Delivery Of ODNs Research Articles

Small EV-based delivery of CpG ODNs for melanoma postsurgical immunotherapy

Blocking programmed cell death protein 1 (PD-1) is an effective therapeutic strategy for melanoma. However, patients often develop tumor recurrence postoperatively due to the low response rate to the anti-PD-1 antibody (aPD-1). In this study, we developed an in situ sprayable fibrin gel that contains cytosine-guanine oligodeoxynucleotides (CpG ODNs)-modified ovalbumin (OVA) antigen-expressing bone marrow dendritic cell (DC)-derived small extracellular vesicles (DC-sEVs) and aPD-1. CpG ODNs can activate DCs, which have potent immunostimulatory effects, by stimulating both the maturation and activation of tumor-infiltrating dendritic cells (TIDCs) and DCs in tumor-draining lymph nodes (TDLNs). In addition, DC-sEVs can deliver OVA to the same DCs, leading to the specific expression of tumor antigens by antigen-presenting cells (APCs). In brief, the unique synergistic combination of aPD-1 and colocalized delivery of immune adjuvants and tumor antigens enhances antitumor T-cell immunity, not only in the tumor microenvironment (TME) but also in TDLNs. This effectively attenuates local tumor recurrence and metastasis. Our results suggest that dual activation by CpG ODNs prolongs the survival of mice and decreases the recurrence rate in an incomplete tumor resection model, providing a promising approach to prevent B16-F10-OVA melanoma tumor recurrence and metastasis.

Aminated yeast β-D-glucan for macrophage-targeted delivery of CpG oligodeoxynucleotides and synergistically enhanced cancer immunotherapy

CpG oligodeoxynucleotides (CpG ODNs) activate immune system and show strong potential in cancer immunotherapy. However, therapeutic efficacy of CpG ODNs is hampered due to rapid nuclease degradation and insufficient cellular uptake. Delivery of CpG ODNs into antigen presenting cells (APCs) is vital to enhance their therapeutic efficacy. Herein, we developed a super-convenient yet efficient strategy for macrophage-targeted delivery of CpG ODNs and synergistically enhanced cancer immunotherapy. Aminated yeast β-D-glucan (NH2-Glu) was simply synthesized through functionalization of β-D-glucan with DETA, which exhibited a dendrimer-like shape with size of about 80 nm. NH2-Glu complexed negatively-charged CpG ODNs. The as-prepared NH2-Glu/CpG complexes were positively charged, uniformly dispersed and exhibited good stability against nuclease degradation. Due to the specific recognition with dectin-1 expressed on macrophages, NH2-Glu/CpG complexes targeted macrophage and exhibited significantly enhanced cellular uptake due to dectin-1-mediated endocytosis. NH2-Glu/CpG complexes showed potent immunostimulatory activity. Contributed by the inherent immunostimulatory and antitumor activity, yeast β-D-glucan functioned synergistically with CpG ODNs in inducing antitumor immunity. NH2-Glu/CpG complexes remarkably inhibited tumor growth without causing toxic effect. In summary, this work provides a facile yet efficient macrophage-targeted CpG ODNs delivery system for cancer immunotherapy.

Molecular Bottlebrushes for Immunostimulatory CpG ODN Delivery: Relationship among Cation Density, Complex Formation Ability, and Cytotoxicity.

Artificially designed short single-stranded DNA sequences containing unmethylated CG (CpG ODNs) are agonists for toll-like receptor 9 (TLR9); thus, they have great potential as vaccine adjuvants for cancer immunotherapy and preventing infectious diseases. To deliver effectively CpG ODNs into cells bearing TLR9, nanoparticle polyion complexes of cationic polymers that are able to ingest multiple CpG ODN molecules have been developed; however, their structures and synthesized polycations are hard to control and bioincompatible, respectively. To solve these issues, we designed cationic molecular bottlebrushes (CMBs) with branches that are made from copolymers of 2-methacryloyloxyethyl phosphorylcholine and 2-methacryloyloxyethyl trimethylammonium chloride. Several instrumental methods were carried out to determine the structure of a CMB and its complex with CpG ODNs. The complexation did not change the overall shape of the original CMB, and the bound CpG ODNs were captured by the outer layer of the CMB. The moderation of cations was important to reduce toxicity and improve secretion of inflammatory cytokines.

