Cellular delivery of antisense oligonucleotides.

Antisense oligonucleotides can be successfully employed to inhibit specifically gene expression. However, many oligonucleotide classes are polyanions and cannot passively transit the cell membrane. Thus, the use of naked oligonucleotides for antisense purposes poses some rather stringent challenges, and it is not a trivial task to appropriately interpret the data derived from experiments in which they have been used. Multiple methods have been developed to improve intracellular, and in particular, intranuclear oligonucleotide delivery, and in doing so, to maximize the performance of the antisense technologies that are currently available. This review discusses the use of cationic lipids, protein and peptide delivery agents, and several novel chemical and viral methods that have recently been explored as delivery vehicles, focussing not only on their strengths, but also on their limitations.     

Novel non-endocytic delivery of antisense oligonucleotides

Antisense oligonucleotides (ONs) have several properties that make them attractive as therapeutic agents. Hybridization of antisense ONs to their complementary nucleic acid sequences by Watson-Crick base pairing is a highly selective and efficient process. Design of therapeutic antisense agents can be made more rationally as compared to most traditional drugs, i.e., they can be designed on the basis of target RNA sequences and their secondary structures. Despite these advantages, the design and use of antisense ONs as therapeutic agents are still faced with several obstacles. One major obstacle is their inefficient cellular uptake and poor accessibility to target sites. In this article, we will discuss key barriers affecting ON delivery and approaches to overcome these barriers. Current methods of ON delivery will be reviewed with an emphasis on novel non-endocytic methods of delivery. ONs are taken up by cells via an endocytic process. The process of ON release from endosomes is a very inefficient process and, hence, ONs end up being degraded in the endosomes. Thus, ONs do not reach their intended site of action in the cytoplasm or nucleus. Delivery systems ensuring a cytoplasmic delivery of ONs have the potential to increase the amount of ON reaching the target. Here, we shall examine various ON delivery methods that bypass the endosomal pathway. The advantages and disadvantages of these methods compared to other existing methods of ON delivery will be discussed.     

Enhanced transfection of tumor cells in vivo using "Smart" pH-sensitive TAT-modified pegylated liposomes

Liposomes have been prepared loaded with DNA (plasmid encoding for the green fluorescent protein, GFP) and additionally modified with TATp and PEG, with PEG being attached to the liposome surface via both pH-sensitive hydrazone and non-pH-sensitive bonds. The pGFP-loaded liposomal preparations have been administered intratumorarly in tumor-bearing mice and the efficacy of tumor cell transfection was followed after 72 h. The administration of pGFP-TATp-liposomes with non-pH-sensitive PEG coating has resulted in only minimal transfection of tumor cells because of steric hindrances for the liposome-to-cell interaction created by the PEG coat, which shielded the surface-attached TATp. At the same time, the administration of pGFP-TATp-liposomes with the low pH-detachable PEG resulted in at least three times more efficient transfection since the removal of PEG under the action of the decreased intratumoral pH leads to the exposure of the liposome-attached TATp residues, enhanced penetration of the liposomes inside tumor cells and more effective intracellular delivery of the pGFP. This result can be considered as an important step in the development of tumor-specific stimuli-sensitive drug and gene delivery systems.     

Oral delivery of antisense oligonucleotides in man

Treatment of systemic disease with phosphorothioate antisense oligonucleotides (PS ASOs) has been accomplished using local or parenteral routes of administration to date. This report describes, for the first time, the effective oral delivery of a second generation oligonucleotide where significant milligram amounts of intact drug are absorbed in human subjects. In this study, a variety of oral solid dosage formulations were evaluated and it was determined that pulsing the delivery of sodium caprate (C10), a well-known permeation enhancer, in a novel manner may provide optimal ASO plasma bioavailability. Further, these dosage forms, containing C10 and ASO, were well tolerated in both fasted and fed volunteers. Oral absorption of the 2'-O-(2-methoxyethyl) modified antisense oligonucleotide (2'-MOE ASO), ISIS 104838, was demonstrated in healthy volunteers with an average 9.5% plasma bioavailability across four formulations tested. The greatest average performance achieved in this study for a single formulation was 12.0% bioavailability within an individual dose and subject range of 1.96-27.5%. The totality of the data suggests that formulations can be devised that allow oral administration of oligonucleotides that maintain systemic concentrations associated with inhibition of targeted human mRNA.     

