Tumor microenvironment-responsive spherical nucleic acid nanoparticles for enhanced chemo-immunotherapy

Characterization of MPLA-CpG-sMMP9-DOX

To ensure the accurate loading of chemotherapeutic agents and adjuvants and fabricate the nanoparticles by self-assembly, we synthesized an amphiphilic molecule of MPLA-CpG-sMMP9-DOX via three steps reaction (Fig. 1A). First, DOX was covalently bound to sMMP9 through the reaction of an N-hydroxysuccinimide (NHS) ester and amines. Then, the resulting sMMP9-DOX was activated with SPDP and linked with sulfhydryl-modified CpG ODN to yield CpG-sMMP9-DOX conjugate through a disulfide exchange reaction. Lastly, MPLA was conjugated with CpG-sMMP9-DOX through an amidation reaction to yield the MPLA-CpG-sMMP9-DOX [20]. To confirm the successful synthesis, the purified conjugates of each step were characterized with Fourier‐transform infrared spectroscopy (FTIR). As shown in Fig. 1B-a and b, compared to sMMP9, newly appeared stretching vibration peaks of the benzene ring skeleton (1617.7 cm−1, 1576.9 cm−1, and 1282.7 cm−1) in sMMP9-DOX proved that DOX was successfully conjugated with sMMP9. In Fig. 1B-b, 1092.5 cm−1 and 1062.4 cm−1 were C–O stretching vibration peaks of deoxyribose in CpG, and the absorption peak at 519.8 cm−1 indicated that CpG and sMMP9 are successfully connected via disulfide bond generating CpG-sMMP9-DOX conjugate. In Fig. 1B-d, the methylene characteristic peaks (2923.1 cm−1 and 2852.6 cm−1) belonging to MPLA indicated that the amphiphilic molecule MPLA-CpG-sMMP9-DOX (MCMD) was successfully synthesized. The results of Gel retardation of CpG-sMMP9-DOX and MPLA-CpG-sMMP9-DOX further confirmed the successful synthesis of MPLA-CpG-sMMP9-DOX (Fig. 1C and D).

Fig. 1figure 1

Characterization of MCMD NPs. A The detailed synthesis route of MCMD NPs. B The FITR of MCMD (a: sMMP9, b: sMMP9-DOX, c: CpG- sMMP9-DOX, d: MPLA-CpG-sMMP9-DOX). C The agarose gel electrophoresis of free CpG and CpG-sMMP9-DOX. D The agarose gel electrophoresis of CpG-sMMP9-DOX and MPLA-CpG-sMMP9-DOX. E The typical TEM image of MCMD NPs. F The size distribution of MCMD NPs in PBS. G The size distribution of MCMD NPs against the MMP9 enzyme or not. H The DOX release profile from MCMD NPs with MMP9 enzyme or not. Data were expressed in the form of mean ± SD (n = 3)

MCMD NPs preparation and characterization

Amphiphilic MCMD has dimensionless packing parameter P according to Israelachvili's surfactant theory. The “P” value of amphiphilic molecules is related to the morphology of these molecules self-assembled, and when P < 1/3, spherical morphologies are produced. In this study, the “P” value of MCMD was 0.325, which suggests that these molecules were inclined to self-assemble into a spherical structure [21]. TEM results showed that MCMD assemblies in water did display spherical morphology with uniform distribution and good dispersibility (Fig. 1E). The size of MCMD nanoparticles (MCMD NPs) in aqueous solution was measured by DLS with a diameter of 159.3 ± 0.8 nm and the polydispersity Index of 0.26 ± 0.02 (Additional file 1: Fig. S1). Zeta potential measurements indicate that the MCMD NPs have a negative charge (− 36.5 ± 0.2 mV) in pure water (Additional file 1: Table S1). To investigate the stability of MCMD NPs, they were kept in PBS for 25 days at 4 ℃. The size change of MCMD NPs was monitored using the DLS. It is found that no momentous change in the diameter of MCMD NPs is observed over 15 days whereas the apparent change occurred after 15 days (Fig. 1F), demonstrating relatively good stability within 15 days.