Yeast β-D-glucan functionalized graphene oxide for macrophage-targeted delivery of CpG oligodeoxynucleotides and synergistically enhanced antitumor immunity

Immunostimulatory CpG oligodeoxynucleotides (CpG ODNs) show strong potential in cancer immunotherapy. However, therapeutic efficacy of CpG ODNs is hindered due to rapid nuclease degradation and insufficient cellular uptake. Transfecting CpG ODNs into antigen presenting cells (APCs) is vital to enhance their therapeutic efficacy while reduce the potential side effects. Herein, a multifunctional CpG ODNs vector was fabricated through functionalization of graphene oxide (GO) with yeast β-D-glucan, and its potential in cancer immunotherapy was further investigated. GO-β-D-glucan protected CpG ODNs from nuclease digestion. β-D-glucan endowed the delivery system with targeting ability for macrophage due to its recognition with dectin-1. Thus, GO-β-D-glucan enhanced the delivery of CpG ODNs into RAW264.7 cells due to dectin-1-mediated endocytosis. More importantly, β-D-glucan functioned synergistically with CpG ODNs in inducing antitumor immunity. GO-β-D-glucan/CpG ODNs inhibited the tumor cells growth more effectively. This work provides a macrophage-targeted CpG ODNs delivery system for cancer immunotherapy.Graphic abstract.

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-L-Lysine Induce Antibody Production as Vaccine Adjuvants.

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-β. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.

Targeted Delivery of Immunostimulatory CpG Oligodeoxynucleotides to Antigen-Presenting Cells in Draining Lymph Nodes by Stearic Acid Modification and Nanostructurization.

Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.

Reversing tumor immunosuppressive microenvironment via targeting codelivery of CpG ODNs/PD-L1 peptide antagonists to enhance the immune checkpoint blockade-based anti-tumor effect

In order to reverse tumor immunosuppressive microenvironment and improve antitumor immune effect based on immune checkpoint blocking, a mannose-modified liposome-based CpG ODNs and PD-L1 antagonistic peptides (P) co-delivery system (HA/M-Lipo CpG-P) was constructed, in which hyaluronic acid (HA) coating was supposed to improve the systemic circulation stability and thereby promote its accumulation in tumor tissues. When the HA/M-Lipo CpG-P complexes enter the tumor tissues, HA will be hydrolyzed under the action of hyaluronidase, exposing P peptides. Then, P peptides linked by octapeptides that can be cleaved by matrix metalloproteinases (MMPs) are released into tumor tissues under the action of MMPs, exerting a blocking effect in the PD-1/PD-L1 pathway. The M-Lipo CpG complexes can recognize macrophage surface mannose receptors through its surface modified mannose molecules, and promote the intracellular delivery of CpG ODNs, thereby activating macrophages. The results showed that HA/M-Lipo CpG-P complexes successfully reversed M2-type macrophages in tumor microenvironment (TME) to M1, thereby activating anti-tumor related immune cells and inhibiting tumor growth. Moreover, the HA/M-Lipo CpG-P complexes showed a better tumor inhibitory effect than the HA/M-Lipo CpG or the HA/M-Lipo-P (monotherapy) treatment groups. Overall, HA/M-Lipo CpG-P complexes provide a promising co-delivery strategy for targeting tumors to improve the antitumor effect based on immune checkpoint blockade.

Chitosan nanoparticles fabricated through host-guest interaction for enhancing the immunostimulatory effect of CpG oligodeoxynucleotide

CpG oligodeoxynucleotides (CpG ODNs) which can induce innate immune responses and promote adaptive immune responses, are powerful tools in defeating diseases. Here, a novel chitosan nanoparticle (CS-NPs) based on host-guest interaction has been designed for encapsulation and delivery of CpG ODNs for the first time. The CS-NPs exhibited high encapsulation efficiency (98.3%) of CpG ODNs and remained stable in storage under room temperature for at least 7 days. CS-NPs can also prevent CpG ODN diffusion at pH 7. The results of confocal laser scanning microscope images and flow cytometry show that CS-NPs can also be efficiently delivered into living cells. Furthermore, CpG@CS-NPs can increase the immunostimulatory activity of CpG ODNs. Raw 264.7 cells treated with CpG@CS-NPs demonstrated upregulation of both TNF-α and IL-6 cytokines by 13% and 40%, respectively. The newly developed CpG@CS-NPs were thus identified as an efficient system to deliver CpG-ODNs to treat various diseases.

