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The objective of this investigation was to develop a self-assembled, dual-functionalized delivery system that could effectively transport doxorubicin (DOX) to cancer cells through the use of AS1411 aptamer and hyaluronic acid polymer (HA). The ultimate goal is an improved targeting approach for more efficient treatment. The core of this system comprised polyethylenimine (PEI) and FOXM1 aptamer, which was coated by HA. Next, nucleolin targeting aptamers (AS1411) were loaded onto the nanocomplex. Afterward, DOX was added to Aptamers (Apts)-HA-PEI-FOXM1 NPs to create the DOX-AS1411-HA-PEI-FOXM1 NPs for better treatment of cancer cells. The cytotoxic effect of the nanocomplex on L929, 4T1, and A549 cells showed that cell mortality in target cancer cells (4T1 and A549) was considerably enhanced compared to nontarget cells (L929, normal cells). The findings from the flow cytometry analysis and fluorescence imaging demonstrated the cellular absorption of DOX-Apts-HA-PEI-FOXM1 NPs in target cells was significantly enhanced when compared to L929 cells. Furthermore, in vivo antitumor study exhibited that DOX-Apts-HA-PEI-FOXM1 NPs rendered specific tumor accumulation and increasing of the anti-tumor effects., Competing Interests: Declaration of competing interest There is no conflict of interest about this article., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6C hi CD11b hi CD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response., Competing Interests: Declaration of competing interest All authors declared that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Liposomal spherical nucleic acids (LSNAs) modified with polyethylene glycol (PEG) units are studied in an attempt to understand how the circulation time and biodistribution of the constructs can be manipulated. Specifically, the effect of (1) PEG molecular weight, (2) PEG shell stability, and (3) PEG modification method (PEG in both the core and shell versus PEG in the shell only) on LSNA blood circulation, biodistribution, and in vivo cell internalization in a syngeneic, orthotopic triple-negative breast cancer mouse model is studied. Generally, high PEG molecular weight extends blood circulation lifetime, and a more lipophilic anchor stabilizes the PEG shell and improves circulation and tumor accumulation but at the cost of cell uptake efficiency. The PEGylation strategy has a minor effect on in vitro properties of LSNAs but significantly alters in vivo cell uptake. For example, surface-only PEG in one design contributed to higher in vivo cell internalization than its counterpart with PEG both in the shell and core. Taken together, this work provides guidelines for designing LSNAs that exhibit maximal in vivo cancer cell uptake characteristics in the context of a breast cancer model.

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg 2+ . The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C 8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A photo-triggerable aptamer nanoswitch was proposed for spatiotemporal regulation of siRNA delivery. Recognition between AS1411 and nucleolin was effectively blocked by a photo-labile complementary oligonucleotide, which could be reactivated with photo-irradiation, resulting in efficient tumor-targeted siRNA internalization and gene silencing in vitro and in vivo.

Background and Aims: The topically applied Toll-like receptor 9 [TLR9] agonist cobitolimod is a first-in-class DNA-based oligonucleotide with demonstrated therapeutic efficacy in clinical trials with ulcerative colitis [UC] patients. We here characterized its anti-inflammatory mechanism in UC., Methods: Luminal cobitolimod administration was evaluated in an experimental dextran sodium sulfate [DSS]-induced colitis model. Cultured blood and mucosal cells from UC patients were treated with cobitolimod and analysed via microarray, quantitative real-time PCR, ELISA and flow cytometry. Intestinal slides of cobitolimod-treated UC patients were analysed by immunohistochemistry., Results: Cobitolimod administration markedly suppressed experimental colitis activity, and microarray analyses demonstrated mucosal IL10 upregulation and suppression of IL17 signalling pathways. Cobitolimod treatment was associated with significant induction of mucosal IL10+Tr1 and Treg cells and suppression of Th17 cells. TLR9 knockout mice indicated that cobitolimod requires TLR9 signalling for IL10 induction. In UC patients, mucosal TLR9 levels correlated with severity of inflammation. Cobitolimod inhibited IL17A and IL17F, but increased IL10 and FoxP3 expression in cultured intestinal UC T cells. Cobitolimod-mediated suppression of intestinal IL17+T cells was abrogated by IL10 blockade. Furthermore, cobitolimod led to heightened IL10 production by wound healing macrophages. Immunohistochemistry in intestinal biopsies of cobitolimod-treated UC patients indicated increased presence of IL10+mononuclear and regulatory T cells, as well as reduction of IL17+cells., Conclusion: Activation of TLR9 via cobitolimod might represent a novel therapeutic approach in UC, as it suppresses Th17 cells and induces anti-inflammatory IL10+macrophages and regulatory T cells, thereby modifying the dysregulated intestinal cytokine balance., Podcast: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast., (© European Crohn’s and Colitis Organisation (ECCO) 2019.)

Palladium nanosheets (Pd NSs) have recently attracted increasing research interest in the biomedical field due to their excellent near-infrared absorption, photothermal conversion capability and biocompatibility. However, the application of Pd NSs in immunotherapy has not been reported. Here, Pd NSs were used as the carriers of immunoadjuvant CpG ODNs for not only efficient delivery of CpG but also for enhancing the immunotherapeutic effects of CpG by the Pd NS-based photothermal therapy (PTT). Pd NSs had no influence on the immune system, and the prepared Pd-CpG nanocomposites, especially Pd(5)-CpG(PS), could significantly increase the uptake of CpG by immune cells and enhance the immunostimulatory activity of CpG in vitro and in vivo. With the combination of Pd(5)-CpG(PS) mediated PTT and immunotherapy, highly efficient tumor inhibition was achieved and the survival rate of the tumor-bearing mice was greatly increased depending on Pd(5)-CpG(PS) with safe near-infrared (NIR) irradiation (808 nm laser, 0.15 W cm -2 ). Importantly, the combination therapy induced tumor cell death and released tumor-associated antigens, which could be effectively taken up and presented by antigen presenting cells with the assistance of CpG, leading to increased TNF-α and IL-6 production and enhanced cytotoxic T lymphocyte (CTL) activity. This work provides a new paradigm of utilizing photothermal nanomaterials for safe and highly efficient cancer photothermal combined immunotherapy.

Objectives: Carbon dots (CDs), as a fascinating class of fluorescent carbon nanomaterials, have been proven to be powerful tools in the field of bioimaging and biosensing due to their small size, suitable photostability and favourable biocompatibility. However, the cellular uptake of free CDs lacks selectivity and the same negative charges as cell membranes may cause inefficient cell internalization. In this study, an efficient detecting and targeting nanosystem was developed based on the DNA aptamer AS1411 modified CDs with polyethyleneimine (PEI) as connecting bridge., Materials and Methods: Hydrothermally prepared CDs were assembled with positive-charged PEI, followed by conjugation with AS1411 through electrostatic interaction to form CDs-PEI-AS1411 nanocomplexes. The CDs, CDs-PEI and CDs-PEI-AS1411 were characterized by transmission electron microscopy (TEM), fourier transform infrared (FTIR) spectra, UV-vis spectra, zeta potential measurements and capillary electrophoresis characterizations. The cytotoxicity investigation of the CDs-PEI-AS1411 and CDs-PEI in both MCF-7 and L929 cells was carried out by the CCK-8 assay. The cellular uptake of the CDs-PEI-AS1411 was studied with confocal microscopy and flow cytometry., Results: The as-prepared nanosystem possessed good photostability and no obvious cytotoxicity. On the basis of the confocal laser scanning microscope observation and the flow cytometry studies, the cellular uptake of CDs-PEI-AS1411 nanosystem in MCF-7 cells was significantly higher than that of L929 cells, which revealed the highly selective detection ability of nucleolin-positive cells., Conclusions: The results of this study indicated that the CDs-PEI-AS1411 nanosystem had a potential value in cancer cell targeted imaging., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