To assess whether MCMD NPs could release DOX, MCMD NPs were co-incubated with the MMP9 enzyme (0.2 µg/mL) [22]. Then the size of nanoparticles was measured with DLS, and the release amount of DOX in the dialysate was detected by UV–Vis at preset timepoints. DLS data displayed that the nanoparticles aggregated, and their size increased after co-incubating with the MMP9 enzyme for 4 days (Fig. 1G). The release results showed that the cumulative release amount of DOX in the MMP9 enzyme-added group was up to 76.5 ± 2.8% in the first 4 days, which was much faster than that of the enzyme-free group (47.0 ± 5.7%) (Fig. 1H), indicating that sMMP9 was cleaved by MMP9 enzyme which led to the release of DOX from MCMD NPs. These results suggested that MCMD NPs response to the MMP9 enzyme could accelerate the DOX release from the nanoparticles.

In vitro experimentsCell viability

Free DOX was usually administrated by intravenous injection, which results in a high degree of cardiovascular toxicity [23]. To evaluate whether MCMD NPs could decrease the toxic side effects of DOX on normal tissues, human umbilical vein endothelial cells (HUVECs) were incubated with various concentrations of MCMD NPs for 24 h. The cell viability of HUVECs was assessed by CCK-8 assay. HUVECs treated with MCMD NPs showed low cell viability even the DOX concentration reached up to 1.25 µg/mL (Fig. 2A). In contrast, high cytotoxicity was exhibited when the cells were treated with free DOX even at a low concentration of 0.04 µg/mL. Additionally, when MCMD NPs were pretreated with the MMP9 enzyme, it could cause a significant viability decrease of mouse T lymphoma cells (E.G7-OVA) (Fig. 2B).

Fig. 2figure 2

The effect of MCMD NPs on cell viability, DC maturation, and activation. The cell viability of A HUVECs and B E.G7-OVA treated with free DOX or MCMD NPs. C Quantitative analysis of average fluorescence intensity of CpG in DCs. D CLSM images of DCs after co-incubation with free CpG or MC NPs. Red: lysosome. Green: Cy5-labeled CpG. Blue: nucleus. The cytokine secretion levels of E IFN-α, F IFN-β, and G TNF-α were measured with ELISA assays. The expression levels of H MHC-I, I CD86, and J CD40 were measured with flow cytometers. Data were expressed in the form of mean ± SD (n = 5). * P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001

Cellular uptake and DCs activation

We next investigated whether dual-adjuvant MC NPs as the core of MCMD NPs could drive the internalization of adjuvants into dendritic cells (DCs) and stimulate DCs maturation and activation which could initiate a cascade of anti-tumor immune responses. After Cy5-labeled MCMD NPs were pretreated with MMP9 enzyme and ultracentrifuged to remove the supernatant, the remaining nanoparticles were incubated with DCs for 6 h. DCs were imaged with a confocal laser scanning microscope (CLSM) and the fluorescence intensity in cells was quantified using ImageJ software. The results showed that the fluorescent signal in MC NPs group was 2.6-fold stronger than the free CpG group (Fig. 2C and D). Additionally, MC NPs could increase the expressions of MHC-II and CD80 molecules on the surface of BMDCs which could present antigens to T cells (Additional file 1: Figs. S2 and S3). Meanwhile, the results of enzyme-linked immunosorbent assay (ELISA) demonstrated that MC NPs could significantly stimulate DCs secretion of TNF-α and Type I interferon (including IFN-α and IFN-β) compared with free adjuvants and PBS (Fig. 2E–G). TNF-α can induce immature DC maturation and differentiation. IFN-α and IFN-β can activate T, B, and natural killer cells (NK cells) against tumor cells [24].