Oral delivery of siRNA and antisense oligonucleotides

Inhibition of gene expression with antisense oligonucleotides or RNA interference (RNAi) mediated gene silencing by small interfering RNA (siRNA) has tremendous potential to silence the expression of disease-causing genes in the clinic. A major hurdle to their widespread clinical use is the safe and efficient delivery to target cells in vivo. Delivery via the oral route is considered the holy grail for small molecule and macromolecular drug delivery as it has the advantages of ease of administration, increased patient compliance, and cost-effectiveness. However, the harsh biological milieu of the acidic stomach and enzyme-rich gastrointestinal tract make efficient delivery of oligonucleotides and siRNA via the oral route difficult. Nonetheless, the first studies on the oral delivery of siRNA in animals and antisense oligonucleotides in humans suggest that significant oral delivery of these nucleic acids can be achieved across the gut wall. This can occur either by encapsulating siRNA within biodegradable particles that protect them from degradation and target them to M cells in intestinal Peyer’s patches or by using chemically stabilized antisense oligonucleotides together with a penetration enhancer. This article reviews these studies as they mark important advances in the delivery of gene silencing nucleic acids and have heralded a new wave of enthusiasm that might lead to a significant expansion of the therapeutic options available for gene silencing drugs in the clinic.     

The cellular delivery of antisense oligonucleotides and ribozymes

The design and development of antisense oligonucleotides and ribozymes for the treatment of diseases arising from genetic abnormalities has become a real possibility over the past few years. Improvements in oligonucleotide chemistry have led to the synthesis of nucleic acids that are relatively stable in the biological milieu. However, advances in cellular targeting and intracellular delivery will probably lead to more widespread clinical applications. This review looks at recent advances in the in vitro and in vivo delivery of antisense oligodeoxynucleotides and ribozymes.     

Nanoparticulate systems for the delivery of antisense oligonucleotides

Antisense oligonucleotides are molecules that are able to inhibit gene expression being therefore potentially active for the treatment of viral infections or cancer. However, because of their poor stability in biological medium and their weak intracellular penetration, colloidal drugs carriers such as nanoparticles were developed for the delivery of oligonucleotides (ODN). ODN associated to nanoparticles were shown to be protected against degradation and to penetrate more easily into different types of cells. As a consequence, nanoparticles were shown to improve the efficiency of ODNs for the inhibition of the proliferation of cells expressing the point mutated Ha-ras gene. In vivo, polyalkylcyanoacrylate (PACA) nanoparticles were able to efficiently distribute the ODNs to the liver whereas the alginate nanosponges could concentrate the ODNs in the lungs. Finally, ODN loaded to PACA nanoparticles were able to improve in mice, the treatment of RAS cells expressing the point mutated Ha-ras gene.     

Intradermal delivery of antisense oligonucleotides by the pulse depolarization iontophoretic system

The intradermal delivery of an antisense oligonucleotide was examined by iontophoresis. In this experiment, the antisense sequence of [(32)P]-labeled phosphodiester oligonucleotide ([(32)P]D-oligo, 18-mer) hybridizing to mouse interleukin 10 (IL-10) mRNA was used as a model D-oligo. In in vitro iontophoretic experiments, isolated hairless mouse skin was used with a horizontal diffusion cell. The enhancing effect of pulse depolarization (PDP) iontophoresis on the [(32)P]D-oligo permeation through the skin was better, and the skin irritation was less, than those of constant direct current (CDC) iontophoresis. The apparent fluxes of [(32)P]D-oligo were enhanced with the increasing current densities and [(32)P]D-oligo concentrations in the donor solution, whereas the enhanced flux decreased with the increasing NaCl concentrations in the donor solution. An optimum electric current was observed for the intradermal delivery of [(32)P]D-oligo, and intact [(32)P]D-oligo was detected within the skin after iontophoresis for 6 h. These results suggest that PDP iontophoresis may be useful for the intradermal delivery of antisense oligonucleotides.     