New type of nanocapsules based on carboxymethyl chitosan functionalized with AS1411 aptamer and poly(N-vinylpyrrolidone-alt-itaconic anhydride) loaded with 5-Fluorouracil (5-FU) were developed, with the potential to improve the treatment of cancer. Functionalization of nanocapsules with AS1411 aptamer will enhance their recognition by tumor cells, due to the interaction with nucleolin, and subsequent endocytosis. Nanocapsules were prepared by interfacial condensation method in the absence of any toxic crosslinking agents. The condensation reaction took place at the interface between the organic and aqueous phases by opening the anhydride cycles from the copolymer, under the action of the NH 2 groups from mixture of chitosan/aptamer-functionalized carboxymethyl chitosan. The nanocapsules diameter varied between 100 and 267 nm as a function of the molar ratio of the polymers. SEM images have revealed that nanocapsules were spherical and presented relatively low dimensional polydispersity. Nanocapsules swelling degree was found between 1000 and 1680% in PBS solution (pH = 7.4) and they allowed the encapsulation of an important amount of 5-Fluorouracil (5-FU). The release efficiency of 5-FU was studied, the processes being controlled by the drug diffusion through the polymeric membrane, as confirmed by the theoretical analysis of the drug release. The cytotoxicity and haemolysis tests performed on the nanocapsules proved their lack of toxicity and their excellent hemocompatibility. The obtained results were encouraging, showing that these original 5-FU-loaded nanocapsules were able to induce a more pronounced cytotoxic effect on neoplastic MCF-7 cells, the occurrence of dead cells being more rapidly than in the case of free 5-FU., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein, we report the synthesis of 4'-C-trifluoromethyl (4'-CF3) thymidine (T4'-CF3) and its incorporation into oligodeoxynucleotides (ODNs) through solid-supported DNA synthesis. The 4'-CF3 modification leads to a marginal effect on the deoxyribose conformation and a local helical structure perturbation for ODN/RNA duplexes. This type of modification slightly decreases the thermal stability of ODN/RNA duplexes (-1 °C/modification) and leads to improved nuclease resistance. Like the well-known phosphorothioate (PS) modification, heavy 4'-CF3 modifications enable direct cellular uptake of the modified ODNs without any delivery reagents. This work highlights that 4'-CF3 modified ODNs are promising candidates for antisense-based therapeutics, which will, in turn, inspire us to develop more potent modifications for antisense ODNs and siRNAs.

TLR21 can recognize unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN) and activates NF-κB immune signaling pathway. However, the function of TLR21 in duck remains largely unclear. Here, the complete duck TLR21 (duTLR21) cDNA was cloned from Cherry Valley duck for the first time, and its immune response was preliminarily studied. Tissue specificity analysis showed duTLR21 was higher expressed in the peripheral blood, spleen, bursa of Fabricius and cecum. The expression of duTLR21 was significantly upregulated after stimulation with CpG-ODN or duck plague virus (DPV), but not Tembusu virus (TMUV), LPS or Poly (I:C). In addition, the transfection of DEF with duTLR21 stimulated by CpG-ODN activated NF-κB, through this signal pathway, the transcription of IL-1β, IL-6 and IFN-α were promoted, whereas knockdown of duTLR21 impaired the transcription of these genes. Furthermore, the overexpression of duTLR21 inhibited the replication of the DPV and the knockdown of duTLR21 by shRNA significantly promoted DPV replication in vitro. Altogether, these results indicate that duTLR21 can be activated by CpG-ODN, which mediates activation of NF-κB signaling pathway, and plays an important role in the host defence of DPV infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates, but further research is required to enhance their efficiency. The trimeric structure of the HIV-1 envelope glycoprotein provides a structural basis for multivalent drug design. In this work, a "multi-domain" strategy was adopted for design of an oligodeoxynucleotide with assembly, linkage, and activity domains. Built on the self-assembly of higher-order nucleic acid structure, a novel category of multivalent DNA helix-based HIV-1 fusion inhibitor could be easily obtained by a simple annealing course in solution buffer, with no other chemical synthesis for multivalent connection. An optimized multivalent molecule, M4, showed significantly higher anti-HIV-1 fusion activity than did corresponding monovalent inhibitors. Examination of the underlying mechanism indicated that M4 could interact with HIV-1 glycoproteins gp120 and gp41, thereby inhibiting 6HB formation in the fusion course. M4 also showed anti-RDDP and anti-RNase H activity of reverse transcriptase. Besides, these assembled molecules showed improved in vitro metabolic stability in liver homogenate, kidney homogenate, and rat plasma. Moreover, little acute toxicity was observed. Our findings aid in the structural design and understanding of the mechanisms of DNA helix-based HIV-1 inhibitors. This study also provides a general strategy based on a new structural paradigm for the design of other multivalent nucleic acid drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: GTI-2040 is a novel antisense oligonucleotide inhibitor of the R2 subunit of ribonucleotide reductase. This phase I study assessed safety and maximum tolerated dose (MTD) of GTI-2040 in combination with weekly gemcitabine in patients with advanced solid tumors., Methods: GTI-2040 was given as a 14-day continuous intravenous infusion, while gemcitabine was administered on days 1, 8, and 15. This combination was repeated every 4 weeks and study followed a modified 3 + 3 Fibonacci schema. Incidence, severity of adverse events, pharmacokinetics (PK), and pharmacodynamics (PD) was assessed. Responses were assessed using RECIST criteria version 1.0 with CT scans performed after every other cycle., Results: A total of 16 patients received at least one dose of GTI-2040/gemcitabine and were included in the safety analysis. The MTD of this regimen is 100 mg/m 2 /day of GTI-2040 over 14 days combined with 400 mg/m 2 /day of gemcitabine administered weekly on days 1, 8, and 15. The dose-limiting toxicities (DLTs) included grade 3 fatigue and thrombocytopenia with hematemesis (both at 100/600 mg/m 2 /day). The most common adverse events were grade 1/2 fatigue, nausea, vomiting, diarrhea, and anorexia. There was no evidence of alteration in gemcitabine PKs. PD modulation of R2mRNA expression in peripheral blood mononuclear cells was observed. No objective tumor response was observed although stable disease was seen in 25% patients., Conclusions: The combination of GTI-2040 and gemcitabine has an acceptable safety profile in a heavily pre-treated patient population with advanced solid tumors. No clear signal of anti-tumor activity was observed; however, several patients had prolonged stable disease.

