Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how oligonucleotides function within living organisms is still rather limited. A major concern in ...this regard is the mechanisms of cellular uptake and intracellular trafficking of both ‘free’ oligonucleotides and oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand oligonucleotide delivery. In particular we discuss opportunities for escape of oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance oligonucleotide effects.
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Short DNA or RNA oligonucleotides have tremendous potential as therapeutic agents. Because of their ability to engage in Watson-Crick base pairing, they can interact with mRNA or pre-mRNA targets ...with high selectivity. As a result, they could precisely manipulate gene expression. This possibility has engendered extensive efforts to develop oligonucleotides as drugs, and many candidates are already in clinical trials. However, a major impediment to the maturation of this field of oligonucleotide-based therapeutics remains: these relatively large and often highly charged molecules don't easily cross cellular membranes, making it difficult for them to reach their sites of action in the cytosol or nucleus. In this Account, we summarize some basic features of the biology of antisense and siRNA oligonucleotides. We then discuss chemical conjugation as an approach to improving the intracellular delivery and therapeutic potential of these agents. Instead of focusing on the details of conjugation chemistry, we emphasize the pharmacological ramifications of oligonucleotide conjugates. In one important approach to improving delivery and efficacy, researchers have conjugated oligonucleotides with ligands designed to bind to particular receptors and thus provide specific interactions with cells. In another strategy, researchers have coupled antisense or siRNA with agents such as cell penetrating peptides that are designed to provoke escape of the conjugate from intracellular vesicular compartments. Although both of these strategies have had some success, further research is needed before oligonucleotide conjugates can find an important place in human therapeutics.
We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by ...doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)β(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)β(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.
Cellular interactions with the extracellular matrix and with neighboring cells profoundly influence a variety of signaling events including those involved in mitogenesis, survival, and ...differentiation. Recent advances have provided insights into mechanisms underlying the ability of integrins, cadherins, selectins, and other cell adhesion molecules to regulate signal transduction cascades. These mechanisms often involve the ability of cell adhesion molecules to initiate the formation of organized structures or scaffolds that permit the efficient flow of information in signaling pathways.
One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of ...cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed.
The objectives were to study the diversity of Enterococcus spp. isolated from mastitis cases, milking equipment and the environment of dairy cows; and to determine in vitro resistance of isolates to ...antimicrobials that are relevant to human and animal health. Ten dairy farms were visited to collect samples from mastitis cases, faeces, bedding, aisles, water and milking equipment. Identification of Enterococcus at the species level and antimicrobial resistance testing was performed by MALDI‐TOF and the disk‐diffusion method, respectively. Of 365 isolates, Enterococcus hirae was the most prevalent, being more likely to be isolated from faeces than from milk (odds ratio (OR) = 39·2), liners (OR = 5·4) or bedding (OR = 2·2). Enterococcus saccharolyticus was the most prevalent in milk samples. The chances of isolating Enterococcus faecalis from milk were higher than from aisles (OR = 12·5), faeces (OR = 5·3), bedding (OR = 3·6) or liners (OR = 3·0). The odds of isolating Enterococcus faecium from faeces were higher than from liners (OR = 7·3), bedding (OR = 2·5) or aisles (OR = 2·4). Of 360 tested isolates, 1·9, 0·3 and 0·6% were resistant to penicillin, vancomycin and teicoplanin, respectively. Our results suggest that Enterococcus species can occupy specific ecological niches on dairy farms and pose a risk to public and animal health.
Significance and Impact of the Study: Enterococcus spp. can cause diseases in both humans and animals and are widely found in the environment. Results of this study show that the distribution of Enterococcus species was not similar between farm sources, such as mastitis cases, milking equipment and the environment of the cows, suggesting that species can occupy specific ecological niches. We also observed isolates that were resistant to antimicrobials that are relevant to animal and human health. Thus, this study highlights the risk of Enterococcus for animal and public health and the need for epidemiological monitoring of such pathogens on dairy farms.
Sarcopenia, cachexia, and atrophy due to inactivity and disease states are characterized by a loss of skeletal muscle mass, often accompanied by reduced levels of anabolic hormones (e.g. ...testosterone). These conditions are associated with an increase in mortality, hospitalization and worsening in quality of life. Both physical exercise (EX) and anabolic–androgenic steroid (AAS) administration can improve the prognosis of patients as they increase physical functionality. However, there is a gap in the literature as to the impact of these therapies on the gains in strength and muscle mass and their implications for patient safety. Accordingly, we performed a random-effects meta-analysis to elucidate the effects of AAS and/or EX interventions on lean body mass (LBM) and muscle strength in conditions involving muscle loss. A systematic search for relevant clinical trials was conducted in MEDLINE, EMBASE, SCOPUS, Web of Science, and SPORTDiscus. Comparisons included AAS
vs.
Control, EX
vs.
Control, AAS
vs.
EX, AAS + EX
vs.
AAS and AAS + EX
vs.
EX. A total of 1114 individuals were analyzed. AAS increased LBM (effect size ES: 0.46; 95% CI: 0.25, 0.68,
P
= 0.00) and muscle strength (ES: 0.31; 95% CI: 0.08, 0.53,
P
= 0.01) when compared to a control group. EX promoted an increase in muscular strength (ES: 0.89; 95% CI: 0.53, 1.25,
P
= 0.00), with no effect on LBM when compared to the control group (ES: 0.15; 95% CI: -0.07, 0.38,
P
= 0.17). AAS did not demonstrate statistically significant differences when compared to EX for LBM and muscle strength. The combination of EX + AAS promoted a greater increase in LBM and muscular strength when compared to AAS or EX in isolation. Qualitatively, AAS administration had relatively few side effects. Significant heterogeneity was found in some analyses, which may be explained by the use of different AAS types and EX protocols. Our findings suggest that AAS administration in cachectic and sarcopenic conditions may be a viable interventional strategy to enhance muscle function when exercise is not a possible approach. Moreover, combining AAS with exercise may enhance positive outcomes in this population.
The problem of targeted delivery of antisense and siRNA oligonucleotides can be resolved into two distinct aspects. The first concerns devising ligand-oligonucleotide or ligand-carrier moieties that ...bind with high selectivity to receptors on the cell type of interest and that are efficiently internalized by endocytosis. The second concerns releasing oligonucleotides from pharmacologically inert endomembrane compartments so that they can access RNA in the cytosol or nucleus. In this review, we will address both of these aspects. Thus, we present information on three important receptor families, the integrins, the receptor tyrosine kinases, and the G protein-coupled receptors in terms of their suitability for targeted delivery of oligonucleotides. This includes discussion of receptor abundance, internalization and trafficking pathways, and the availability of suitable high affinity ligands. We also consider the process of oligonucleotide uptake and intracellular trafficking and discuss approaches to modulating these processes in a pharmacologically productive manner. Hopefully, the basic information presented in this review will be of value to investigators involved in designing delivery approaches for oligonucleotides.
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