Increased uptake of antisense oligonucleotides by delivery as double stranded complexes

Antisense oligonucleotides were potentially very powerful tools to modulate gene expression. Progress in chemical modification of oligonucleotides to enhance the strength and stability of interaction, without loosing specificity, has made the antisense strategy very attractive for therapeutic manipulation of the gene expression. However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells. In this study we evaluated a new concept for antisense delivery in cellular systems. We have shown that formation of a duplex between the active oligonucleotide (with a chemically modified backbone) and an easily degradable complementary oligodeoxynucleotide in the presence of Lipofectamine 2000 leads to better intracellular uptake and more significant pharmacological effect of the active oligonucleotide. To evaluate our approach we targeted the MDR1 gene, which coded for P-glycoprotein, a membrane ATPase associated with multi-drug resistance in tumor cells. The 2'-O-methyl gapmer antisense RNA (active component of the duplex) was complementary to a site flanking the AUG of the MDR1 message. Effective inhibition of P-glycoprotein expression was attained with sub-micromolar concentrations of duplexes under serum-replete conditions and was much stronger than with traditional single stranded antisense delivery. The results obtained suggested that double stranded delivery could provide a simple and effective means for enhancing cell uptake of pharmacologically active oligonucleotides.     

Cellular delivery of siRNA and antisense oligonucleotides via receptor-mediated endocytosis

INTRODUCTION: There is great potential for antisense and siRNA oligonucleotides to become mainstream therapeutic entities thanks to their high specificity and wide therapeutic target space compared with small molecules. Despite this potential, the pharmacological targets within the cells are less accessible to oligonucleotides that are hydrophilic and often charged. Oligonucleotides access their intracellular targets mainly by means of endocytosis, but only a fraction of them reach their targets, as delivery requires functional synergy of cellular uptake and intracellular trafficking. AREAS COVERED: This review provides an update on the progress of receptor-targeted delivery of oligonucleotides over the last 15 years and summarizes various targeting moieties for oligonucleotide delivery and coupling strategies. To inspire new strategies that can lead to oligonucleotides in the clinic, this review highlights how oligonucleotides successfully reach their intracellular targets by means of receptor-mediated endocytosis. EXPERT OPINION: Understanding the mechanisms of oligonucleotide internalization has led to greater cellular uptake and superior endosomal release through the rational design of receptor-targeted delivery systems. Further improvements will again depend on a better understanding of the intracellular trafficking of oligonucleotides.     

Cellular delivery of siRNA and antisense oligonucleotides via receptor-mediated endocytosis

Introduction: There is great potential for antisense and siRNA oligonucleotides to become mainstream therapeutic entities thanks to their high specificity and wide therapeutic target space compared with small molecules. Despite this potential, the pharmacological targets within the cells are less accessible to oligonucleotides that are hydrophilic and often charged. Oligonucleotides access their intracellular targets mainly by means of endocytosis, but only a fraction of them reach their targets, as delivery requires functional synergy of cellular uptake and intracellular trafficking.

Areas covered: This review provides an update on the progress of receptor-targeted delivery of oligonucleotides over the last 15 years and summarizes various targeting moieties for oligonucleotide delivery and coupling strategies. To inspire new strategies that can lead to oligonucleotides in the clinic, this review highlights how oligonucleotides successfully reach their intracellular targets by means of receptor-mediated endocytosis.

Expert opinion: Understanding the mechanisms of oligonucleotide internalization has led to greater cellular uptake and superior endosomal release through the rational design of receptor-targeted delivery systems. Further improvements will again depend on a better understanding of the intracellular trafficking of oligonucleotides.     

反义寡核苷酸类化合物的体内转运研究进展

反义寡聚核苷酸已成功用于对基因表达的特异性调控与抑制。但是,大多数此类化合物是呈负电性的,不能有效跨越细胞膜。到目前为止,已发展了数种提高寡聚核苷酸类化合物细胞内转运的方法。现对提高寡聚核苷酸细胞内转运的载体和方法作一较详尽的归纳。     

pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system

The lack of stability in blood and the short blood circulation time of pH-sensitive liposomes are major drawbacks for their application in-vivo. To develop pH-sensitive, serum-stable and long-circulating liposomes as drug delivery systems, the impact of polyethylene glycol-derived phosphatidylethanolamine (DSPE-PEG) on the properties of pH-sensitive liposomes was investigated. pH-sensitive liposomes were prepared with dioleoylphosphatidylethanolamine (DOPE) and oleic acid (DOPE/oleic acid liposome) or DOPE and 1,2-dipalmitoylsuccinylglycerol (DOPE/DPSG liposome). The inclusion of DSPE-PEG enhanced the serum stability of both DOPE/oleic acid and DOPE/DPSG liposomes, but also shifted the pH-response curve of pH-sensitive liposomes to more acidic regions and reduced the maximum leakage percentage. The impact of DSPE-PEG, however, was much lower in the DOPE/DPSG liposomes than in the DOPE/oleic acid liposomes. In tumour tissue homogenates, where the pH is lower than normal healthy tissues, the pH-sensitive DOPE/DPSG liposomes released the entrapped markers rapidly, in comparison with pH-insensitive dipalmitoylphosphatidylcholine/cholesterol/DSPE-PEG liposomes. Moreover, the release rate was not affected by the content of DSPE-PEG. The blood circulation time of methotrexate incorporated in DOPE/UDPSG liposomes was significantly prolonged with increasing content of DSPE-PEG. Taken together, the liposomes composed of DOPE, DPSG and DSPE-PEG (up to 5%) were pH sensitive, plasma stable and had a long circulation time in the blood. The complete destabilization of the liposomes at tumour tissues suggests that the liposomes might be useful for the targeted delivery of drugs such as anticancer agents.     

Enhancement of the transdermal delivery of catechins by liposomes incorporating anionic surfactants and ethanol

The aim of this study was to develop and evaluate liposomal formulations encapsulating tea catechins, which possess antioxidant and chemopreventive activities. Liposomes were characterized for size, zeta potential, and entrapment efficiency. Both in vitro and in vivo skin permeation were examined using nude mouse skin as a model. The results suggested that the liposomal composition plays an important role in affecting the efficiency of transdermal catechin delivery. Incorporation of anionic surfactants such as deoxycholic acid (DA) and dicetyl phosphate (DP) in the liposomes in the presence of 15% ethanol increased the (+)-catechin permeation by five to seven-fold as compared to the control. The flexibility of bilayers is suggested as an important factor governing the enhancing effect of liposomes. Intercellular spaces within the stratum corneum but not shunt routes are the major pathways for catechin delivery from liposomes. (+)-Catechin and (−)-epicatechin are isomers which showed similar encapsulation efficiencies and skin permeation in liposomes. (−)-Epigallocatechin-3-gallate showed the highest encapsulation rate and in vivo skin deposition level in liposomes among all catechins tested. The stability and in vitro tranepidermal water loss test indicated the safety of the practical use of liposomes developed in this study.     

A review of mechanistic insight and application of pH-sensitive liposomes in drug delivery

Abstract

The pH-sensitive liposomes have been extensively used as an alternative to conventional liposomes in effective intracellular delivery of therapeutics/antigen/DNA/diagnostics to various compartments of the target cell. Such liposomes are destabilized under acidic conditions of the endocytotic pathway as they usually contain pH-sensitive lipid components. Therefore, the encapsulated content is delivered into the intracellular bio-environment through destabilization or its fusion with the endosomal membrane. The therapeutic efficacy of pH-sensitive liposomes enables them as biomaterial with commercial utility especially in cancer treatment. In addition, targeting ligands including antibodies can be anchored on the surface of pH-sensitive liposomes to target specific cell surface receptors/antigen present on tumor cells. These vesicles have also been widely explored for antigen delivery and serve as immunological adjuvant to enhance the immune response to antigens. The present review deals with recent research updates on application of pH-sensitive liposomes in chemotherapy/diagnostics/antigen/gene delivery etc.     

Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: benefits over classical vesicles

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes [SPC:CHOL:PEG-750-DSPE (47:47:6 molar% ratio)] or pH-sensitive stealth liposomes [DOPE:CHEMS:CHOL:PEG₇₅₀-DSPE (43:21:30:6 molar% ratio)] were used to deliver a therapeutic peptide to its nuclear site of action. The benefit of using stealth pH-sensitive liposomes was investigated and formulations were compared to classical liposomes in terms of size, shape, charge, encapsulation efficiency, stability and, most importantly, in terms of cellular uptake. Confocal microscopy and flow cytometry were used to evaluate the intracellular fate of liposomes themselves and of their hydrophilic encapsulated material. Cellular uptake of peptide-loaded liposomes was also investigated in three cell lines: Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was investigated as a function of time. Characterization studies showed that both formulations possess acceptable size, shape and encapsulation efficiency but cellular uptake studies showed the important benefit of the pH-sensitive formulation over the classical one, in spite of liposome PEGylation. Indeed, stealth pH-sensitive liposomes were able to deliver hydrophilic materials strongly to the cytoplasm. Most importantly, when encapsulated in pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells.     

pH-sensitive liposomes: mechanism of triggered release to drug and gene delivery prospects

A growing amount of literature describes the development and applications of novel targeting and/or release triggering schemes to improve the therapeutic index of drugs encapsulated within liposomes. The composition of liposomal systems can be modified to facilitate site specific release in response to environmental conditions, namely at the gross anatomical level, at the cellular or sub-cellular level. They are designed in order to release their contents in response to acidic pH within the endosomal system while remaining stable in plasma thus improving the cytoplasmic delivery of various polar materials and macromolecules such as anti-tumor drugs, toxins, proteins and DNA. This review covers various aspect of research on pH-sensitive liposomes.     