CpG oligodeoxynucleotides (CpG-ODN), a common immune stimulator and vaccine adjuvant, was reported to switch Tumor Associated Macrophages (TAMs) from M2 to M1 phenotype inducing anti-tumor responses. Liposomes are of the successfully applied carriers for CpG-ODN. The aim of present study was design and preparation of a liposomal formulation containing phosphodiester CpG-ODN, evaluation of its effect on macrophages responses, and subsequent antitumor responses in mice. Liposomal formulations containing phosphodiester CpG-ODN or non-CpG-ODN were prepared and characterized. MTT reduction assay in four different cell lines, uptake, arginase and iNOS activity evaluation in macrophage cell lines, biodistribution study and therapeutic anti-tumor effects of formulations in mice bearing C26 colon carcinoma or B16F0 melanoma were carried out. The size of liposomes containing CpG-ODN was ~200 nm with the encapsulation efficiency of 33%. The iNOS activity assay showed high nitric oxide (NO) level in M2 phenotype of macrophage cell lines treated by liposomes containing CpG-ODN. In mice which received liposomes containing CpG-ODN as a monotherapy, maximum tumor growth delay with remarkable survival improvement was observed compared to control groups. Biodistribution study showed the accumulation of liposomal formulation in tumor micro-environment. In conclusion, considerable anti-tumor responses observed by liposomes containing CpG-ODN was due to enhanced delivery of CpG-ODN to immune cells and subsequent initiation of anti-tumoral immune responses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Transfection of NF κB synthetic decoy Oligodeoxynucleotides (ODNs) has been proposed as a promising therapeutic strategy for a variety of diseases arising from constitutive activation of the eukaryotic transcription factor NF κB. The decoy approach faces some limitations under physiological conditions notably nuclease-induced degradation., Results: In this work, we show how a systems pharmacology model of NF κB regulatory networks displaying oscillatory temporal dynamics, can be used to predict quantitatively the dependence of therapeutic efficacy of NF κB synthetic decoy ODNs on dose, unbinding kinetic rates and nuclease-induced degradation rates. Both deterministic mass action simulations and stochastic simulations of the systems biology model show that the therapeutic efficacy of synthetic decoy ODNs is inversely correlated with unbinding kinetic rates, nuclease-induced degradation rates and molecular stripping rates, but is positively correlated with dose. We show that the temporal coherence of the stochastic dynamics of NF κB regulatory networks is most sensitive to adding NF κB synthetic decoy ODNs having unbinding time-scales that are in-resonance with the time-scale of the limit cycle of the network., Conclusions: The pharmacokinetics/pharmacodynamics (PK/PD) predicted by the systems-level model should provide quantitative guidance for in-depth translational research of optimizing the thermodynamics/kinetic properties of synthetic decoy ODNs.

The main roles of equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) are to transfer single nucleosides and analogues for the nucleic acid salvage pathway. Oligodeoxyribonucleotides (ODNs) can be transported into the cytoplasm or nucleus of cells under certain conditions. Among ODNs composed of a single type of nucleotide, the transport efficiency differs with the length and nucleotide composition of the ODNs and varies in different types of leukaemia cells; among the 5 tested random sequence ODNs and 3 aptamers with varying sequences, the data showed that some sequences were associated with significantly higher transport efficiency than others. The transport of ODNs was sodium, energy, and pH-independent, membrane protein-dependent, substrate nonspecific for ODNs and 4-nitrobenzylthioinosine (NBMPR)-insensitive, but it showed a low sensitivity to dipyridamole (IC50 = 35.44 µmol/L), distinguishing it from ENT1-4 and CNTs. The delivery efficiency of ODNs was superior to that of Lipofection and Nucleofection, demonstrating its potential applications in research or therapeutics. Moreover, this process was associated with p38 mitogen activated protein kinase (p38MAPK) instead of c-Jun N-terminal kinase (JNK) signalling pathways. We have denoted ODN transmembrane transport as equilibrative nucleic acid transport (ENAT). Overall, these findings indicate a new approach and mechanism for transmembrane transport of ODNs.

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo., Statement of Significance: We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics. Although, the siRNA constructs have already given very promising results in the cancer therapy, the in vivo application of RNA-based oligonucleotide therapeutics still is limited due to their sensitivity to serum nucleases and some toxicity. We propose a carrier for RNA-based therapeutics that is made of bioengineered spider silk. We showed that functionalized bioengineered spider silk spheres not only protected RNA-based therapeutics from degradation by serum nucleases, but what is more important the embedding of siRNA into silk spheres delayed and extended target gene silencing compared with naked oligonucleotides. Moreover, we showed that plain silk spheres did not have unspecific effect on target gene levels proving not only to be non-cytotoxic but also very neutral vehicles in terms of TLR9/STAT3 activation in macrophages. We demonstrated advantages of novel delivery technology in safety and efficacy comparing with delivery of naked CpG-STAT3siRNA therapeutics., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Chemotherapy is still the main adjuvant strategy after surgery in glioblastoma therapy. As the main obstacles of chemotherapeutic drugs for glioblastoma treatment, the blood brain barrier (BBB) and non-specific delivery to non-tumor tissues greatly limit the accumulation of drugs into tumor tissues and simultaneously cause serious toxicity to nearby normal tissues which altogether compromised the chemotherapeutic effect. In the present study, we established an aptamer AS1411-functionalized poly (l-γ-glutamyl-glutamine)-paclitaxel (PGG-PTX) nanoconjugates drug delivery system (AS1411-PGG-PTX), providing an advantageous solution of combining the precisely active targeting and the optimized solubilization of paclitaxel. The receptor nucleolin, highly expressed in glioblastoma U87 MG cells as well as neo-vascular endothelial cells, mediated the binding and endocytosis of AS1411-PGG-PTX nanoconjugates, leading to significantly enhanced uptake of AS1411-PGG-PTX nanoconjugates by tumor cells and three-dimension tumor spheroids, and intensive pro-apoptosis effect of AS1411-PGG-PTX nanoconjugates. In vivo fluorescence imaging and tissue distribution further demonstrated the higher tumor distribution of AS1411-PGG-PTX as compared with PGG-PTX. As a result, the AS1411-PGG-PTX nanoconjugates presented the best anti-glioblastoma effect with prolonged median survival time and most tumor cell apoptosis in vivo as compared with other groups. In conclusion, the AS1411-PGG-PTX nanoconjugates exhibited a promising targeting delivery strategy for glioblastoma therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cancer vaccine has the ability to directly eradicate tumor cells by creating and activating cytotoxic T lymphocytes (CTLs). To achieve efficient CTL activity and to induce Th1 responses, it is essential to administer an appropriate adjuvant as well as an antigen. CpG-ODN is known as a ligand of Toll-like receptor 9 (TLR9) and strongly induces Th1 responses. In our previous study, we developed a CpG-ODN delivery system by use of the formation of complexes between ODN and a β-glucan SPG, denoted as CpG/SPG, and demonstrated that CpG/SPG induces high Th1 responses. In this study, we created a nanogel made from CpG/SPG complexes through DNA-DNA hybridization (cross-linked (CL)-CpG). Immunization with CL-CpG induced much stronger antigen-specific Th1 responses in combination with the antigenic protein ovalbumin (OVA) than that with CpG/SPG. Mice preimmunized with CL-CpG and OVA exhibited a long delay in tumor growth and an improved survival rate after tumor inoculation. These immune inductions can be attributed to the improvement of cellular uptake by the combination of increased size and the cluster effect of the β-glucan recognition site in the nanogel structure. In other words, the particle nature of CL-CpG, instead of the semiflexible rod conformation of CpG/SPG, enhanced the efficacy of a cancer vaccine. The present results indicate that CL-CpG can be used as a potent vaccine adjuvant for the treatment of cancers and infectious diseases.

ISIS 388626 is an antisense sodium-glucose cotransporter 2 (SGLT2) inhibitor designed to treat type 2 diabetes mellitus by induction of glucosuria. ISIS 388626 was demonstrated to be safe and effective in preclinical trails in several species. We undertook the present study to evaluate the safety and efficacy of 13 weekly doses of 50, 100, and 200 mg of ISIS 388626 in humans. ISIS 388626 increased 24-hour urinary glucose excretion dose dependently with 508.9 ± 781.45 mg/day in the 100-mg and 1299.8 ± 1833.4 mg/day in the 200-mg cohort, versus 88.7 ± 259.29 mg/day in the placebo group. ISIS 388626 induced a reversible increase in serum creatinine, with the largest effect after eight doses of ISIS 388626 (200 mg; 0.38 ± 0.089 mg/dl; 44% increase over baseline). Three subjects were discontinued as a result of creatinine increases. The renal clearance test revealed no indications for impairment of glomerular filtration or renal perfusion. The creatinine increases were accompanied by a rise in the levels of urinary renal damage markers [β-2-microglobulin (B2M), total protein, kidney injury molecule (KIM1), α-glutathione S-transferase (aGST), N-acetyl-β-(d)-glucosaminidase (NAG)]. Other treatment-related adverse events included mild injection site reactions occurring in 8-19% of the subjects. In conclusion, ISIS 388626 treatment induced glucosuria at a dose level of 200 mg/week. This intended pharmacological effect was small, amounting to approximately 1% of the total amount of filtered glucose. Changes in serum and urinary markers were indicative of transient renal dysfunction, most probably of tubular origin. Whether the glucosuria is caused by specific SGLT2 inhibition or general tubular dysfunction or a combination remains uncertain., (Copyright © 2016 by The Author(s).)