Enhanced Immunostimulatory Activity of CpG Oligodeoxynucleotide by the Combination of Mannose Modification and Incorporation into Nanostructured DNA.

The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5'-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.

Development of a Gene Delivery System of Oligonucleotides for Fibroses by Targeting Cell-Surface Vimentin-Expressing Cells with N-Acetylglucosamine-Bearing Polymer-Conjugated Polyethyleneimine.

Targeting myofibroblasts and activated stellate cells in lesion sites of fibrotic tissues is an important approach to treat fibroses. Herein, we focused on targeting the cytoskeletal proteins vimentin, which are reportedly highly expressed on the surface of these cells and have N-acetylglucosamine (GlcNAc)-binding activity. A GlcNAc-bearing polymer synthesized via radical polymerization with a reversible addition-fragmentation chain transfer reagent has been previously found to interact with cell-surface vimentin-expressing cells. We designed a GlcNAc-bearing polymer-conjugated polyethyleneimine (PEI), as the gene carrier to target cell-surface vimentin-expressing cells and specifically deliver nuclear factor-κB decoy oligonucleotides (ODNs) and heat shock protein 47 (HSP47)-small interfering RNA (siRNA) to normal human dermal fibroblasts (NHDFs) that express cell-surface vimentin. The results showed that the expression of tumor necrosis factor-α in lipopolysaccharide-stimulated NHDFs and HSP47 in transforming growth factor-β1-stimulated NHDFs was suppressed by cellular uptake of the GlcNAc-bearing polymer-conjugated PEI/nuclear factor (NF)-κB decoy ODNs and HSP47-siRNA complexes through cell-surface vimentin, respectively. These findings suggest that the effective and specific delivery of ODNs and siRNA for cell-surface vimentin-expressing cells such as myofibroblasts and activated stellate cells can be achieved using GlcNAc-bearing polymer-conjugated PEI. This therapeutic approach could prove advantageous to prevent the promotion of various fibroses.

Construction of Monomeric and Dimeric G-Quadruplex-Structured CpG Oligodeoxynucleotides for Enhanced Uptake and Activation in TLR9-Positive Macrophages.

The G-quadruplex (GQ) structure has potential applications in nucleic acid drug delivery because of its superior stability. In this study, we added one G-tract (five guanines) to an unmethylated phosphodiester-linked cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), a potential immune adjuvant, to construct a GQ-structured CpG ODN with precise structural properties, increased biological stability, and efficient delivery to Toll-like receptor 9 (TLR9)-positive immune cells. A G-tract was added to phosphodiester-backboned CpG1668 at the 5'-end [1668(5'-G5)], 3'-end [1668(3'-G5)], or within the sequence [1668(mid-G5)]. Circular dichroism analysis showed that all CpG ODNs with a G-tract formed parallel GQ structures, irrespective of its position. Electrophoresis showed that 1668(5'-G5) formed a GQ dimer, whereas others remained GQ monomers. GQ-structured CpG ODNs induced greater tumor necrosis factor-α and interleukin-6 secretion from TLR9-positive mouse macrophage-like RAW264.7 cells than single-stranded CpG ODNs, with the highest for 1668(3'-G5). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3'-G5) decomposed more slowly in serum than 1668(5'-G5). Thus, GQ formation with one G-tract is a simple and efficient strategy for CpG ODN delivery to TLR9-positive cells, and addition of a G-tract to the 3'-end is effective in obtaining monomeric GQ-structured CpG ODN with high biological stability and immunostimulatory activity.

