Immunomodulatory Nanodiamond Agents For Cancer Immunotherapy

Therapeutic nucleic acids, including functional oligonucleotide(ODNs), aptamers, small interfering RNAs(si RNAs), holds great promise for treating diseases ranging from inherited disorders to acquired conditions and cancers. Unmethylated cytosine-phosphate-guanine(Cp G) ODNs are a type of immunostimulatory nucleic acids that are commonly found in viral and bacterial DNA. Synthetic Cp G ODNs have been exploited as immune modulators for many applications including vaccine adjuvants and immunotherapy for cancers, infectious and allergic diseases. Clinical trials of Cp G ODNs have been performed from phase I to phase III. However, the observation of several adverse effects(e.g. ‘sepsis-like events’) did not support continuation of the study. These side effects are largely associated with the use of high-dose Cp G ODNs and repeated administration, which are possibly due to low cellular uptake and rapid clearance of naked ODNs. Hence, it is highly demanding to develop a new delivery system that can increase both the cellular internalization and the stability against nuclease degradation for sustained functioning of Cp G ODNs.Nanomaterials have shown great potential as controllable carriers for drug delivery. Various types of nanomaterials ranging from inorganic, organic to biomolecular structures, have been employed to carry Cp G ODNs to stimulate immunological reactions in cells. However, the high cost and potential toxicological concerns of these nanomaterials largely hamper their in vivo applications. Among a wide spectrum of nanomaterials-based vehicles, nanodiamonds(NDs) have shown remarkable delivery ability, flexible functionality and excellent biocompatibility. In particular, NDs can spontaneously form porous nanoclusters under physiological conditions, which show “sponge-like” effects for the delivery of small-molecule chemotherapeutic drugs as well as biomacromolecules. In this work, we employed poly-D-lysine(PDL)-coated functional NDs(f NDs) for intracellular and in-vivo delivery of Cp G ODNs and explored its immunostimulatory effects for cancer therapy. The main contents and results of this paper are summarized as following:(1) To design a therapeutic delivery vector for ODNs, we first coated NDs with a cationic polymer, poly-D-lysine(PDL), to form PDL-NDs, which was subsequently complexed with negatively charged unmethylated Cp G ODNs via the simple electrostatic interaction. After the PDL coating, the average size of functionalized NDs(f NDs) was increased from ~247 nm to ~325 nm, and their zeta potential was shifted from 33.0 e V to 67.3 e V. After the DNA adsorption, the mean size of ND clusters was further increased to ~338 nm, whereas the zeta potential was decreased to 40.6 e V.(2) Then, we evaluated the cellular uptake of Cp G-f NDs by using confocal microscopy. Quantitative analysis showed that the mean fluorescence intensity(MFI) of internalized Cp G was enhanced by nearly three orders of magnitudes as compared with that of naked one. We further investigate the clearance of Cp G-f NDs in cells. After the initial 6-h incubation with Cp G-f NDs, cells were fluorescently imaged at 24, 48 and 72 h, respectively. We also note that the MFI for intracellular Cp G-f NDs was ~900-, 830- and 400- fold higher than that for naked Cp G at 24, 48 and 72 h, respectively.(3) We next assessed the in vitro immunostimulatory activity induced by Cp G-f NDs. We found that Cp G-f NDs dramatically induced the production of tumor necrosis factor-alpha(TNF-α) and interleukin-6(IL-6) in RAW264.7 cells at 6-h incubation. Compared with naked Cp G ODNs, Cp G-f NDs increased the secreted levels of TNF-α and IL-6 by 59- and 20- fold, respectively. Then we measured the concentrations of secreted cytokines at the intervals of 24, 48 and 72 h. We still observed substantial amount of secreted TNF-α/IL-6 at 72 h, which were 13- and 26-folds higher than those induced by naked Cp G ODNs. The nanoagents exhibited long-term immunoregulatory activities lasting at least three days at the cellular level.(4) To further assess the therapeutic potential of Cp G-f NDs, we next investigated their in vivo immunostimulatory activity. ICR mice administrated with Cp G-f NDs via intravenous injection were analyzed by measuring their secreted levels of cytokines in serum. We found that Cp G-f NDs dramatically induced the production of serum IL-12 and IL-6 at 3 h. Compared with the treatment with naked Cp G ODNs of the same amount, the secreted levels of IL-12 and IL-6 were enhanced by 19 and 61 times, respectively. Even at 48 h after administration of Cp G-f NDs, the secreted levels of IL-12 were still 4- fold higher than that with naked Cp G ODNs. The nanoagents exhibited long-term immunoregulatory activities lasting two days in mice.Their in vivo immunostimulatory activity. ICR mice administrated with Cp G-f NDs via intravenous injection were analyzed by measuring their secreted levels of cytokines in serum. We found that Cp G-f NDs dramatically induced the production of serum IL-12 and IL-6 at 3 h. Compared with the treatment with naked Cp G ODNs of the same amount, the secreted levels of IL-12 and IL-6 were enhanced by 19 and 61 times, respectively. Even at 48 h after administration of Cp G-f NDs, the secreted levels of IL-12 were still 4- fold higher than that with naked Cp G ODNs. The nanoagents exhibited long-term immunoregulatory activities lasting two days in mice.