The present study generated a novel DNA complex to specifically target endothelial NF-κB to inhibit cerebral vascular inflammation. This DNA complex (GS24-NFκB) contains a DNA decoy which inhibits NF-κB activity, and a DNA aptamer (GS-24), a ligand of transferrin receptor (TfR), which allows for targeted delivery of the DNA decoy into cells. The results indicate that GS24-NFκB was successfully delivered into a murine brain-derived endothelial cell line, bEND5, and inhibited inflammatory responses induced by tumor necrosis factor α (TNF-α) or oxygen-glucose deprivation/re-oxygenation (OGD/R) via down-regulation of the nuclear NF-κB subunit, p65, as well as its downstream inflammatory cytokines, inter-cellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1). The inhibitory effect of the GS24-NFκB was demonstrated by a significant reduction in TNF-α or OGD/R induced monocyte adhesion to the bEND5 cells after GS24-NFκB treatment. Intravenous (i.v.) injection of GS24-'NFκB (15mg/kg) was able to inhibit the levels of phoseph-p65 and VCAM-1 in brain endothelial cells in a mouse lipopolysaccharide (LPS)-induced inflammatory model in vivo. In conclusion, our approach using DNA nanotechnology for DNA decoy delivery could potentially be utilized for inhibition of inflammation in ischemic stroke and other neuro-inflammatory diseases affecting cerebral vasculature., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Magnetic resonance imaging (MRI) with high spatial resolution has been widely used in clinics as a noninvasive diagnostic technology, and MRI diagnosis-based theranostic nanomaterials have attracted increasing attention. Herein, we report on the fabrication of a GO/BSA-Gd2O3/AS1411-DOX theranostic nanocomplex with BSA-Gd2O3 nanoparticles for use as an MRI contrast agent (CA), with graphene oxide (GO) nanoplates as the CA and drug nanocarrier, as well as an aptamer, AS1411, as the targeting molecule. The proposed theranostic nanocomplex not only provided stronger MR contrast enhancement but also inhibited the growth of 786-0 human renal carcinoma cells with the help of the AS1411 aptamer, while sparing the normal cells from harm, thus demonstrating their specific drug delivery capability. Additionally, 786-0 cells could be specifically recognized using the GO/BSA-Gd2O3/AS1411-DOX theranostic nanocomplex with MRI both in vitro and in vivo. Notably, most of the nanocomplex that was injected into the tail vein was excreted by the kidneys and bladder, while MRI signals from the nanocomplexes that accumulated specifically in the tumor region could remain as long as 24 hours, which is beneficial for future clinical diagnosis and therapy.

The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that β-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

CpG oligonucleotides (ODN) stimulate the innate immune system by triggering cells that express TLR9. The resulting response promotes tumor regression, an effect optimized by delivery of CpG ODN to the tumor site. This work examines the effect of instilling CpG ODN adsorbed onto polyketal microparticles (CpG-MP) into the lungs of mice with non-small cell lung cancer. Intrapulmonary delivery of CpG-MP improved ODN uptake and retention at the tumor site, thereby inducing a stronger Th1 response than systemically administered or unadsorbed CpG ODN. CpG-MP reversed the immunosuppression that characterized the tumor microenvironment by (i) decreasing the number of immunosuppressive Tregs and M2 macrophages while (ii) increasing the number of tumoricidal CD8(+) T cells and M1 macrophages. These effects promoted tumor regression and culminated in 82% permanent survival of mice with otherwise fatal Lewis lung cancer., (©2015 American Association for Cancer Research.)

The synthetic cytosine-phosphate-guanosine oligodeoxynucleotide 107 (CpG ODN107) is a novel radiosensitizer for glioma treatment. However, the information related to its pharmacokinetics and toxicity remains unclear. Therefore, the plasma pharmacokinetics, distribution, elimination, and acute toxicity of CpG ODN107 in mice were investigated in the present experiments. The results from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that the plasma elimination half-life (t1/2β) of CpG ODN107 in BALB/c mice varied slightly with the dose, and it was 0.65, 0.49, and 0.50 h at the intravenous doses of 2.5, 5, and 10 mg/kg, respectively. CpG ODN107 rapidly and widely distributed in organs/tissues, except the brain and testes. The highest concentrations were found in the liver (28.6% of the administered dose after 0.5 h) and the kidneys (5.7% of the administered dose after 1 h). CpG ODN107 (0.3, 3, and 30 μg/mL) could highly bind to human and mouse plasma proteins in vitro. CpG ODN107 in the forms of prototype was excreted in urine (1.79%) and feces (0.91%), and its shortened metabolites were excreted in urine (2.1%) and feces (2.2%) within the first 24 h. The mice in vivo optical image showed CpG ODN107 labeled with Alexa Fluor 680 fluorochrome (AF680) accumulated in the brain after orthotopic injection, eliminated very slowly, and excreted in urine compared with poly T labeled with AF680. The median lethal dose (LD50) of CpG ODN107 was 75.7 mg/kg for mice; this dose only could produce apparent spleen and liver damage, in line with the distribution features of CpG ODN. In conclusion, our present pharmacokinetic and toxicity investigation will provide helpful information to further pharmacodynamic and pharmacokinetic research of CpG ODN107 and other oligodeoxynucleotide drugs in the future.

Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed "cell box" was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain.

Synthetic oligodeoxyribonucleotides (ODNs) containing CpG (unmethylated deoxycytidylyl-deoxyguanosine dinucleotide) motifs activate endosomal TLR9. The nucleotide sequence, length, and dimerization properties of ODNs modulate their activation of TLR9. We performed a systematic investigation of the sequence motifs of B-class and C-class phosphodiester ODNs to identify the sequence properties that govern TLR9 activation. ODNs shorter than 21 nt and with the adenosine adjacent to the cytidine-guanosine (CG) dinucleotide motif led to a significant loss of the propensity to activate TLR9. The distance between the stimulatory CpG motifs within the ODN fine-tunes the activation of B cells. The minimal ODNs that activate human TLR9 comprise 2 CG dinucleotides separated by 6-10 nt, where the first CpG motif is preceded by the 5'-thymidine and the elongated poly-thymidine tail at the 3' end of the ODN. The minimal sequence provides insight into the molecular mechanism of TLR9 ligand recognition. On the basis of sequence requirements, we conclude that two binding sites with different affinities for CG are formed in the human TLR9 dimer, with a very stringent binding site interacting with the 5' CpG motif., (Copyright © 2015 by The American Association of Immunologists, Inc.)

AS1411, a 26-base guanine-rich oligonucleotide aptamer, has high affinity to nucleolin, mainly on tumor cell surfaces. In this study, a modified AS1411 was labeled with (99m)Tc and evaluated as a potential tumor-targeting agent for imaging. The AS1411 aptamer was conjugated with HYNIC and labeled with (99m)Tc in the presence a co-ligand. Radiochemical purity and stability testing of the (99m)Tc-HYNIC-AS1411 aptamer were carried out with thin layer chromatography and a size-exclusion column in normal saline and human serum. Cellular nucleolin-specific binding, cellular internalization in DU-145 cells, as high levels of nucleolin expression, were performed. Additionally, biodistribution in normal mice and DU-145 tumour-bearing mice was assessed. Radiolabeling of the aptamer resulted in a reasonable yield and radiochemical purity after purification. The aptamer was stable in normal saline and human serum, and cellular experiments demonstrated specific binding of the AS1411 aptamer to the nucleolin protein. Based on biodistribution assessment of (99m)Tc-HYNIC-AS1411, rapid blood clearance was seen after injection and it appears that the excretion route was via the urinary system at 1 h post-injection. Tumours also showed a higher accumulation of radioactivity with this labeled aptamer. (99m)Tc-AS1411 can be a potential tool for the molecular imaging of nucleolin-overexpressing cancers.

