,31]. Innate immune cells are then recruited to present the antigen, eventually priming and boosting adaptive immunity [32,33]. These complicated steps have emphasized the critical roles of macrophages, DCs and T cells [34]. Consequently, the regulation of immune cells by Nano PCPT+PIMDQ was further studied in vivo. After four administrations, the mice were sacrificed in each group, and the lymphocytes of spleens and TDLNs were harvested to evaluate the immune response. It has been reported that most of the T cells in tumors are derived from TDLNs, followed by their expansion in tumor tissue [35]. Thus, the elevated percentage of CD8 + T cells in TDLNs revealed that Nano PCPT+PIMDQ might increase T-cell activity at the tumor site. In addition, CD8 + T cells showed upregulated protein expression of granulomycin B (GrB) and tumor necrosis factor α (TNF-α) upon treatment with Nano PCPT+PIMDQ . Nano PCPT+PIMDQ also elicited a higher percentage of interferon-γ (IFNγ)-producing CD4 + T cells. These results indicated that Nano PCPT+PIMDQ enhanced antigen presentation and promoted T-cell effector function (Fig. 6a-f ). In mice receiving Nano PCPT+PIMDQ , the total percentage of macrophages (F4/80 + cells) was upregulated (Fig. 6g). In particular, although the amount of M2 macrophages (CD206 + ) did not change obviously, the proportion of M1 macrophages (iNOS + ) and the M1/M2 ratio were increased, indicating that macrophages had transformed from M2 to M1-like macrophages. In addition, MHC-I and MHC-II were expressed to a greater extent on the surface of DCs treated with Nano PCPT+PIMDQ than on the surface of DCs treated with control agents (Fig. 6h). Moreover, the number of Treg cells (CD4 + Foxp3 + T cells) decreased in the mice receiving the CPT-containing treatment, demonstrating the Treg inhabitation (Fig. 6i).
The spleen, the largest immune organ, plays a critical role in systemic immunity [36][37][38], and the lymphocytes isolated from it were used here to evaluate the immune response of Nano PCPT+PIMDQ . The results demonstrated that Nano PCPT+PIMDQ effectively boosted the beneficial immune function of the spleen. Increased CD8 + T and CD4 + T cell numbers, along with elevated GrB and IFN-γ respectively expression in CD8 + T and CD4 + T cells, suggested a cytolytic role (Fig. 7a-e). CD103 + DCs are a core subgroup in antigen-presenting cells to promote the production of tumor antigen-specific CD8 + T cells, and Fig. 7f showed that administration of Nano PCPT+PIMDQ generated more CD103 + DCs than other treatments. The costimulatory molecule CD86 and the antigen-presenting molecules MHC-I and MHC-II on the surface of DCs verified their activation in the spleen. The number of Treg cells (CD4 + Foxp3 + T cells) were also decreased in mice receiving CPT-containing treatment (Fig. 7g). Fortunately, we also observed the formation of effector memory T cells (Tem, CD44 + CD62L − T cells) gated on CD4 + T and CD8 + T cells in spleens upon the Nano PCPT+PIMDQ group (Fig. 7h). These results demonstrated that Nano PCPT+PIMDQ effectively primed the immune response after administration and subsequently promoted antitumor effects. To investigate the immunocomponent within tumors, we performed a large tumor model (> 200 mm 3 ) and pretreated with different formulations via intratumoral injection. Compared with other groups, Nano PCPT+PIMDQ treatment elevated the infiltration of CD8 + T cells within tumors. The expression or secretion of GrB, TNF-α, IFN-γ on CD8 + T cells were upregulated, demonstrating their activation (Fig. 8a,  b). Meanwhile, CD4 + T cells proportion were also elevated after treatment with Nano PCPT+PIMDQ and the expression or secretion of ICOS, IFN-γ and TNF-α were upregulated (Fig. 8c). Treg cells are an essential subset of immunosuppressive cells. Foxp3, as the master gene of Treg cells, is required for immunosuppressive function. The accumulation of Foxp3 + Treg cells is linked to disease progression and immunosuppression and may alter CD8 + T-cell activity [39]. Hence, cancer immunotherapy may be improved by specifically  3). b, c The frequency of CD8 + T cells and the expression of GrB in CD8 + T cells from spleens (n = 3). d, e The frequency of CD4 + T cells and the expression of IFN-γ in CD4 + T cells from spleens (n = 3). f Flow cytometry analysis of CD103 + CD11c + , CD86 + CD11c + , MHC-I + CD11c + , and MHC-II + CD11c + cells in spleens. g Tabulated flow cytometric data for CD4 + Foxp3 + expression in the spleen is shown. h Flow cytometry results showing the Tem ratios of CD4 + T cells or CD8 + T cells in spleens. *p < 0.05, **p < 0.01, ***p < 0.001 inhibiting Treg cells [40]. As shown in Fig. 8d, the number of Treg cells (CD4 + Foxp3 + T cells) decreased in tumors in mice receiving the CPT-containing treatment, which was in line with earlier research [9]. Subsequently, CD11c antibody was used to label DC to investigate their activation status. Nano PCPT+PIMDQ treatment increased the expression of CD40 and CD80 in comparison with other groups (Fig. 8e). Additionally, Nano PCPT+PIMDQ induced the re-polarization of TAMs based on the downregulation of M2 TAMs and upregulation of M1 TAMs (Fig. 8f ). These results indicated that Nano PCPT+PIMDQ hold promise for triggering robust antitumor immune response within tumors.