To mimic the TME and further study whether MCMD NPs could efficiently promote BMDCs to present tumor antigens in tumor tissue, BMDCs were incubated with MC NPs and tumor cell fragments obtained from treated E.G7 tumor cells with DOX, which was named MC + group, and then evaluated the expression level of MHC-I/II and co-stimulated molecules. The results showed that not only the expression level of CD40 but also MHC-I and CD86 on DCs were up-regulated (Fig. 2 H-J), indicating that MCMD NPs could promote tumor antigens presented via the MHC-II pathway to activate CD4+ T cells as well as cross-presented via the MHC-I pathway to activate CD8+ T cells [25].

In vivo experimentsBiodistribution

Nanoparticles are expected to prolong the blood circulation of drugs and enhance their tumor-targeting capability [26, 27]. To study the biodistribution of MCMD NPs, Cy7 labeled MCMD NPs (Cy7-MCMD NPs) were injected into C57BL/6 mice bearing subcutaneous E.G7 OVA xenografts through the tail vein. The Cy7 fluorescence in the mouse model was detected by in vivo imaging system (CRI Maestro EX, USA). Free Cy7 rapidly accumulated at the tumor site in the first 3 h but rapidly disappeared after 24 h, whereas Cy7-MCMD NPs gradually accumulated in tumors and showed a strong fluorescence signal even after 48 h (Fig. 3A). These results indicated that MCMD NPs could increase the accumulation of DOX and adjuvants in tumors.

Fig. 3figure 3

Biodistribution and therapeutic effect of MCMD NPs. A The images of free Cy7 and Cy7-MCMD NPs accumulated at the tumor site at selected time-points. B The fluorescence images of tumors and organs from mice at 48 h after injection. C Average fluorescence intensity of organs and tumors in each group. D Tumor images. E Average tumor weight of each group. F The curves of tumor growth of tumor-bearing mice. G The survival curves of tumor-bearing mice. Data were expressed in the form of mean ± SD (n = 5). *P < 0.05, **P < 0.01, ****P < 0.0001

To further observe the biodistribution and clearance of the drug, main organs, and tumors were collected after 48 h post administration and imaged by CRI. Compared to free Cy7, Cy7-MCMD NPs remarkably decreased the amount of Cy7 fluorescence in the kidney (Fig. 3B and C), indicating that these nanoparticles could hinder renal excretion. This can be attributed that renal excretion of molecules or nanoparticles was size-dependent and the nanoparticles larger than 30 nm could diminish the clearance through kidneys [28]. Of note, despite the fact that MCMD NPs decreased the renal accumuation to a greater extents compared to free DOX, the possibility of sustained and slow release of DOX might lead to potential renal toxicity [29, 30]. To prevent the DOX-induced renal toxicity, some protective adjuvants like atorvastatin and/or theanine may be adopted as a component against DOX toxicity, via antioxidant, anti-nitrosative, anti-inflammatory and anti-apoptotic mechanisms [31, 32]. Additionally, MCMD NPs significantly enhanced drug accumulation in tumors to 3.2-fold higher compared to free Cy7, likely due to the enhanced permeability and retention (EPR) effect. On the other hand, Cy7-MCMD NPs significantly decreased drug accumulation in the heart compared with free Cy7, suggesting that the nanoparticles might attenuate DOX-induced cardiotoxicity which is the most noteworthy side effect of DOX [33]. However, many MCMD nanoparticles were still located in the lung, showing no significant selectivity between tumors and the lung. To further achieve a better targeting capability, the nanoparticles should be modified with tumor-specific antibodies or aptamers with high binding affinity [34, 35]. Collectively, these results showed that MCMD NPs could promote drug accumulation at the tumor site and decrease heart accumulation as well.