Preparation, characterization, and evaluation of chitosan-based nanoparticles as CpG ODN carriers

Chitosan-based particles are desirable materials for drug delivery because of their muco-adhesiveness in tissues, biocompatibility, low toxicity and effectiveness in antigen transport. It is still a challenge to prepare chitosan-based particles with high stability and by an effective method. Here we developed chitosan-based nanoparticles for the delivery of toll-like receptor 9 ligands, cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs), which can induce the change from Th-2 type to Th-1 type immune response. Because of the low tolerance to DNase and less uptake into the cells of the free CpG ODNs, the development of positively charged carriers is necessary for the effective delivery of CpG ODNs into immune cells. Chitosan nanoparticles were prepared by ionic gelation, and the nanoparticles size and surface charge were measured by dynamic light spectroscopy and zeta potential analyzer, respectively. The synthesized chitosan nanoparticles showed two dispersion peaks, at 196 ± 29 nm and 1.33 ± 0.36 μm, with a zeta potential of +3.3 ± 0.4 mV. The toxicity of the chitosan nanoparticles to murine RAW264 macrophages was measured by formazan dye assay utilizing the water-soluble tetrazolium salt WST-8. Our chitosan nanoparticles exhibited no cytotoxicity to RAW264 cells. Finally, we evaluated the immunostimulatory activity of CpG ODNs loaded on chitosan nanoparticles using human peripheral blood mononuclear cells. CpG ODNs significantly enhanced the secretion level of interleukin-6 and interferon-γ by cells compared to the free CpG ODNs. These results indicated that chitosan nanoparticles could be a good candidate for the delivery of CpG ODNs.

Inorganic kernel - Supported asymmetric hybrid vesicles for targeting delivery of STAT3-decoy oligonucleotides to overcome anti-HER2 therapeutic resistance of BT474R

As a recombinant humanized monoclonal antibody that targets the extracellular region of HER2 tyrosine kinase receptor, trastuzumab (TRAZ) has demonstrated comparable clinical efficacy and improved survival in patients with HER2-positive breast cancer. Nevertheless, the therapeutic potential of TRAZ is often limited due to its frequent resistance to anti-HER2 therapy. Therefore, we investigate the reversal effect of STAT3-specific decoy oligonucleotides (STAT3-decoy ODNs) on TRAZ resistance, which contain the consensus sequence within the targeted gene promoter of STAT3. Considering the shortcomings of poor cellular permeability and rapid degradation in vivo limit the further clinical applications of ODNs, we report here an asymmetric hybrid lipid/polymer vesicles with calcium phosphate as the solid kernel (CaP@HA). Through hyaluronan-mediated CD44 targeting, the constructed vesicles can specifically carry STAT3-decoy ODNs into TRAZ-resistant breast cancer cells and then regulate TRAZ-induced apoptosis. In comparison with the native ones, ODNs packaged with CaP@HA showed significantly increased serum stability, cellular transfection, synergistic cytotoxicity and apoptosis in vitro. The improved TRAZ sensitization is attributed to the blockade of STAT3 signaling as well as the expression of downstream target genes associated with TRAZ resistance. With the synergistic action of STAT3-decoy ODNs loaded CaP@HA, TRAZ inhibited the growth of its resistant breast cancer xenograft dramatically and induced significant tumor cell apoptosis in vivo. These results suggested that CaP@HA mediated targeted delivery of STAT3-decoy ODNs might be a promising new strategy to overcome anti-HER2 resistance in breast cancer therapy.

IL-4Rα aptamer-liposome-CpG oligodeoxynucleotides suppress tumour growth by targeting the tumour microenvironment