Local drug delivery depots have significant clinical utility, but there is currently no noninvasive technique to refill these systems once their payload is exhausted. Inspired by the ability of nanotherapeutics to target specific tissues, we hypothesized that blood-borne drug payloads could be modified to home to and refill hydrogel drug delivery systems. To address this possibility, hydrogels were modified with oligodeoxynucleotides (ODNs) that provide a target for drug payloads in the form of free alginate strands carrying complementary ODNs. Coupling ODNs to alginate strands led to specific binding to complementary-ODN-carrying alginate gels in vitro and to injected gels in vivo. When coupled to a drug payload, sequence-targeted refilling of a delivery depot consisting of intratumor hydrogels completely abrogated tumor growth. These results suggest a new paradigm for nanotherapeutic drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.

IMT504 is a non-CpG 24-mer oligodeoxynucleotide (ODN) with immunomodulatory as well as tissue repair activity. IMT504 has been previously proven to be effective in animal models of vaccine potency, chronic lymphocytic leukemia, tissue regeneration, and sepsis. Here, we assessed the safety, including pharmacokinetics and toxicity studies in rats and monkeys, of IMT504 in a single- or repeated-dose administration by the subcutaneous (SC) or intravenous (IV) routes. In rats, the maximum tolerated dose was determined to be 50 mg/kg when administered SC. Adverse effects at 50 mg/kg were mild and reversible liver injury, revealed as lobular inflammation, focal necrosis, and small changes in the transaminase profile. Dose-dependent splenomegaly and lymphoid hyperplasia, most probably associated with immune stimulation, were commonly observed. Rats and monkeys were also IV injected with a single dose of 10 or 3.5 mg/kg, and no adverse effects were observed. Rats injected IV with 10 mg/kg showed a transient increase in spleen weight, together with a slight increase in the marginal zone of the white pulp and in leukocyte count 2 days post-administration. In monkeys, this dosage caused slight changes in total serum complement and leukocyte count on day 14. No adverse effects were observed at 3.5 mg/kg IV in rats or monkeys. Therefore, this dose was defined as the "no observed adverse effect level" for this route. Furthermore, repeated-dose toxicity studies were performed in these species using 3.5 or 0.35 mg/kg/day IV for 6 weeks. A transient increase in the spleen and liver weight was observed at 3.5 mg/kg/day only in female rats. No changes in clotting time and activation of the alternative complement pathway were observed. The toxicity profile of IMT504 herein reported suggests a dose range in which IMT504 can be used safely in clinical trials.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

We hypothesized that GTI-2040, a 20-mer oligonucleotide complementary to the R2 subunit mRNA of ribonucleotide reductase, combined with high dose cytarabine (HiDAC) would result in enhanced cytotoxicity by favoring Ara-CTP DNA incorporation. In a phase I dose escalation trial, adults (≥ 60 years) with refractory or relapsed acute myeloid leukemia (AML) received daily HiDAC plus infusional GTI-2040. Using a novel assay, evidence of intracellular drug accumulation and target R2 down-regulation was observed. GTI-2040/HiDAC can be administered safely. However, with no complete remissions observed, alternative doses and schedules may need to be investigated to achieve clinical activity in older patients with AML.

Purpose: To evaluate the internalization and subcellular fate of AS1411 aptamer (for glioma targeting) and TGN peptide (for blood-brain barrier targeting)-modified nanoparticles (AsTNPs), which was important for optimizing targeted delivery systems and realizing the potential toxicity to cells., Methods: Organelles were labelled with specific markers. Several uptake inhibitors were used to determine the endocytosis pathways. Transmission electron microscopy (TEM) was utilized to directly observe the endocytosis procedure and subcellular fate of AsTNPs., Results: Subcellular localization demonstrated that endosomes and mitochondria were involved in the uptake of AsTNPs by both C6 and bEnd.3 cells, however, lysosomes and Golgi apparatus were only involved in the internalization by C6 cells rather than bEnd.3 cells. Uptake mechanism study demonstrated the clathrin- and caveolae-mediated endocytosis were the main pathways in the uptake of AsTNPs by C6 and bEnd.3 cells. However, other pathways, including clathrin- and caveolae-independent endocytosis and macropinocytosis are also involved in the uptake by C6 cells and not by bEnd.3 cells. TEM directly demonstrated the involvement of these pathways. Particles could be found mostly in endosomes., Conclusion: Compared to unmodified nanoparticles, AsTNPs displayed different internalization pathways involved in several cell organelles.

The adjuvants approved in human vaccine with recombinant/purified antigens induce weak cellular immune response and so the development of new adjuvant strategies is critical. CpG-ODN has successfully been used as an adjuvant (phase I-III clinical trials) but its bioavailability needs to be improved. We investigated the adjuvant ability of CpG-ODN formulated with a liquid crystal nanostructure of 6-O-ascorbyl palmitate (Coa-ASC16). Mice immunized with OVA/CpG-ODN/Coa-ASC16 elicited a potent specific IgG1, IgG2a, Th1 and Th17 cellular response without systemic adverse effects. These responses were superior to those induced by OVA/CpG-ODN (solution of OVA with CpG-ODN) and to those induced by the formulation OVA/CpG-ODN/Al(OH)3. Immunization with OVA/CpG-ODN/Coa-ASC16 resulted in a long-lasting cell-mediated immune response (at least 6.5 months). Furthermore, Coa-ASC16 alone allows a controlled release of CpG-ODN in vitro and induces local inflammatory response, independent of TLR4 signaling, characterized by an influx of neutrophils and Ly6C(high) monocytes and pro-inflammatory cytokines. Remarkably, the adjuvant capacity of CpG-ODN co-injected with Coa-ASC16 (OVA/CpG-ODN plus Coa-ASC16) was similar to the adjuvant activity of OVA/CpG-ODN, supporting the requirement for whole formulation to help CpG-ODN adjuvanticity. These results show the potential of this formulation, opening a new avenue for the development of better vaccines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The development of functional nanocarriers that can enhance the cellular delivery of a variety of nucleic acid agents is important in many biomedical applications such as siRNA therapy. We report the synthesis of large pore mesoporous silica nanoparticles (LPMSN) loaded with iron oxide and covalently modified by polyethyleneimine (denoted PEI-Fe-LPMSN) as carriers for gene delivery. The LPMSN have a particle size of ∼200 nm and a large pore size of 11 nm. The large pore size is essential for the formation of large iron oxide nanoparticles to increase the magnetic properties and the adsorption capacity of siRNA molecules. The magnetic property facilitates the cellular uptake of nanocarriers under an external magnetic field. PEI is covalently grafted on the silica surface to enhance the nanocarriers' affinity against siRNA molecules and to improve gene silencing performance. The PEI-Fe-LPMSN delivered siRNA-PLK1 effectively into osteosarcoma cancer cells, leading to cell viability inhibition of 80%, higher compared to the 50% reduction when the same dose of siRNA was delivered by a commercial product, oligofectamine.