反义寡核苷酸阳离子脂质体的制备和体外细胞摄取研究反义寡核苷酸阳离子脂质体的制备和体外细胞摄取研究

目的制备高效促进细胞摄取反义寡核苷酸(ASON)和保护ASON的脂质体。方法以3β［n-(n′,n′-二甲氨基乙基)氨甲酰基-胆固醇(DC-Chol)为类脂成分制备阳离子脂质体(以下简称DC-Chol脂质体),与ASON混合得到载药脂质体,测定载药率。用琼脂糖凝胶电泳分析载药脂质体的结构特点;流式细胞仪检测不同条件下细胞摄取荧光标记ASON的情况;变性聚丙烯酰胺电泳考察DC-Chol脂质体对ASON的保护作用。结果载药率与DC-Chol脂质体和药物的+/-电荷比有关,当+/-电荷比大于2时,载药率达90%以上;琼脂糖凝胶电泳显示ASON同时存在于DC-Chol脂质体的周围和包裹于其内部的两种形式;流式细胞仪测定结果表明,DC-Chol脂质体可明显增加细胞对ASON的摄取,阳性细胞染色率和胞内平均荧光强度均较对照组有明显增加,增加程度主要取决于+/-电荷比例,血清可降低细胞的摄取;变性聚丙烯酰胺电泳证实DC-Chol脂质体具有保护ASON的作用。结论DC-Chol脂质体具有显著增加细胞摄取ASON和保护ASON的作用,有望成为反义类药物的高效传递系统。     

Drug and gene delivery by "bubble liposomes" and ultrasound

Gene therapy has a potentiality for treatment of cancer and diseases from genomic defects. It is important to select a vector which has good potency in terms of gene transduction efficiency, and is safe and easy to apply. Many researchers have attempted to develop an effective gene delivery carrier. Recently, it was reported that microbubbles, which are ultrasound (US) contrast agents, improved the transfection efficiency by cavitation with US exposure. However, microbubbles had problems with stability and targeting ability. To solve these problems, we focused on liposomes that had many advantages such as being stable and safe in vivo and easily modifying targeting ligand. We succeeded in preparing the liposomes ("Bubble liposomes" (BLs)) entrapping perfluoropropane gas which was utilized for contrast enhancement in ultrasonography. In this study, we assessed the feasibility of BLs as gene delivery carrier utilized cavitation by US exposure. BLs could deliver plasmid DNA to various cell types in vitro by combining with US without cytotoxicity. To evaluate the ability of BLs to in vivo gene delivery, we attempted to deliver plasmid DNA into the femoral artery. The gene expression at this artery treated with BLs and US combination was higher than with US only, BLs without US or Lipofectamine 2000. This result suggested that Bubble liposomes could quickly deliver plasmid DNA into the artery even under conditions of short contact time between BLs and the endothelial cells and the existence of the bloodstream and serum. These results suggested that BLs might be a non-invasive and effective carrier for gene delivery.     

Controlled delivery of antisense oligonucleotides: a brief review of current strategies

Antisense therapy has been investigated extensively over the past two decades, either experimentally for gene functional research or clinically as therapeutic agents owing to the conceptual simplicity, ease of design and low cost. The concept of this therapeutic approach is promising because short antisense oligonucleotides (ASOs) can be delivered into target cells for specific hybridisation with target mRNA, resulting in the inhibition of the expression of pathogenic genes. However, the efficient delivery of the ASO molecules into target cells remains challenging; this bottleneck together with several other technical hurdles need to be overcome before this approach becomes effective and widely adopted. A variety of vectors such as lipids, polymers, peptides and nanoparticles have been explored. This review outlines the recent advances of the non-viral ASO delivery strategies. Several recent scientific studies, including authors' contributions, have been selected to highlight the technical aspects of ASO delivery.