Therapeutic effect of MCMD NPs

To further assess the in vivo therapeutic effect of MCMD NPs, we established an E.G7-OVA tumor model using C57BL/6 mice. The tumor size was measured regularly to draw a tumor growth curve after mice were treated with various formulations. When the tumor volume exceeded 2000 mm3, the mice were regarded as dead. The results showed that free DOX and MC NPs inhibited tumor growth to some extent compared with PBS, while MCMD NPs exhibited the most powerful capability of retarding tumor growth, even though some tumors almost completely stop growing. The average tumor weight of the mice treated with MCMD NPs was 0.28 ± 0.19 g, which was much smaller than that of the PBS group (5.70 ± 0.95 g), the free DOX group (and 2.00 ± 0.94 g) and the MC NPs group (1.79 ± 0.62 g) (Fig. 3D–F). However, the body weight of the mice treated with MCMD NPs was not affected during the treatment process, demonstrating the biological safety of MCMD NPs (Additional file 1: Fig. S4).

Meanwhile, the results of the survival period showed a similar trend to the tumor volume curve. The mice treated with PBS gradually died from 16 to 25 days, while free DOX prolonged the survival time slightly, and the MC NPs group prolonged the survival period moderately. In contrast, MCMD NPs significantly prolonged the survival time of tumor-bearing mice, with 33% of the mice surviving more than 45 days (Fig. 3G).

The hematoxylin–eosin (H&E) staining results demonstrated that, compared with other groups, MCMD NPs induced a larger area of typical apoptosis in tumor tissues, such as a reduction in cell size, separation from surrounding cells, nucleus concentration, and nuclear karyoplasm [36]. Moreover, the pathological analysis of heart tissue also showed that MCMD NPs effectively reduced cardiotoxicity compared to free DOX (Additional file 1: Fig. S5). These results were consistent with the previous study which demonstrated that nanoparticle-based chemotherapy was superior to conventional DOX chemotherapy in terms of reduction in the risk of cardiotoxicity [37, 38].

The above in vivo results showed that MCMD NPs had a good capability to enhance drug accumulation in tumors, induce tumor apoptosis, and then inhibit tumor growth, and significantly prolonged the survival period with reduced side toxicity.

Antitumor immune responses in vivo

To further explore whether MCMD NPs could efficiently enhance the local anti-tumor immune response in tumor tissue and stimulate systemic immunity. The immune levels in tumors, lymph nodes, and spleens collected from mice treated with various formulations were evaluated. The results revealed that not only the proportion of CD4+ T cells in tumor tissues treated with MCMD NPs was about ninefold, fivefold, and twofold compared to PBS, free DOX, and MC NPs, respectively, but also the proportion of CD8+ T cells was eightfold, 2.5-fold and 1.4-fold compared with the three control groups, respectively (Fig. 4A–D). The results suggested that MCMD could significantly amplify the DOX-induced tumor-specific immune responses. T cell expansion was important in overcoming the suppressive TME and yielding long-term improved therapeutic outcomes [39]. Besides within the tumor tissue, MCMD NPs also significantly increased the proportion of T cells in the adjacent lymph nodes. The proportion of CD4+ and CD8+ T cells in lymph nodes increased over fourfold in MCMD NPs group compared with the PBS group. The percentage of CD4+ T cells increased from 10.9% to 46.8%, and CD8+ T cells increased from 8.8 to 37.1% (Additional file 1: Fig. S6). Moreover, MCMD NPs increased the percentage of CD4+ T cells even in the spleens from 5.3 to 19.3%, and CD8+ T cells from 3.9 to 12.8% which both were close to fourfold compared to the PBS group (Fig. 4E–H). Therefore, the nanoparticles could induce synergistic anti-tumor immune responses in terms of T cell proliferation. These results indicated that MCMD NPs could not only enhance the anti-tumor immune responses at tumor tissue leading to the local tumor destruction but also could stimulate the systemic immune responses leading to the distant lesion of metastatic tumors.