Introduction: AS1411 is a 26-base guanine-rich oligonucleotide aptamer shown binding to surface nucleolin, a protein over-expressed in multiple cancer cells, thus AS1411 labeled with a PET isotope can be explored as a potential diagnostic imaging agent. Our objective was to perform preliminary biological characterization of (64)Cu-labeled AS1411 in vitro and in vivo., Methods: Four chelators (DOTA, CB-TE2A, DOTA-Bn and NOTA-Bn) were selected to label AS1411 with Cu-64. 185kBq (5μCi) of each tracer was incubated in each well with H460 cells at 37°C for 1, 3, 6, 12, 24 and 48h, respectively (n=4). For microPET/CT imaging, 7.4MBq (200μCi) of AS1411 labeled with either (64)Cu-DOTA or (64)Cu-CB-TE2A was I.V. injected and multiple scans were obtained at 1, 3, 6 and 24h post injection. Afterward in vivo biodistribution studies were performed., Results: Percent uptake of (64)Cu-DOTA-AS1411 and (64)Cu-CB-TE2A-AS1411 was significantly higher than that of (64)Cu-DOTA-Bn-AS1411 and (64)Cu-NOTA-Bn-AS1411. About 90% of uptake for (64)Cu-DOTA-AS1411 and (64)Cu-CB-TE2A-AS1411 was internalized into cells within 3h and the internalization process was completed before 24h. Both tracers demonstrated reasonable in vivo stability and high binding affinity to the cells. MicroPET imaging with (64)Cu-CB-TE2A-AS1411 showed clear tumor uptake at both legs from 1 to 24h post injection, whereas both tumors were undetectable for up to 24h with (64)Cu-DOTA-AS1411. In addition, (64)Cu-CB-TE2A-AS1411 had faster in vivo pharmacokinetics than (64)Cu-DOTA-AS1411 with lower liver uptake and higher tumor to background contrast., Conclusion: CB-TE2A is a preferred chelator with higher tumor-to-background ratio, lower liver uptake and faster clearance than DOTA. Aptamer imaging with (64)Cu-CB-TE2A-AS1411 may be feasible for detecting lung cancer, if an appropriate chelator can be identified and further validation can be performed with a known control oligonucleotide. It may also be used as a companion diagnostic imaging agent for AS1411 in the treatment of cancer., (© 2013.)

Background: DNA aptamers represent a novel strategy in anti-cancer medicine. AS1411, a DNA aptamer targeting nucleolin (a protein which is overexpressed in many tumor types), was evaluated in patients with metastatic, clear-cell, renal cell carcinoma (RCC) who had failed treatment with ≥1 prior tyrosine kinase inhibitor., Methods: In this phase II, single-arm study, AS1411 was administered at 40 mg/kg/day by continuous intravenous infusion on days 1-4 of a 28-day cycle, for two cycles. Primary endpoint was overall response rate; progression-free survival (PFS) and safety were secondary endpoints., Results: 35 patients were enrolled and treated. One patient (2.9 %) had a response to treatment. The response was dramatic (84 % reduction in tumor burden by RECIST 1.0 criteria) and durable (patient remains free of progression 2 years after completing therapy). Whole exome sequencing of this patient's tumor revealed missense mutations in the mTOR and FGFR2 genes which is of interest because nucleolin is known to upregulate mTOR pathway activity by enhancing AKT1 mRNA translation. No other responses were seen. Thirty-four percent of patients had an AS1411-related adverse event, all of which were mild or moderate., Conclusions: AS1411 appears to have minimal activity in unselected patients with metastatic RCC. However, rare, dramatic and durable responses can be observed and toxicity is low. One patient in this study had an excellent response and was found to have FGFR2 and mTOR mutations which will be of interest in future efforts to discover and validate predictive biomarkers of response to nucleolin targeted compounds. DNA aptamers represent a novel way to target cancer cells at a molecular level and continue to be developed with a view to improving treatment and imaging in cancer medicine.

CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli currently under investigation as antimicrobial agents for different species. Toll-like receptor (TLR) 9 and TLR21 are the cellular receptors of CpG-ODN in mammals and chickens, respectively. The avian genomes lack TLR9, whereas mammalian genomes lack TLR21. Although fish contain both of these genes, the biological functions of fish TLR9 and TLR21 have not been investigated previously. In this study, we comparatively investigated zebrafish TLR9 (zebTLR9) and TLR21 (zebTLR21). The two TLRs have similar expression profiles in zebrafish. They are expressed during early development stages and are preferentially expressed in innate immune function-related organs in adult fish. Results from cell-based activation assays indicate that these two zebrafish TLRs are functional, responding to CpG-ODN but not to other TLR ligands. zebTLR9 broadly recognized CpG-ODN with different CpG motifs, but CpG-ODN with GACGTT or AACGTT had better activity to this TLR. In contrast, zebTLR21 responded preferentially to CpG-ODN with GTCGTT motifs. The distinctive ligand recognition profiles of these two TLRs were determined by their ectodomains. Activation of these two TLRs by CpG-ODN occurred inside the cells and was modulated by UNC93B1. The biological functions of these two TLRs were further investigated. The CpG-ODNs that activate both zebTLR9 and zebTLR21 were more potent than others that activate only zebTLR9 in the activation of cytokine productions and were more bactericidal in zebrafish. These results suggest that zebTLR9 and zebTLR21 cooperatively mediate the antimicrobial activities of CpG-ODN. Overall, this study provides a molecular basis for the activities of CpG-ODN in fish.

Background: Tremelimumab, a fully human cytotoxic T-lymphocyte antigen 4 monoclonal antibody, and PF-3512676, a Toll-like receptor-9 agonist, are targeted immune modulators that elicit durable single-agent antitumour activity in advanced cancer., Methods: To determine the maximum tolerated dose (MTD) of these agents combined during this phase I study, patients received intravenous tremelimumab (6.0, 10.0, or 15.0 mg kg(-1)) every 12 weeks plus subcutaneous PF-3512676 (0.05, 0.10, or 0.15 mg kg(-1)) weekly. Primary end points were safety and tolerability; secondary end points included pharmacokinetics and antitumour activity., Results: Twenty-one patients with stage IV melanoma (n=17) or advanced solid tumours (n=4) were enrolled. Injection-site reactions (n=21; 100%), influenza-like illness (n=18; 86%), and diarrhoea (n=13; 62%) were the most common treatment-related adverse events (TAEs). Grade ≥3 TAEs were reported (n=7; 33%). Dose-limiting toxicities (prespecified 6-week observation) occurred in one of the six patients in the 10 mg kg(-1) tremelimumab plus 0.05 mg kg(-1) PF-3512676 cohort (grade 3 hypothalamopituitary disorder) and two of the six patients in the 15 mg kg(-1) tremelimumab plus 0.05 mg kg(-1) PF-3512676 cohort (grade 3 diarrhoea). Consequently, 15 mg kg(-1) tremelimumab plus 0.05 mg kg(-1) PF-3512676 exceeded the MTD. Two melanoma patients achieved durable (≥170 days) partial response. No human antihuman antibody responses to tremelimumab were observed., Conclusion: Weekly PF-3512676 (≤0.15 mg kg(-1)) plus tremelimumab (≤10 mg kg(-1) every 12 weeks) was tolerable.

An estrone phosphoramidite, synthesized in a single step, was directly incorporated at the 5' ends of small interfering RNAs (siRNAs). The resulting estrone-conjugated siRNAs readily pass through cellular membranes and down-regulate the target protein, and show specific distributions in vivo.