Tumour immunosuppressive microenvironments inhibit antigen-specific cellular responses and interfere with CpG-mediated immunotherapy. Overcoming tumour microenvironment (TME) immunosuppression is an important strategy for effective therapy. This study investigated the ability of a tumour-targeting IL-4Rα aptamer-liposome-CpG ODN delivery system to introduce CpG into tumours and overcome the immunosuppressive TME. The IL-4Rα-liposome-CpG delivery system was prepared. FAM-CpG visualisation was used to demonstrate tumour targeting in vitro and in vivo. Anti-tumour effects of this delivery system were evaluated in CT26 tumour-bearing mice. Mechanisms for conquering the TME were investigated. FAM-CpG was better distributed into the tumours upon treatment with IL-4Rα-liposome-FAM-CpG compared to distribution in the control group in vitro and in vivo. IL-4Rα-aptamer-liposome-CpG treatment inhibited distinct myeloid-derived suppressor cell populations in tumours and bone marrow. Similar profiles were observed for regulatory T cells in tumours. In CT26 tumour-bearing mice, IL-4Rα-liposome-CpG treatment exhibited enhanced anti-tumour activity. Increased mRNA levels of TNF-α, IL-2, and IL-12, and decreased mRNA levels of VEGF, IL-6, IL-10, MMP9, arginase-1, inducible NOS, CXCL9, p-Stat3, and NF-κB were observed in tumours upon IL-4R-liposome-CpG-treatment. The results suggested that pharmacologic targeting by the IL-4R aptamer-liposome-CpG system improves TME therapeutic benefit and provides a rationale for cancer immunotherapies.

Induction of a balanced Th1/Th2 immune responses by co-delivery of PLGA/ovalbumin nanospheres and CpG ODNs/PEI-SWCNT nanoparticles as TLR9 agonist in BALB/c mice

To develop effective and safe vaccines with reduced dose of antigen and adjuvant, intelligent delivery systems are required. Many delivery systems have been developed to enhance the biological activity of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODN) as both immunotherapeutic agents and vaccine adjuvants. In this study we designed a novel CpG ODN delivery system based on single-walled carbon nanotube (SWCNT) functionalized with polyethylenimine (PEI) and alkylcarboxylated PEI (AL-PEI). The physicochemical characteristics, cytotoxicity and cellular uptake studies of these carriers were performed.All carriers were conjugated with CpG ODN followed by co-delivery with ovalbumin (OVA) encapsulated into poly (lactic-co-glycolic acid) nanospheres (PLGA NSs) to enhance the induction of immune responses. The effect of these formulations on antibody (IgG1, IgG2a) and cytokine (IL-1β, IFN-γ, IL-4) production was evaluated in an in vivo experiment. The results showed that all nano-adjuvant formulations had a strong influence in up-regulation of IFN-γ and IL-4 in parallel with high IgG1-IgG2a isotype antibody titers in mice. In particular, SWCNT-AL-PEI nano-adjuvant formulation generated a balanced Th1 and Th2 immune response with more biased toward Th1 response without exhibiting any inflammatory and toxic effects. Therefore this nano-adjuvant formulation could be used as an efficient prophylactic immune responses agent.

Nanodelivery systems for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Synthetic oligodeoxynucleotides containing immunostimulatory CpG motif mimic bacterial DNA and are potent activator of innate and adaptive immune responses. Therefore, CpG ODNs have significant potentials as immunotherapeutic agent for treatment of infectious diseases, allergy and cancer. Many clinical trials involving CpG ODNs either used alone or as adjuvant have been initiated. However, delivery of CpG ODNs to target sites still remains a great challenge due to their extreme susceptibility to nuclease degradation in serum and poor cellular uptake. Chemical modification of CpG ODNs backbone can protect them against degradation by nucleases, but have raised concern regarding several severe side effects. Development of efficient CpG ODNs delivery systems to address these issues and enhance their immunostimulatory effect are highly desirable. In recent years, the emergence of nanotechnology has provided unprecedented opportunities to encapsulate CpG ODN into various nanocarriers or synthesize CpG ODNs nanostructures. This review provides an overview of the delivery systems based on nanomaterials and nanostructures newly developed for enhancing the immunostimulatory effect of CpG ODNs, together with a brief discussion on perspectives for future studies in this field.