Conclusions
In summary, we developed a sequential pH/GSHresponsive triblock polymeric nanomicelle system that codelivers the chemotherapeutic drug CPT and the TLR7/8 agonist IMDQ, inducing amplified chemoimmunotherapy of colorectal cancer. First, Nano PCPT+PIMDQ responding to the tumor acidic microenvironment achieved charge reversal and dispersed into smaller micelles, consequently increasing intracellular uptake and improving the penetration of Nano PCPT+PIMDQ inside tumor tissue. Subsequently, GSH-triggered CPT was released from PCPT in tumor cells. In addition to the chemotherapeutic killing effect, CPT lowered the number of Treg cells (CD4 + Foxp3 + ) in TDLNs and spleens, allowing the infiltration of cytotoxic T cells into tumors. The exposed PIMDQ remodeled M2 macrophages into M1 macrophages and activated DCs to prime T cells, which further stimulated tumor-specific immune responses. In the CT26 colon tumor model, we showed that Nano PCPT+PIMDQ markedly inhibited the growth of primary and distal tumors. As a consequence, our strategy provides a promising nanoplatform for the codelivery of specific drugs to orchestrate chemoimmunotherapy.

Cell lines
A murine colorectal cell line (CT26) and macrophages were obtained from the School of Pharmacy, Shandong University. CT26 cells were cultured with RPMI-1640 complete growth medium supplemented with 10% fetal bovine serum and 2% penicillin/streptomycin and incubated at 37 °C with 5% CO 2 saturation. Macrophages were cultured with DMEM complete growth medium supplemented with 10% fetal bovine serum and incubated at 37 °C with 5% CO 2 saturation. Bone marrowderived DCs (BMDCs) were obtained from the bone marrow cells of five-week-old male C57BL/6 mice and cultured in 1640 containing recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4.

Animals
Healthy male BALB/c mice (3-5 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. The Medical Animal Test Center at Shandong University provided male C57BL/6 mice (5-7 weeks old).

Synthesis of N-ethyl-N-propyl-aminoethanol methacrylate (EPEMA)
EPEMA was synthesized according to the reported literature with some modifications [41]. After dissolving N-ethyl-N-propylethanolamine (3 g, 22.9 mmol) and triethylamine (2.317 g, 22.9 mmol) in acetonitrile (20 mL), the mixture was cooled to 0 ℃. Methacryl chloride (2.394 g, 22.9 mmol) was added dropwise. The mixture was stirred at 0 ℃ for 2 h and at room temperature for another 12 h. The mixture was filtered, and the obtained filtrate was extracted with DCM and evaporated under reduced pressure to give the monomer EPEMA (Additional file 1: Fig. S3). 1

Synthesis of acrylyl acetone oxime (AA)
AA was synthesized according to the reported literature [42]. First, acetone oxime (3.98 g, 54.4 mmol) was dissolved in Milli-Q water (32.5 mL) and cooled to 0 ℃ in an ice bath. Acryloyl chloride (5 g, 55.2 mmol) was added slowly into the mixture at 0 ℃. Then, the reaction mixture was stirred at room temperature for 1 h. Next, the layers were separated, and the aqueous phase was extracted with DCM (3 × 50 mL). After being concentrated under reduced pressure, the organic phase was washed with saturated NaHCO 3 aqueous solution (3 × 50 mL) and then water (50 mL). The organic phase was dried over Na 2 SO 4 , and the obtained filtrate was evaporated in vacuo to afford AA (Additional file 1: Fig. S4), which was used directly without purification. 1

Synthesis of PEG-DCT
The macro-CTA PEG-DCT was synthesized as previously reported [44]. DCT (162.68 mg, 0.403 mmol), DMAP (49.234 mg, 0.403 mmol), and DCC (83.15 mg, 0.403 mmol) were dissolved in dichloromethane (DCM) (30 mL). PEG (1.55 g, 0.31 mmol) dissolved in DCM (10 mL) was added to the above mixture. The mixture was stirred at room temperature for 36 h. The mixture was filtered, and then, the obtained filtrate was concentrated and purified by triple precipitation in cold ether.
The product was then dried under vacuum overnight to obtain a pale yellow powder (Additional file 1: Fig. S6).