Plasmacytoid dendritic cells (pDCs) produce a vast amount of interferon (IFN)-α in response to nucleic acids from viruses and damaged self-cells through Toll-like receptor (TLR)7 and TLR9. Pharmaceutical agents that suppress IFN-α production by pDCs are instrumental in elucidating the mechanisms behind IFN-α production, and in developing novel therapies for inflammatory disorders that involve pDCs. Here, we show that a tyrosine kinase inhibitor for chronic myeloid leukemia with multiple targets, dasatinib, strongly suppresses production of IFN-α and proinflammatory cytokines by human pDCs stimulated with multimeric CpG oligodeoxynucleotides (CpG-A) without reducing viability. In contrast, other tyrosine kinase inhibitors, imatinib, and nilotinib, did not suppress the cytokine production at clinically relevant concentrations. Inhibitors of SRC family kinases (SFKs), which are prominent targets of dasatinib, also suppressed the cytokine production. Notably, however, dasatinib, but not SFK inhibitors, abrogated prolonged localization of CpG-A in early endosomes, which is a critical step for pDCs to produce a large amount of IFN-α. This study suggests that dasatinib suppresses IFN-α production by pDCs by inhibiting SFK-dependent pathways and SFK-independent endosomal retention of CpG DNA. Kinases controlling the distinctive endosomal trafficking in pDCs may be exploited as targets to develop novel therapies for pDC-related inflammatory disorders., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Cytosine-phosphate-guanine (CpG) oligodeoxynucleotides activate Toll-like receptor 9, leading to induction of proinflammatory cytokines, which play an important role in induction and maintenance of innate and adaptive immune responses. Previously, we have used boron nitride nanospheres (BNNS) as a carrier for delivery of unmodified CpG oligodeoxynucleotides to activate Toll-like receptor 9. However, because CpG oligodeoxynucleotides and BNNS are both negatively charged, electrostatic repulsion between them is likely to reduce the loading of CpG oligodeoxynucleotides onto BNNS. Therefore, the efficiency of uptake of CpG oligodeoxynucleotides is also limited and does not result in induction of a robust cytokine response. To ameliorate these problems, we developed a CpG oligodeoxynucleotide delivery system using chitosan-coated BNNS as a carrier., Methods: To facilitate attachment of CpG oligodeoxynucleotides onto the BNNS and improve their loading capacity, we prepared positively charged BNNS by coating them with chitosan preparations of three different molecular weights and used them as carriers for delivery of CpG oligodeoxynucleotides., Results: The zeta potentials of the BNNS-CS complexes were positive, and chitosan coating improved their dispersity and stability in aqueous solution compared with BNNS. The positive charge of the BNNS-CS complexes greatly improved the loading capacity and cellular uptake efficiency of CpG oligodeoxynucleotides. The loading capacity of the CpG oligodeoxynucleotides depended on the molecular weight of chitosan, which affected the positive charge density on the surface of the BNNS. CpG oligodeoxynucleotides loaded onto BNNS-CS complexes significantly enhanced production of interleukin-6 and tumor necrosis factor-α by peripheral blood mononuclear cells compared with CpG oligodeoxynucleotides directly loaded onto BNNS, or when Lipofectamine™ 2000 was used as the carrier. The molecular weight of the chitosan used to coat the BNNS affected the magnitude of cytokine induction by varying the strength of condensation of the CpG oligodeoxynucleotides., Conclusion: Although the loading capacity of BNNS coated with low molecular weight chitosan preparations was the lowest of all the preparations, they induced the highest levels of cytokines.

CpG oligodeoxynucleotide 107 (CpG ODN107) could be used as a novel radiosensitizer for glioma. Herein, a novel and sensitive reversed-phase HPLC coupled with electrospray triple quadrupole mass spectrometry (LC-MS/MS) following a one-step C18 solid-phase extraction (SPE) for biological matrix removal was developed and fully validated for the determination of CpG ODN107 and its metabolites such as 5'N-1, 3'N-1, 3'N-2, and 3'N-3 in mouse plasma. The analytes were separated on an Extend-C18 analytical column (150 mm × 2.1 mm, 3.5 μm) using an eluent of acetonitrile-0.05% aqueous NH(3) (20:80, v/v) and detected by electrospray ionization (ESI) mass spectrometry in the negative multiple reaction monitoring mode (MRM). The assay was specific, and it showed a good linearity with a determination coefficient (r(2)) that was greater than or equal to 0.998 for CpG ODN107 and its metabolites in the biological matrices. The precision, accuracy, and relative recovery values were found to be <15%, ±15%, and 95-105%, respectively. This method was successfully applied to measure the concentrations of CpG ODN107 and its metabolites in the plasma following the intravenous administration of 15.0 mg/kg of CpG ODN107 in mice; therefore, the method was suitable for preclinical pharmacokinetic studies on CpG ODN107 and its metabolites., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Liposomes and polymers are widely used drug carriers for controlled release since they offer many advantages like increased treatment effectiveness, reduced toxicity and are of biodegradable nature. In this work, anticancer drug-loaded PLGA-lecithin-PEG nanoparticles (NPs) were synthesized and were functionalized with AS1411 anti-nucleolin aptamers for site-specific targeting against tumor cells which over expresses nucleolin receptors. The particles were characterized by transmission electron microscope (TEM) and X-ray photoelectron spectroscopy (XPS). The drug-loading efficiency, encapsulation efficiency and in vitro drug release studies were conducted using UV spectroscopy. Cytotoxicity studies were carried out in two different cancer cell lines, MCF-7 and GI-1 cells and two different normal cells, L929 cells and HMEC cells. Confocal microscopy and flowcytometry confirmed the cellular uptake of particles and targeted drug delivery. The morphology analysis of the NPs proved that the particles were smooth and spherical in shape with a size ranging from 60 to 110 nm. Drug-loading studies indicated that under the same drug loading, the aptamer-targeted NPs show enhanced cancer killing effect compared to the corresponding non-targeted NPs. In addition, the PLGA-lecithin-PEG NPs exhibited high encapsulation efficiency and superior sustained drug release than the drug loaded in plain PLGA NPs. The results confirmed that AS1411 aptamer-PLGA-lecithin-PEG NPs are potential carrier candidates for differential targeted drug delivery., (Copyright © 2012 Wiley Periodicals, Inc.)

ISIS 388626, a 2'-methoxyethyl (MOE)-modified antisense oligonucleotide (ASO) that targets human sodium glucose cotransporter 2 (SGLT2) mRNA, is in clinical trials for the management of diabetes. SGLT2 plays a pivotal role in renal glucose reabsorption, and inhibition of SGLT2 is anticipated to reduce hyperglycemia in diabetic subjects by increasing urinary glucose elimination. To selectively inhibit SGLT2 in the kidney, ISIS 388626 was designed as a "shortmer" ASO, consisting of only 12 nucleotides with two 2'-MOE-modified nucleotides at the termini. Mice and monkeys received up to 30 mg/kg/week ISIS 388626 via subcutaneous injection for 6 or 13 weeks. Dose-dependent decreases in renal SGLT2 mRNA expression were observed, which correlated with dose-related increases in glucosuria without concomitant hypoglycemia. There were no histologic changes in the kidney attributed to SGLT2 inhibition after 6 or 13 weeks of treatment. The remaining changes observed in these studies were typical of those produced in these species by the administration of oligonucleotides, correlated with high doses of ISIS 388626, and were unrelated to the inhibition of SGLT2 expression. The kidney contained the highest concentration of ISIS 388626, and dose-dependent basophilic granule accumulation in tubular epithelial cells of the kidney, which is evidence of oligonucleotide accumulation in these cells, was the only histologic change identified. No changes in kidney function were observed. These results revealed only readily reversible changes after the administration of ISIS 388626 and support the continued investigation of the safety and efficacy of ISIS 388626 in human trials.

