Monomeric G-Quadruplex-Based CpG Oligodeoxynucleotides as Potent Toll-Like Receptor 9 Agonists.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) motifs trigger the immune response by stimulating endosomal Toll-like receptor (TLR) 9. Natural linear ODNs are susceptible to nuclease degradation, thereby limiting their clinical applications. Here, we designed monomeric G-quadruplex-based CpG ODNs (G4 CpG ODNs) containing CpG motifs in the central loop region of the G4 structure. The monomeric G4 CpG ODNs were more stable in serum than the linear ODNs. The monomeric G4 CpG ODNs containing two or three CpG motifs induced the production of immunostimulatory cytokines interleukin (IL)-6, IL-12, and interferon (IFN)-ß in mouse macrophage-like RAW264 cells. We also showed that the number of CpG motifs and the number of nucleotides between the CpG motif and G-tracts define the efficacy of the G4 CpG ODNs in activating TLR9. Incubating human peripheral blood mononuclear cells with G4 CpG ODNs promoted IL-6 and IFN-γ production, confirming their stimulatory effects on human immune cells. Mice given intraperitoneal injections of G4 CpG ODNs produced higher plasma IL-6 compared with injections of linear ODNs. These findings provide further understanding of the parameters governing the immunostimulatory activity of G4 CpG ODNs, thereby providing insights into the rational design of highly potent G4 CpG ODNs for vaccine adjuvants.

Composite-dissolving microneedle patches for chemotherapy and photothermal therapy in superficial tumor treatment.

A combination of chemotherapy and photothermal therapy (PTT) has emerged as a promising strategy for cancer therapy. To ensure that the chemotherapeutic drug and photothermal agent can be simultaneously delivered to the tumor site to exert their synergistic effects, a safe and efficient delivery system is needed. Herein, we fabricated doxorubicin hydrochloride (DOX)- and indocyanine green (ICG)-loaded microneedle (MN) patches (PVP@DOX/MSN@ICG) using a two-step casting process. Mesoporous silica nanoparticles (MSNs) were used to improve the ICG stability and avoid reducing its PTT efficiency in vivo. The MN patches exhibited a good skin penetration ability, and the tips of the MN patches were dissolved by the interstitial fluid to release DOX and ICG at the tumor sites. Under 808 nm laser irradiation within 2 min, the local temperature in the tumor quickly reached 48 °C at a low power of 0.34 W cm-2. A combination of chemotherapy and PTT for PVP@DOX/MSN@ICG MN patches may maximally induce human osteosarcoma MG-63 cells in vitro. Moreover, the in vivo results showed that PVP@DOX/MSN@ICG MN patches had the best antitumor effects because of synergistic chemotherapy and PTT. Therefore, the composite-dissolving MN patch is a promising strategy for enhancing the antitumor effect of chemotherapy alone and shows the potential for the synergistic therapy of superficial tumors.

Double-stranded phosphodiester cytosine-guanine oligodeoxynucleotide complexed with calcium phosphate as a potent vaccine adjuvant for activating cellular and Th1-type humoral immunities.

Conventional class B cytosine-guanine (CpG) (CpG-B) oligodeoxynucleotide (ODNs) consisting of a single-stranded (ss) phosphorothioate (PT) backbone (ss CpG-B-PT) is converted from a proinflammatory cytokine inducer to a type-I interferon (IFN) inducer when complexed with cationic materials. In this study, we designed ss CpG-B and double-stranded (ds) CpG-B ODNs with a phosphodiester (PD) backbone (ss CpG-B-PD and ds CpG-B-PD, respectively) that became type-I IFN inducers upon complexation with Lipofectamine 2000 (Lipo), a cationic liposome. The ds CpG-B-PD complex induced higher IFN-ß expression in mouse macrophage-like RAW264 cells than ss CpG-B-PD and ss CpG-B-PT complexes. The fold induction of IFN-ß increased with the number of CpG motifs in ds CpG-B-PD, and a complex of ds CpG-B-PD consisting of 72 base pairs with nine CpG motifs (ds CpG-B72-PD) and Lipo showed the highest capacity to induce IFN-ß. The materials and method used for complexation influenced the degree of IFN-ß induction: ds CpG-B72-PD entrapped by calcium phosphate (CaP) (ds CpG-B72-PD/CaP) showed a higher induction capacity than ds CpG-B72-PD adsorbed onto the CaP surface. Entrapment of ds CpG-B72-PD by CaP also enhanced the induction of the proinflammatory cytokine interleukin-12. Vaccinating mice with ds CpG-B72-PD/CaP in conjunction with ovalbumin (OVA) increased the ratios of OVA-specific CD8+ T cells to total CD8+ T cells in peripheral blood and of OVA-specific IgG2a associated with helper T (Th)1 cells to OVA-specific IgG1 associated with Th2 cells. These results indicate that ds CpG-B72-PD/CaP is an effective vaccine adjuvant that can activate both cellular and Th1-type humoral immune responses.

