Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice

Abstract

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.

Structure-dependent immunostimulatory effect of CpG oligodeoxynucleotides and their delivery system

“…One of the causes of this high activity is believed to be DL-DNA's resistance to DNase. Recently, Nishikawa et al 54 observed that TNF-α release from RAW264.7 cells at 8 hours after stimulation by CpG motifs contained X-shaped DNA consisting entirely of phosphodiester backbone. This suggests that X-shaped DNA was stable for 8 hours at least, although Y-DNA has no resistance to DNase.…”

“…What is extremely interesting is that Y-DNA has immunostimulatory activity even if it does not contain CpG motifs. 54 This suggests the importance of the higher-order structure of ODNs on immunostimulation.…”

Increased immunostimulatory activity of polypod-like structured DNA by ligation of the terminal loop structures

“…Together, these results indicate that the immunostimulatory activity of tetrapodna is not affected by the presence or absence of terminal loop structures, but is dependent on the activity of the components. In addition, the comparison of the TNF-α release by tetrapodna preparations and their components clearly demonstrates that the tetrapod-like structure markedly increases the immunostimulatory activity of CpG DNA as observed in our previous study [17]. Fig.…”

“…Each tetrapodna was prepared by mixing equimolar amounts of a set of four ODNs as previously reported [17]. Briefly, ODNs dissolved in TE buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8) containing 1.5 M sodium chloride were diluted with sterile Milli-Q water to give a final concentration of 0.1 mM for each ODN and 150 mM for sodium chloride.…”

Nucleic Acid Drugs and DNA-based Delivery Systems

“…Keywords: Nucleic acid drug, DNA-based delivery system (5,6). The physicochemical and biological properties of this DNA hydrogel can be modulated through the use of chemically modified nucleotides, which would greatly increase its therapeutic potency and, inevitably, the cost of its synthesis.…”