Fig. 4figure 4

MCMD NPs significantly enhanced the antitumor immune responses in vivo. Representative FACS plots and histogram of percentage of CD3+ CD4+ and CD3+ CD8+ T cells in AD tumors (A, B: CD3+CD4+; C, D: CD3+CD8+) and EH spleens (E, F: CD3+CD4+; G, H: CD3+CD8+). Cytokines I IL-1β, J IL-12, K IFN-α, and L IFN-γ were evaluated with ELISA kits. Data were expressed in the form of mean ± SD (n = 5). * P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001

Cytokines are derived from activated immune cells and are involved in the whole process of anti-tumor immune responses, including antigen presentation, immune cell interaction and activation. Cytokines were related to the type and intensity of immune responses. DOX-triggered ICD could activate the pyrin domain containing-3 protein (NLRP3)-dependent (inflammasome) pathway releasing some cytokines, such as IL-1β [40, 41]. Our previous study demonstrated that MC NPs could synergistically activate TLR4 and TLR9 signaling pathways and generate robust Th1-biased immune responses, releasing IL-12 and interferons [42]. So, splenic lymphocytes collected from mice treated with various formulations were co-cultured with DOX-treated E.G7 cell debris for 72 h and the supernatants were collected to detect cytokines of IL-1β, IL-12, IFN-α and IFN-γ. As shown in Fig. 4I–J, the level of IL-1β was enhanced by about 6.0-, 2.9-, and 3.4-fold compared with PBS, free DOX, and MC NPs. IL-12 was enhanced about 8.9-, 3.4-, and 3.3-fold compared with PBS, free DOX, and MC NPs. The level of IFN-α secreted by splenic lymphocytes treated with MCMD NPs was 19.6-, 6.3-, and 2.9-fold higher than those of PBS, free DOX, and MC NPs (Fig. 4K). IFN-α can not only regulate the innate immune responses by promoting the function of antigen-presenting cells (APC) and NK cells but also activate the adaptive immune system by promoting antigen-specific T and B cell immune responses and immunological memory [43]. In addition, the level of IFN-γ treated with MCMD NPs was also significantly increased compared with other groups (Fig. 4L). IFN-γ belongs to type II interferons, which can induce Th1 cell differentiation and promote CD8+ cytotoxic T cell response (CTLs). Collectively, these results indicated that MCMD NPs could efficiently induce improved antitumor immune responses.

T cell immune memory

To evaluate whether MCMD NPs could effectively induce the differentiation of memory cells and provide a long-term protective immune response, the spleen lymphocytes from mice treated with various formulations were collected and re-stimulated with E.G7 cell debris in vitro. The proliferation of memory cells including central memory T cells (Tcm) and effector memory T cells (Tem) was detected [44]. The results showed that the percentage of CD4+ Tcm and CD8+ Tcm in the MCMD NPs group was enhanced about 1.7- and 2.5-fold than the PBS group, respectively (Fig. 5A–D), indicating that MCMD NPs could induce an antigen-specific immune response upon encountering the same tumor-related antigens for a long duration, which meant that the nanoparticles could prevent the tumor recurrence to a certain extent. Additionally, after being re-stimulated with E.G7 cell debris, the expansion of T cells was significantly expanded, the percentages of CD4+Ki67+ T cells and CD8+Ki67+ T cells in the MCMD NPs group reached 28.7% and 49.2% respectively, which were significantly higher than those in PBS group (CD4+Ki67+ T cells were 6.9%, CD8+Ki67+ T cells were 23.9%) (Fig. 5E–H). The results further confirmed that MCMD NPs could generate memory cells that could rapidly react upon encountering the same antigen again [45]. All the results indicated that MCMD NPs could effectively produce immune memory which plays a role in preventing cancer recurrence and metastasis.

Fig. 5figure 5

Generation of T cells immune memory. Representative FACS plots and histogram of the percentage of A, B CD4+ and C, D CD8+ T memory cells. The proliferation of E, F CD4+ and G, H CD8+ T cells from mice in different groups after being re-stimulated with tumor debris in vitro. (n = 5). * P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001

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