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      The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

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          Abstract

          An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.

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          Liposomal siRNA nanocarriers for cancer therapy.

          Bulent Ozpolat, Anil Sood, Gabriel Lopez-Berestein (2014)
          Small interfering RNAs (siRNA) have recently emerged as a new class of therapeutics with a great potential to revolutionize the treatment of cancer and other diseases. A specifically designed siRNA binds and induces post-transcriptional silencing of target genes (mRNA). Clinical applications of siRNA-based therapeutics have been limited by their rapid degradation, poor cellular uptake, and rapid renal clearance following systemic administration. A variety of synthetic and natural nanoparticles composed of lipids, polymers, and metals have been developed for siRNA delivery, with different efficacy and safety profiles. Liposomal nanoparticles have proven effective in delivering siRNA into tumor tissues by improving stability and bioavailability. While providing high transfection efficiency and a capacity to form complexes with negatively charged siRNA, cationic lipids/liposomes are highly toxic. Negatively charged liposomes, on the other hand, are rapidly cleared from circulation. To overcome these problems we developed highly safe and effective neutral lipid-based nanoliposomes that provide robust gene silencing in tumors following systemic (intravenous) administration. This delivery system demonstrated remarkable antitumor efficacy in various orthotopic human cancer models in animals. Here, we briefly overview this and other lipid-based approaches with preclinical applications in different tumor models for cancer therapy and potential applications as siRNA-nanotherapeutics in human cancers. © 2014 Elsevier B.V. All rights reserved.
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            CpG Oligonucleotides as Cancer Vaccine Adjuvants

            Hidekazu Shirota, Debra Tross, Dennis Klinman (2015)
            Adjuvants improve host responsiveness to co-delivered vaccines through a variety of mechanisms. Agents that trigger cells expressing Toll-like receptors (TLR) activate an innate immune response that enhances the induction of vaccine-specific immunity. When administered in combination with vaccines designed to prevent or slow tumor growth, TLR agonists have significantly improved the generation of cytotoxic T lymphocytes. Unfortunately, vaccines containing TLR agonists have rarely been able to eliminate large established tumors when administered systemically. To improve efficacy, attention has focused on delivering TLR agonists intra-tumorally with the intent of altering the tumor microenvironment. Agonists targeting TLRs 7/8 or 9 can reduce the frequency of Tregs while causing immunosuppressive MDSC in the tumor bed to differentiate into tumoricidal macrophages thereby enhancing tumor elimination. This work reviews pre-clinical and clinical studies concerning the utility of TLR 7/8/9 agonists as adjuvants for tumor vaccines.
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              Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape.

              Ayman El-Sayed Khalil, Shiroh Futaki, Dania A. Khalil … (2008)
              The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8-liposomes fused efficiently at both acidic and neutral pH, whereas K8-liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Sci Technol Adv Mater
                Journal ID (iso-abbrev): Sci Technol Adv Mater
                Journal ID (archive): TSTA
                Journal ID (publisher-id): tsta20
                Title: Science and Technology of Advanced Materials
                Publisher: Taylor & Francis
                ISSN (Print): 1468-6996
                ISSN (Electronic): 1878-5514
                Publication date Collection: 2016
                Publication date (Electronic): 16 September 2016
                Volume: 17
                Issue: 1
                Pages: 554-562
                Affiliations
                [ a ]Department of Biophysical Chemistry, Kyoto Pharmaceutical University , Kyoto, Japan
                [ b ]Tokushima University Graduate School of Biomedical Sciences , Tokushima, Japan
                Author notes
                [* ]Corresponding author. Email: kogure@ 123456tokushima-u.ac.jp
                Article
                Publisher ID: 1221726
                DOI: 10.1080/14686996.2016.1221726
                PMC ID: 5111564
                SO-VID: 031c6fa0-a292-4394-a673-87b200ef6172
                Copyright © © 2016 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 29 February 2016
                Date revision received : 2 August 2016
                Date accepted : 4 August 2016
                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 20, Pages: 9
                Funding
                Funded by: Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Nanomedicine Molecular Science’’ from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
                Award ID: 2306
                Categories
                Subject: Article
                Subject: Focus issue on Nanomedicine molecular science

                Keywords: ired,4′-thiodna,rnai effect,faint electric treatment,cytoplasmic delivery,30 bio-inspired and biomedical materials,211 scaffold / tissue engineering / drug delivery,600 others: nucleic acids
                Data availability:
                Keywords: ired, 4′-thiodna, rnai effect, faint electric treatment, cytoplasmic delivery, 30 bio-inspired and biomedical materials, 211 scaffold / tissue engineering / drug delivery, 600 others: nucleic acids

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