Synthesis of diblock polymer PEG-PEPEMA
PEG-PEPEMA was synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization technique. Briefly, PEG-DCT (270.24 mg, 0.050 mmol), EPEMA (500 mg, 2.51 mmol), and AIBN (2.47 mg, 0.015 mmol) were dissolved in dioxane and placed into Schlenk tubes. After four freeze-pump-thaw cycles, the mixture was heated to 70 °C to initiate polymerization and stirred for 48 h. The solution was cooled and purified by dialysis against water. PEG-PEPEMA was obtained by lyophilization. 1 HNMR spectroscopy was used to determine the monomer conversion (Additional file 1: Fig. S7).

Synthesis of PEG-PEPEMA-PAA-PIMDQ (PIMDQ)
To connect IMDQ, we employed the amino group on IMDQ and the PAA on the polymer to conduct a substitution process. PEG-PEPEMA-PAA (20 mg, 0.00115 mmol), IMDQ (5.4 mg, 0.0125 mmol), and TEA (3.79 mg, 0.0374 mmol) were dissolved in dioxane and stirred at 50 ℃ for 48 h under N 2 protection. The solution was cooled and dialyzed against water overnight. A white powder was obtained by lyophilization. The IMDQ grafting rate was determined by UV-vis analysis (Additional file 1: Fig. S12).

Synthesis of PEG-PEPEMA-PAA Rho
To fluorescently label the polymer, we employed the amino group on Rho and the PAA on the polymer to conduct a substitution process. In short, PEG-PEPEMA-PAA (0.0034 mmol), Rho (0.67 μmol), and TEA (0.036 mmol) were dissolved in dioxane and stirred at 50 ℃ for 48 h under N 2 protection. The solution was cooled and dialyzed against water for 24 h to remove free Rho. A pink power was obtained by lyophilization.

Synthesis of PEG-PEPEMA-PAA cy5.5
To fluorescently label the polymer, we employed the amino group on Cy 5.5 and the PAA on the polymer to conduct a substitution process. In short, PEG-PEPEMA-PAA (0.003 mmol), NH2-Cy5.5 (0.7 μmol), and TEA (0.038 mmol) were dissolved in dioxane and stirred at 50 ℃ for 48 h under N 2 protection. The solution was cooled and dialyzed against water for 24 h to remove free Cy5.5. A green powder was obtained by lyophilization.

Characterization of Nano PCPT+PIMDQ
We diluted the micelle stock solution with various pH buffers to investigate the pH effect on micelles. After overnight incubation at 37 °C, dynamic light scattering (DLS) was used to determine the count rate and zeta potential, and TEM was used to examine the micelle morphology.

Self-assembly of PEG-PEPEMA-PCPT nanomicelles (Nano. PCPT )
Sonication was also used to prepare Nano PCPT . After dissolving PEG-PEPEMA-PCPT (60 mg) in DMSO (0.5 mL), the above solution was dropped into Milli-Q water (18 mL) under sonication for 30 min. To remove DMSO, the solution was dialyzed against deionized water overnight. Finally, the stock solution was diluted to 3 mg·mL −1 and stored in the dark at 4 °C for further testing.

Self-assembly of PEG-PEPEMA-PIMDQ nanomicelles (Nano. PIMDQ )
After dissolving PEG-PEPEMA-PIMDQ (5 mg) in DMSO (0.1 mL), the above solution was dropped into Milli-Q water (3 mL) under sonication for 30 min. To remove DMSO, the solution was dialyzed against deionized water overnight. Finally, the stock solution was diluted to 1 mg·mL −1 and stored in the dark at 4 °C for further testing.

Preparation of TAMRA-labeled nanomicelles (Nano. Rho )
Similarly, PEG-PEPEMA-PTAMRA was dissolved in DMSO and then added dropwise into Milli-Q water under continuous sonication. After sonication for 30 min, the solution was dialyzed against deionized water for 24 h to remove DMSO. Finally, the stock solution (10 mg·mL −1 ) was stored in the dark at 4 °C for further experiments.