CpG oligonucleotides (CpG ODNs) interact with Toll-like receptor 9 (TLR9), which results in the induction of immunostimulatory cytokines. We delivered CpG ODNs intracellularly using boron nitride nanospheres (BNNS). To enhance the loading capacity of CpG ODNs on BNNS, we used a phage display technique to identify a 12-amino acid peptide designated as BP7, with specific affinity for BNNS, and used it as a linker to load CpG ODNs on BNNS. The tyrosine residue (Y) at the eighth position from the N-terminus played a crucial role in the affinity of BP7 to BNNS. BNNS that bound BP7 (BNNS-BP7) were taken up by cells and showed no cytotoxicity, and CpG ODNs were successfully crosslinked with BP7 to create BP7-CpG ODN conjugates. Using BP7 as a linker, the loading efficiency of CpG ODNs on BNNS increased 5-fold compared to the direct binding of CpG ODNs to BNNS. Furthermore, the BP7-CpG ODN conjugate-loaded BNNS had a greater capacity to induce interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production from peripheral blood mononuclear cells (PBMCs) than that of CpG ODNs directly loaded on BNNS. The higher amount of cytokine induction by BP7-CpG ODN conjugate-loaded BNNS may be attributed to a higher loading capacity and stronger binding to BNNS of the linker BP7. The greater functionality of BP7-conjugated CpG ODNs on BNNS expands the potential of BNNS for drug delivery applications.

Background: Class B CpG oligodeoxynucleotides primarily interact with Toll-like receptor 9 (TLR9) in B cells and enhance the immune system through induction of various interleukins including interleukin-6 in these immune cells. Although free class B CpG oligodeoxynucleotides do not induce interferon (IFN)-α production, CpG oligodeoxynucleotide molecules have been reported to induce IFN-α when loaded onto nanoparticles. Here, we investigated the in vitro induction of IFN-α by a nanocarrier delivery system for class B CpG oligodeoxynucleotide molecules., Methods: For improving the capacity to load CpG oligodeoxynucleotide molecules, flake-shell SiO(2) nanoparticles with a specific surface area approximately 83-fold higher than that of smooth-surfaced SiO(2) nanoparticles were prepared by coating SiO(2) nanoparticles with polyethyleneimine (PEI) of three different number-average molecular weights (Mns 600, 1800, and 10,000 Da)., Results: The capacity of the flake-shell SiO(2) nanoparticles to load CpG oligodeoxynucleotides was observed to be 5.8-fold to 6.7-fold higher than that of smooth-surfaced SiO(2) nanoparticles and was found to increase with an increase in the Mn of the PEI because the Mn contributed to the positive surface charge density of the nanoparticles. Further, the flake-shell SiO(2) nanoparticles showed much higher levels of IFN-α induction than the smooth-surfaced SiO(2) nanoparticles. The highest IFN-α induction potential was observed for CpG oligodeoxynucleotide molecules loaded onto flake-shell SiO(2) nanoparticles coated with PEI of Mn 600 Da, although the CpG oligodeoxynucleotide density was lower than that on flake-shell SiO(2) nanoparticles coated with PEI of Mns 1800 and 10,000 Da. Even with the same density of CpG oligodeoxynucleotides on flake-shell SiO(2) nanoparticles, PEI with an Mn of 600 Da caused a markedly higher level of IFN-α induction than that with Mns of 1800 Da and 10,000 Da. The higher TLR9-mediated IFN-α induction by CpG oligodeoxynucleotides on flake-shell SiO(2) nanoparticles coated with a PEI of Mn 600 Da is attributed to residence of the CpG oligodeoxynucleotide molecules in endolysosomes.

During bacterial and viral infections, unmethylated CpG-DNA released by proliferating and dying microbes is recognized by toll-like receptor (TLR) 9 in host cells, initiating innate immune responses. Many corneal infections occur secondary to epithelial breaches and represent a major cause of vision impairment and blindness globally. To mimic this clinical situation, we investigated mechanisms of TLR9 ligand-induced corneal inflammation in mice after epithelial debridement. Application of CpG oligodeoxynucleotides (ODNs) resulted in neutrophil and macrophage infiltration to the cornea and loss of transparency. By 6 hours after CpG-ODN administration, TLR9 mRNA was increased in the cornea and retina. In vivo clinical examination at 24 hours revealed inflammatory infiltrates in the vitreous and retina, which were confirmed ex vivo to be neutrophils and macrophages, along with activated resident microglia. CpG-ODN-induced intraocular inflammation was abrogated in TLR9(-/-) and macrophage-depleted mice. Bone marrow reconstitution of irradiated TLR9(-/-) mice with TLR9(+/+) bone marrow led to restored corneal inflammatory responses to CpG-ODN. Fluorescein isothiocyanate-CpG-ODN rapidly penetrated the cornea and ocular media to reach the retina, where it was present within CD68(+) retinal macrophages and microglia. These data show that topically applied CpG-ODN induces intraocular inflammation owing to TLR9 activation of monocyte-lineage cells. These novel findings indicate that microbial CpG-DNA released during bacterial and/or viral keratitis can cause widespread inflammation within the eye, including the retina., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

A poly(ethylene glycol) (PEG)-modified dendritic poly(L-lysine) (PEG-WeKG6) containing tryptophan residues in its core was synthesized as an oligonucleotide carrier to tumors after systemic injection. PEG- WeKG6 formed a stable complex with double-stranded deoxyoligonucleotide (ODN). The size and the zeta-potential of the complex were smaller than those of a dendritic poly(L-lysine) without PEG (WeKG6). To study the biodistribution of the complexes in tumor-bearing mice after intravenous injection, the den- drimers and the oligonucleotide were labeled with gadolinium and Cy5, respectively. Our results show that PEG modification of the dendrimer improved the stability of ODN in blood circulation. Effective accumulation of the PEG-WeKG6/ODN complex in the tumor tissue was found 24 h after the injection. These results indicate that PEG-WeKG6 is suitable for forming a complex with any genetic or therapeutic material for efficient delivery to tumors.

Purpose: GTI-2040, a 20-mer phosphorothioate oligonucleotide, was designed to hybridize to the mRNA sequence of human ribonucleotide reductase R2. GTI-2040 has been shown to inhibit human cancer cell proliferation by downregulation of R2 expression in vitro and to significantly inhibit tumor growth in xenograft models of human cancer in mice. As part of the safety evaluation for human clinical trials, the toxicity and toxicokinetics of GTI-2040 were determined in Sprague-Dawley rats and rhesus monkeys., Methods: GTI-2040 was administered to rats at 2, 10, and 50 mg/kg/day by bolus intravenous injection every second day for 21 days with a 21-day recovery. In monkeys, an acute study was performed with single, escalating doses of GTI-2040 ranging from 10 to 80 mg/kg given as a 24-h continuous intravenous infusion. As well, a 21-day, continuous intravenous infusion study with GTI-2040 was conducted in monkeys at 2, 10, and 50 mg/kg/day, with a 3-week recovery. Blood sampling was done to measure GTI-2040 plasma concentrations, metabolites, and pharmacokinetic parameters, and tissues were collected to assess the distribution of GTI-2040 and/or metabolites., Results: The toxicities of GTI-2040 in both rats and monkeys were typical for the phosphorothioate oligonucleotide class of compounds. In monkeys, there was a dose-related increase in GTI-2040 plasma levels with concomitant increase in complement activation and prolongation of activated partial thromboplastin time. In both rats and monkeys, the tissues having the highest concentrations of GTI-2040 (kidney, liver, spleen) had the largest dose-related toxic effects. Adverse effects were diminished or absent in the recovery animals., Conclusions: GTI-2040 was well tolerated when infused over 24 h at doses up to 80 mg/kg in monkeys. In rats and monkeys, GTI-2040 was reasonably well tolerated and showed reversible toxicities when administered at doses up to 50 mg/kg/day for 21 days. The no observed adverse effect dose level for GTI-2040 in both animal species was 2 mg/kg/day. There were no apparent sequence-specific effects related to the interaction of GTI-2040 with the R2 component of the mRNA expressing ribonucleotide reductase.