Influence of short term storage conditions, concentration methodsand excipients on extracellular vesicle recovery and function.

Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage. Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay. Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and -80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.

Applications of cell-penetrating peptides in regulation of gene expression.

CPPs (cell-penetrating peptides) can be defined as short peptides that are able to efficiently penetrate cellular lipid bilayers. Because of this remarkable feature, they are excellent candidates regarding alterations in gene expression. CPPs have been utilized in in vivo and in vitro experiments as delivery vectors for different bioactive cargoes. This review focuses on the experiments performed in recent years where CPPs have been used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA (small interfering RNA) and decoy dsDNA (double-stranded DNA) applications, and as transfection agents for plasmid delivery.

Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.

Cell-penetrating peptides: mechanisms and applications.

A major obstacle in the development of new therapeutic agents is the low bioavailability of hydrophilic substances. Drugs that bind to intracellular targets must penetrate the lipid bilayer surrounding the cell in order to exert their effect. A relatively new research area that addresses this problem by introducing novel transport peptides that facilitate the cellular penetration of potential drugs has emerged. These peptides predominantly have a positive net charge and/or an amphipathic nature, but can otherwise have very different characteristics. This group of peptides, although sometimes called protein transduction domains (PTDs), is here referred to as cell-penetrating peptides (CPPs). For many years it was believed that these peptides were internalized into cells via a non-endocytotic, receptor-independent pathway. However, recent publications have suggested that an endocytotic pathway cannot be ruled out, and that earlier results might be based on artifacts associated with fixation of cells and membrane association of peptides. Although the mechanism of cellular uptake remains unclear, there is an increasing amount of reports on biological effects of CPPs and their cargos. Thus, CPPs are an attractive pharmaceutical and biochemical tool that needs more attention. This review will discuss some recent results in this research field with focus on the cell-penetrating peptide transportan.

TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein.

One approach to investigate gene function, by silencing the activity of certain proteins, is the usage of double stranded decoy oligodeoxynucleotides (ds decoy ODNs). Decoy, in this sense, is ds ODNs bearing the consensus binding sequence for a DNA-binding protein. This can be used in clinical settings to attenuate the effect of overexpressed transcription factors in tumor cells. We here choose to target the oncogenic protein Myc. Since oligonucleotides are poorly internalized to cells, a cell-penetrating peptide, TP10, was coupled to the Myc decoy, using two different strategies. Either TP10 was simply mixed with ds decoy ODNs forming complexes through non-covalent electrostatic interactions, or by having a nona-nucleotide overhang in one of the decoy strands, and adding a complementary PNA sequence coupled to an NLS sequence and TP10, which could hybridize to the Myc decoy. By using these strategies, uptake was significantly enhanced, especially with the co-incubation approach. Interestingly, various endocytosis inhibitors had no effect on the uptake pattern, suggesting that uptake of these complexes is not mediated via endocytosis. Finally, a decreased proliferative capacity was observed when treating the neuroblastoma cell line N2a with TP10-PNA conjugate hybridized to Myc decoy compared to naked Myc decoy and untreated cells. A dose-dependent decrease in proliferation was also observed in MCF-7 cells, when using both strategies. These results suggest an alternative way to efficiently deliver ds ODNs into cells using the cell-penetrating peptide TP10 and prevent tumor growth by targeting the oncogenic protein Myc.

[Heterogeneity of ciguatoxins extracted from fish caught in the French Antilles]. / Hétérogénéité des ciguatoxines extraites de poissons péchés aux Antilles françaises.

Ciguatoxin-like substances were extracted from the viscera or the flesh of eight Caribbean fish species, including small invertebrate feeders and large carnivores. The had similar properties, i.e. pharmacological action, solubility, chromatographic behaviour on silicic acid or Sephadex LH 20 column, stability in a weak acid solution and instability in alkaline medium. However, Florisil column and thin-layer chromatography showed different ciguatoxins whose number depended on tissue or species but not on fish trophic level. Less polar ciguatoxins appeared in salted and dried flesh. Thus, fish ciguatoxins are believed to be closely related substances, possibly changing in structure according to particular experimental conditions.

[Evaluation of different-stage levels of ciguatera toxicity of the marine food fish chain found around Saint Barthélemy Island in French Antilles]. / Evaluation des niveaux de toxicité ciguatérique des différents étages de la chaîne trophique pisciaire marine présente autour de l'île de Saint-Barthélemy aux Antilles Françaises.

A total of 259 specimens of 32 potentially ciguatoxic fish species from St-Barthelemy, Leeward Islands in the Caribbean sea were checked for ciguatoxin. It was found that flesh and viscera from 229 specimens belonging to 27 species (essentially bottom-dwelling species) were ciguatoxic. No correlation between toxicity and species size or diet was demonstrated since five of the six highly ciguatoxic species Seriola dumerili, Caranx latus, Caranx bartholomaei, Caranx ruber and Scomberomorus cavalla are large roving predators feeding mainly on fishes while the last species (Alphestes afer) is a benthic resident of very small size (less than 300 g) which feeds mainly on invertebrates. Small (less than 1 kg) benthic carnivorous fishes (invertebrate feeders), such as Bodianus rufus, Priacanthus arenatus, Mulloidichtys martinicus, Malacanthus plumieri and Halichoeres radiatus, are significantly ciguatoxic and play an important role in the transmission of ciguatoxin in the food chain. Other ciguatoxic species included large carnivorous fishes and the small surgeon Acanthurus bahianus. Furthermore, additional results suggest that most of edible benthic species from Saint-Barthelemy harbour ciguatoxin as well, though at an extremely low level. The ciguatera food chain in the Caribbean sea is therefore comparable with that described in the Pacific Ocean, except concerning small invertebrate feeders since their role in this chain is demonstrated for the first time.

A study of the distribution of ciguatoxin in individual Caribbean fish.

Ciguatera toxins were extracted from the tissues of 36 poisonous fishes including 9 dangerous species collected in the Caribbean. Toxicity assays were carried out in mice and the distinctive symptoms of ciguatera poisoning were observed. In a single fish, ciguatoxin was found in the blood, flesh, gonads, gills, heart, skin and bones. The concentration was highest in the viscera and in particular in the liver, kidney and spleen. The ratios of the toxin concentrations of the liver or viscera to that of the flesh were high and varied with the species suggesting that the toxin is stored in different ways in different fish. Subcellular fractionation of liver cells revealed that most of the ciguatoxin was attached to cytoplasmic proteins and that some toxin was probably bound to the membranes.