Effect of amino groups of mesoporous silica nanoparticles on CpG oligodexynucleotide delivery

In this study, we proposed to modify mesoporous silica nanoparticles (MSNs) with 3-aminopropyltriethoxysilane (NH2-TES), aminoethylaminopropyltriethoxysilane (2NH2-TES) and 3-[2-(2-aminoethylamino)ethylamino] propyl-trimethoxysilane (3NH2-TES) for binding of cytosine-phosphate-guanosine oligodexynucleotides (CpG ODN), and investigated the effect of different amino groups of MSNs on the CpG ODN delivery. Serum stability, in vitro cytotoxicity, and cytokine interleukin-6 (IL-6) induction by MSN-NH2/CpG, MSN-2NH2/CpG and MSN-3NH2/CpG complexes were investigated in detail. The results showed that three kinds of aminated-MSN-based CpG ODN delivery systems had no cytotoxicity to RAW264.7 cells, and binding of CpG ODN to MSN-NH2, MSN-2NH2 and MSN-3NH2 nanoparticles enhanced the serum stability of CpG ODN due to protection by the nanoparticles. However, three aminated MSN-based CpG ODN delivery systems exhibited different CpG ODN delivery efficiency, and MSN-NH2/CpG complexes had the highest ability to induce IL-6 secretion.

Therapeutic Implications of Orally Delivered Immunomodulatory Oligonucleotides

DNA has multiple and complex effects on the immune system. Bacterial DNA contains immunostimulatory CpG motifs that trigger cells expressing Toll-like receptor 9 (TLR9) to proliferate, mature, and support T helper type 1 (Th1)-biased immune responses (reviewed in ref. 1). This response confers a selective advantage to the host by improving resistance to infection. In contrast, mammalian DNA contains sequence motifs that downregulate the immune system. Synthetic oligodeoxynucleotides that mimic this inhibitory activity (iODNs) slow or prevent the development of inflammatory and autoimmune diseases, consistent with the release of DNA from dying host cells inhibiting autoaggressive immune responses (reviewed in ref. 2). As reported in this issue, Wang et al. break new ground in the study of immunomodulatory ODNs by showing that the severity of chemically induced atopic dermatitis can be reduced by oral delivery of iODNs and worsened by CpG ODNs.3

Microfluidic assembly of lipid nanoparticles for delivery of antisense oligonucleotides.

Microfluidically (MF) synthesized lipid nanoparticles (LNPs) for antisense oligonucleotides (ODN) delivery have been shown to be superior to those prepared by bulk mixing (BM). In this study, a 5-inlet MF chip was used to synthesize LNPs loaded with LOR-2501, an antisense ODN targeting the ribonucleotide reductase R1 subunit. The size distribution of ODN- LNPs was measured by dynamic light scattering. The cytotoxicity of ODN- LNPs was determined by MTS assay. Gene silencing activity of ODN- LNPs was investigated by qRT-PCR and by Western blot. Results showed that MF synthesis produced ODN-LNPs that have lower average size and polydispersity values. The highest antisense activity was shown by LNs synthesized by the MF T2 chip, with downregulation of R1 mRNA by 32.5%. In conclusion, given their simplicity, affordability and reproducibility, MF is an attractive method for synthesis of LNs for ODN delivery.

CpG ODN delivery systems based on nanomaterials

Artificially synthesized CpG oligodeoxynucleotides (CpG ODNs) can imitate bacterial DNA and effectively stimulate the mammalian immune system in response to signals resulting from hazardous invasion. For this reason, CpG ODNs represent promising materials with a range of applications in the treatment of different infections, allergies and cancers. Unfortunately, however, unmodified CpG ODNs alone can be readily degraded by enzymes, resulting in a reduced level of uptake into cells. Repeated high-dose drug administration is therefore required in these treatments, which largely limits their application. In recent years, significant progress in nanomaterial technology has provided a range of new opportunities for the development of effective delivery systems for nucleotide drugs. Based on reports from the literature, a variety of different nanomaterial-based CpG delivery systems have been developed that show a range of advantages, such as high levels of activity, low toxicity and good biocompatibility, making them excellent candidates for use in therapeutic reagents in immune-related diseases. Herein, we provide an introduction to the background and development history of CpG ODN drugs, including their mechanisms of action and the requirements for successful delivery. Particular emphasis has been given to newly developed CpG delivery systems based on different nanomaterials, with a summary of their respective principles and features. Furthermore, we have provided a sketch of the prospects and future trends expected of these materials.