Polyethyleneimine-functionalized boron nitride nanospheres as efficient carriers for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides.

CpG oligodeoxynucleotides (ODNs) stimulate innate and adaptive immune responses. Thus, these molecules are promising therapeutic agents and vaccine adjuvants against various diseases. In this study, we developed a novel CpG ODNs delivery system based on polyethyleneimine (PEI)-functionalized boron nitride nanospheres (BNNS). PEI was coated on the surface of BNNS via electrostatic interactions. The prepared BNNS-PEI complexes had positive zeta potential and exhibited enhanced dispersity and stability in aqueous solution. In vitro cytotoxicity assays revealed that the BNNS-PEI complexes with concentrations up to 100 µg/mL exhibited no obvious cytotoxicity. Furthermore, the positively charged surface of the BNNS-PEI complexes greatly improved the loading capacity and cellular uptake efficiency of CpG ODNs. Class B CpG ODNs loaded on the BNNS-PEI complexes enhanced the production of interleukin-6 and tumor necrosis factor-α from peripheral blood mononuclear cells compared with CpG ODNs directly loaded on BNNS. Contrary to the free CpG ODNs or CpG ODNs directly loaded on BNNS, class B CpG ODNs loaded on the BNNS-PEI complexes induced interferon-α simultaneously. PEI coating may have changed the physical form of class B CpG ODNs on BNNS, which further affected their interaction with Toll-like receptor 9 and induced interferon-α. Therefore, BNNS-PEI complexes can be used to enhance the immunostimulatory effect and therapeutic activity of CpG ODNs and the treatment of diseases requiring interleukin-6, tumor necrosis factor-α, and interferon-α.

Mesoporous silica nanoparticles for enhancing the delivery efficiency of immunostimulatory DNA drugs.

We developed a potential immunostimulatory double-stranded DNA (dsDNA) delivery system by the binding of dsDNA to amino-modified mesoporous silica nanoparticles (MSNs) to form MSN-NH2/dsDNA complexes. Serum stability, in vitro cytotoxicity, cell uptake, and type I interferon-α (IFN-α) induction of MSN-NH2/dsDNA complexes were evaluated. The results showed that MSN-NH2 nanoparticles had no cytotoxicity to Raw 264.7 cells, and MSN-NH2/dsDNA complexes enhanced the serum stability of dsDNA due to the protection by nanoparticles and exhibited a high efficiency of cell uptake due to a small particle size and excellent dispersity. Most importantly, MSN-NH2/dsDNA complexes significantly enhanced the level of IFN-α induction, triggered by cytosolic DNA sensor proteins. Therefore, binding of immunostimulatory DNA to MSNs would play a promising role for enhancing the delivery efficiency of immunostimulatory DNA drugs.

Elemental distribution analysis of type I collagen fibrils in tilapia fish scale with energy-filtered transmission electron microscope.

Elemental distribution of calcium, phosphorus, oxygen, and carbon in a single collagen fibril obtained from tilapia fish scales was identified with an electron energy-loss spectroscopy and an energy-filtered transmission electron microscopy, for the first time. The carbon intensity profile of the single collagen fibril showed the specific D-periodic pattern at 67 nm of type I collagen fibrils. The calcium L(2,3)-edge and oxygen K-edge peak positions were detected at 347/350 eV and 137 eV, respectively, and these positions were identical to those of hydroxyapatite. Calcium, phosphorus, and oxygen were present in the hole zones as the amorphous phase, while carbon was present in the overlap zone. Our results indicated that the hole zones preferentially attract calcium and phosphate ions and thus serve as possible nucleation sites for mineralization.