Preparation of Cy5.5-labeled nanomicelles (Nano. cy5.5 )
The Cy5.5-labeled polymer was dissolved in DMSO and then added dropwise into Milli-Q water under constant sonication. After 30 min, the solution was dialyzed against deionized water for 24 h using a dialysis bag (3500 Da MWCO) to remove DMSO and obtain Nano cy5.5 . All the above experiments were carried out under dark conditions.

Drug grafting rate
According to Lambert-Beer law, under the same experimental conditions, the absorbance (A) is proportional to the concentration (C). The certain concentrations of CPT and IMDQ were used as the controls (C control ) and absorbance were determined by UV-visible (UV-vis) spectrophotometry at 365 nm and 326 nm, respectively (A sample ). The drug-grafting content (DGC) were calculated as follows formula: The DGC results of CPT and IMDQ were 34.09 ± 0.46%, 13.62 ± 0.19% respectively (Additional file 1: Figs. S11-S12).

In vitro drug release of CPT triggered by the reducing agent dithiothreitol (DTT)
In a shaking incubator at 37 °C, a Nano PCPT dispersion in pH 7.4 phosphate-buffered saline (PBS) was placed into a dialysis bag (3500 Da MWCO), which was then dialyzed against the buffered medium in the absence or presence of DTT (10 mM). An aliquot of the external medium was withdrawn and replaced with an equal volume of new buffer solution at various periods. HPLC was used to measure CPT concentrations in the media at different time intervals using a 365 nm absorption wavelength.

Cultivation of tumor spheroids
As reported in the literature, the hanging drop technique was used to prepare tumor spheroids [45]. CT26 cells were resuspended at 400,000·mL −1 in 0.25% methylcellulose, and 20 μL of suspension was transferred dropwise to the lid of the 48-well plate, with PBS (200 μL·well −1 ) supplied to each well to maintain the humidity of the hanging drop. After incubating at 37 °C with 5% CO 2 for 4 days, the tumor spheroids were transferred to a 48-well plate containing fresh culture medium for further study.

In vitro evaluation of tumor penetration of nanomicelles
The obtained tumor spheroids were transferred to 48-well plates with fresh media (pH 7.4 or 6.5) containing Nano cy5.5 (Cy5.5 = 2 μg·mL −1 ). After incubation for 6 h, the tumor spheroids were washed twice with ice-cold PBS and fixed with 1% paraformaldehyde. Subsequently, z-stack images were obtained with a confocal laser scanning microscope.

In vitro effect of pH on intracellular trafficking of nanomicelles
CT26 cells were plated in a 48-well plate with 100,000 cells per well and incubated for 12 h at 37 °C with 5% CO 2 . The culture media were removed, and the cells were treated with fresh media (pH 7.4 or 6.5) containing Nano cy5.5 . After 12 h, the supernatant was removed, the cells were separated, washed with cold PBS, and resuspended in PBS, and the sample was measured using flow cytometry.

In vitro Cytotoxicity Assay by CCK-8
CT26 cells were plated at 5000 cells per well in a 96-well plate and incubated for 12 h. Afterward, the supernatant was removed by pipette aspiration and replaced with a series of media containing free CPT, Nano PCPT , and Nano blank polymer at a series of concentrations, followed by incubation for another 24 h. Subsequently, the drugcontaining medium was removed by pipette aspiration, replaced with fresh medium, and then incubated for 48 h. Next, CCK-8 (10 μL) was added to each well and incubated for 40 min, and the absorption was measured at 465 nm. The other operations were the same as those for CT26 cells, with the exception of incubating macrophages with 3 μM CPT and Nano PCPT containing the same quantity of CPT.

In vitro BMDC uptake
Murine BMDCs were obtained and cultured from C57BL/6 mice. BMDCs were cultured with medium containing Nano Rho for 2, 6, 12, 24 h. Afterward, the cells were collected, washed with cold PBS twice, and resuspended in PBS, and the sample was measured using flow cytometry.

In vitro BMDC activation
To explore the effect of polymer-modified IMDQ on the activation of BMDCs, we incubated BMDCs with different concentrations of free IMDQ and Nano PIMDQ in a 48-well plate for 48 h. Cells were then collected and stained with anti-CD11c, anti-CD86, anti-CD80, anti-MHC-II-BV786, and anti-CD40 to determine the specific DC maturation marker expression by flow cytometry (FACS Celesta) and analyzed using FlowJo software.

