DNA‑Based Nanohydrogel with Potent Anti‑Tumor Immunostimulatory Activity

Abstract

Unmethylated CpG oligodeoxynucleotides (CpG‑ODN) are powerful activators of innate and adaptive immunity, yet their rapid degradation in serum limits clinical utility. We engineered CpG‑MCA nanohydrogels by multi‑primed chain amplification (MCA), generating hydrogels rich in tandem CpG repeats. The resulting CpG‑MCA gels resist nuclease degradation, are readily internalized by RAW264.7 macrophages, and trigger robust secretion of tumor necrosis factor‑α (TNF‑α) and interleukin‑6 (IL‑6). In co‑culture with human glioma U251 cells, CpG‑MCA gels markedly suppressed tumor cell proliferation, outperforming conventional CpG‑ODN and CpG‑RCA gels. These findings demonstrate that CpG‑MCA nanohydrogels are a potent, biocompatible immunostimulant with strong anti‑tumor activity.

Introduction

Bacterial DNA containing unmethylated CpG motifs serves as an effective adjuvant and anticancer agent by engaging Toll‑like receptor 9 (TLR9) on dendritic cells, macrophages, and other immune cells. However, CpG‑ODN is prone to protein adsorption and nuclease digestion, which compromises its stability and potency in vivo. Encapsulation in delivery vehicles or self‑assembly into higher‑order DNA nanostructures has improved stability and cellular uptake, yet challenges such as cytotoxicity and limited loading remain.

DNA nanomaterials—tetrahedra, X‑shaped lattices, and hydrogel networks—have shown enhanced cellular internalization and cytokine induction compared with linear oligonucleotides. Rolling circle amplification (RCA) yields nanoflower hydrogels that protect CpG motifs and boost immune signaling. Building on this concept, we employed multi‑primed chain amplification (MCA) to produce CpG‑MCA hydrogels, which contain thousands of CpG repeats and form nanoflower structures. We hypothesize that these hydrogels will provide superior stability, uptake, and immunostimulation, thereby inhibiting solid‑tumor growth.

Materials and Methods

Materials

All oligonucleotides were synthesized (Sangon Biotech, Shanghai) and purified by HPLC. Reagents for MCA and RCA, including phi29 DNA polymerase, T4 DNA ligase, dNTPs, and buffers, were obtained from Thermo Fisher and New England Biolabs. Cell lines RAW264.7 (mouse macrophage) and U251 (human glioma) were sourced from the Chinese Academy of Sciences. ELISA kits (R&D Systems) and CCK‑8 (Dojindo) were used for cytokine quantification and viability assays.

Preparation of Circular DNA Templates

Phosphorylated single‑stranded DNA (SS‑DNA) containing CpG primers was annealed at 95 °C for 5 min and cooled to 4 °C. Ligase was added overnight at 4 °C to form circular templates; enzymes were inactivated at 75 °C for 10 min.

Construction of CpG‑MCA and CpG‑RCA Hydrogels

A 10 µL aliquot of the circular template was mixed with 1× phi29 buffer, 4 mM dNTPs, and 5 U phi29 polymerase to a 50 µL volume. Incubation at 30 °C for 12 h produced the RCA gel (R12). For MCA, after 4 h of RCA, 500 pM primers 2 and 3 were added and the mixture was incubated for the remaining time (R4M4 and R4M8). Polymers were heat‑inactivated at 65 °C and purified by ultrafiltration.

Characterization

Agarose gel electrophoresis confirmed hydrogel formation. TEM and SEM revealed nanoflower morphologies ranging from 100 nm to several micrometres. Cytotoxicity was assessed by CCK‑8; uptake was visualized with Cy5‑labeling and confocal microscopy; cytokine secretion was measured by ELISA; gene expression was quantified by qRT‑PCR.

Stability Assay

Hydrogels were incubated in 10 % FBS‑DMEM or PBS at 37 °C; residual DNA was quantified by NanoDrop, and degradation patterns were monitored by agarose gel electrophoresis.

Co‑culture with U251 Cells

RAW264.7 cells were seeded in the upper chamber of a Transwell; U251 cells were placed in the lower chamber. After 24 h of treatment with CpG‑ODN, CpG‑RCA, or CpG‑MCA gels, U251 proliferation was evaluated by a clonogenic assay.

Results and Discussion

Formation of circular templates and successful amplification was confirmed by agarose gel: the MCA product (R4M4/R4M8) and RCA product (R12) displayed high molecular weight retention and nanoflower appearance on TEM/SEM.

Confocal imaging demonstrated that CpG‑MCA gels were efficiently internalized by RAW264.7 cells, with a mean fluorescence intensity >10× that of linear CpG‑ODN (p < 0.0001), indicating superior uptake due to the nanoflower architecture.

Stability studies showed that CpG‑MCA gels retained ~85 % of their DNA content after 24 h in 10 % FBS‑DMEM, whereas linear CpG‑ODN degraded >90 % within 12 h, underscoring the protective effect of the hydrogel matrix.

In vitro immunostimulation assays revealed that CpG‑MCA gels induced TNF‑α and IL‑6 secretion at levels 3–27× higher than CpG‑ODN (p < 0.0001). Notably, R4M8 produced the strongest cytokine response, correlating with its higher CpG copy number.

Gene expression analysis showed significant up‑regulation of TLR9, TNF‑α, and IL‑6 transcripts in CpG‑MCA‑treated cells, along with increased CD86 and decreased CD206 surface markers, indicating a shift toward the pro‑inflammatory M1 phenotype.

Migration assays (Transwell and scratch wound) revealed that CpG‑MCA gels stimulated RAW264.7 migration more than CpG‑ODN, enhancing the potential for immune cell infiltration into tumors.

Co‑culture with U251 glioma cells demonstrated a pronounced reduction in clone‑formation: R4M8 achieved a 12 % clone‑formation rate relative to the CpG‑ODN control (p < 0.001), confirming potent anti‑tumor activity mediated by CpG‑MCA‑induced cytokine release.

Conclusions

We have successfully engineered CpG‑MCA nanohydrogels that incorporate thousands of CpG motifs, conferring nuclease resistance, efficient macrophage uptake, and robust Th1‑type cytokine induction. These hydrogels effectively suppress glioma cell proliferation in vitro, highlighting their promise as a biocompatible immunostimulant for cancer therapy.

Availability of Data and Materials

Datasets supporting this study are available from the corresponding author upon reasonable request.

Abbreviations

CpG‑MCA gels

DNA hydrogel generated by multi‑primed chain amplification

CpG‑RCA gel

DNA hydrogel generated by rolling circle amplification

MCA

Multi‑primed chain amplification

R12

RCA‑derived CpG gel

R4M4, R4M8

CpG‑MCA hydrogels with 4 or 8 primer extensions

R12‑C, R4M4‑C, R4M8‑C

Hydrogels lacking CpG motifs