In vitro macrophage repolarization
RAW264.7 macrophage cells were seeded in a 12-well plate at a density of 500,000 cells/well overnight. After adding IL-4 for 24 h to simulate polarization into M2 macrophages, various concentrations of IMDQ and Nano PIMDQ were added for incubation for another 24 h. Cells were then collected and stained with anti-F4/80, anti-CD200R, and anti-iNOS to determine the expression of specific macrophage repolarization markers by flow cytometry (FACS Celesta) and analyzed using FlowJo software.

IVIS imaging and biodistribution in vivo
CT26-bearing mice were obtained by inoculating 800,000 CT26 cells on the backs of male BALB/c mice. When tumor sizes grew to 100-200 mm 3 , mice were used for biodistribution studies. After Nano cy5.5 intratumoral injection, real-time fluorescence images of the anesthetized mice were captured by an in vivo imaging system at different time points (1,24,48,96, and 120 h) to investigate the time-dependent retention of Nano cy5.5 in tumors. The mice were sacrificed, and their major organs were excised for in vitro imaging. The excised livers and tumors were used to extract liver and tumor cells for flow cytometry quantification, respectively.

Subcutaneous Tumor Model Evaluation
CT26 cells (750,000) in PBS were injected subcutaneously into the right back of BALB/c mice (5-7 weeks old), followed three days later by subcutaneous injection of the same density of CT26 cells in the left back. When the proximal tumors grew to 50-60 mm 3 , the mice bearing CT26 tumors were intratumorally injected with five different groups of medicine: 1. PBS; 2. CPT; 3. Nano PCPT ; 4. Nano PIMDQ ; and 5. Nano PCPT+PIMDQ . CPT was administered at a dose of 10 mg·kg −1 , while IMDQ was given at a dose of 0.5 mg·kg −1 body weight. The mice received a total of four treatments, and they were given drugs every three days. The body weight and tumor volumes were recorded regularly, and the tumor volumes (V) were calculated from the following equation: V = LW 2 /2 (L = length of tumor longest axis; W = length of the axis perpendicular to the longest axis of the tumor). The survival of the mice was closely monitored throughout the experiment.

In vivo antitumor immune response post-administration
To assess whether our mixed nanomicelles could elicit an improved immune response, mice from each group were dissected after four intratumoral injections. Subsequently, tumor-draining lymph nodes and spleen were removed from the mice to prepare a single immune cell suspension. Anti-CD3, anti-CD4, anti-CD8, anti-GrB, anti-TNF-α, and anti-IFN-γ were labeled to measure cytotoxic T-cell activation in TDLNs, and anti-CD4, anti-CD8, anti-CD3, and anti-GrB, anti-IFN-γ were labeled to measure cytotoxic T-cell activation in the spleen. Anti-CD11c, anti-MHC-I, and anti-MHC-II were labeled to assess DC cell maturation in TDLNs, and anti-CD86, anti-CD103, anti-MHC-I, and anti-MHC-II were labeled to assess DC cell maturation in spleens. Anti-F4/80, anti-CD206, and anti-iNOS were labeled to evaluate macrophage repolarization from M2 to M1 in TDLNs.
Finally, the cells were analyzed by flow cytometry (FACS Celesta) and FlowJo software.

Antitumor immune response in vivo at the end of treatment
We prepared single-cell suspensions from the tumors, tumor-draining lymph nodes and spleen for immunological analysis and evaluation. Single-cell suspensions of tumors, lymph nodes and spleens were stained with anti-CD4, anti-Foxp3 for the analysis of Treg cells. A singlecell suspension of spleens was stained with anti-CD44 and anti-CD62L to analyze immune memory and the effector memory T cells were based on the CD44 + CD62L − phenotype.

Anti-tumor immune response within tumors
To investigate the immune response within tumors, we performed a large tumor model (> 200 mm 3 ) and pretreated with different formulations via intratumoral injection. After treatment, single-cell suspensions collected from tumors were labeled with anti-CD3e, anti-CD4, anti-CD8 to evaluate T cell infiltration; anti-GrB, anti-TNF-α, anti-IFN-γ, and anti-ICOS to evaluate the activation of T cells; anti-CD4 and anti-Foxp3 to evaluate Treg cells; anti-CD11c, anti-CD40, and anti-CD80 to evaluate DCs maturation; anti-F4/80, anti-CD206, and anti-iNOS to evaluate macrophage repolarization. These cells were performed via flow cytometry (FACS Celesta) and analyzed via FlowJo software.

Immunohistochemical analysis
Main organs and tumors were taken and fixed with 4% formaldehyde before being stained with hematoxylin and eosin (H&E) for an in vivo safety investigation, and tumors were further stained with Ki67 and TUNEL for further research.