PASOS PARA LA ELABORACIÓN DE TRUCHA AHUMADA

The second main method of loading cargo into EVs is ex vitro. In method, EVs are first isolated from cell media by a variety of techniques. The current most widely used method, because of its lack of expense and time, is ultracentrifugation. Other methods include sucrose cushion density grade ultracentrifugation, filtration, size exclusion chromatography and chemical commercial methods which are sold as kits and usually involve precipitating out the EVs. Sucrose cushion density grade ultracentrifugation is considered the most clinically clean method, enriching samples in exosomes rather than other EVs. Once the EVs have been isolated and characterised, they can be stored at -80 °C over long periods with minimal changes to vesicle size and number, at -20 °C with small structural changes or at 4°C or room temperature overnight without loss of function (Lőrincz et al., 2014). However, it is recommended that fresh isolations are used whenever possible to reduce the risk of losing function or the characteristics of EVs changing. External loading of the EVs can now be attempted by several methodologies.

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1.14.1 Electroporation

Electroporation is one such method where EVs are suspended in fluid between two conducting plates (figure 1-16) and an electrical potential applied. The electrical potential, in an attempt to conduct through the fluid and to the opposite conducting plate, presumably punches a hole in the outer membrane allowing the movement of particles into the EV. If the concentration of cargo around the EVs at the time of electroporation is high, then theoretically a sample of the cargo should enter the EV. Once the EVs have resealed some of them should contain cargo. Due to the nature of electroporation there will likely be a proportion of EVs that do not reseal or a population of EVs that have increased in size due to fusion. There has been a high profile study claiming the successful integration of siRNA by electroporation into EVs (Alvarez-Erviti et al., 2011). In contrast to the claims of success, there has been a study published looking at the effects of electroporation on siRNA which indicates that siRNA aggregates to form large particles rather than be loaded into EVs (Kooijmans et

al., 2013). Whilst a study preventing the aggregation of EVs during electroporation has been published

(Hood, Scott, & Wickline, 2013), there is yet to be an answer to preventing siRNA aggregation.

Figure 1-16: diagram demonstrating the action of electroporation. The sample is placed in a cuvette with 2 metal plates on opposite sides. These plates allow the conductance of an electrical potential into the sample fluid where the potential causes the formation of holes in the vesicles. The cargo (being in a high concentration) should naturally diffuse into the vesicle and become encapsulated.

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1.14.2 Sonication

A second method for ex vitro loading of EVs is sonication. Sonication is perhaps even more destructive than electroporation, however more success has been seen with this method. During sonication, the sample is once again suspended in a small volume of fluid with a high concentration of the cargo of choice. A probe is used to transmit ultrasound energy waves and focus them on a point in the fluid (figure 1-17). Microbubbles are formed during the energy transfer which both expand and collapse causing extremely high pressures and temperatures inside of the microbubbles (Gong, 1998). These extreme conditions cause vapour to be formed inside the microbubbles and the release of free radicals to occur for use in chemical synthesis reactions (Gong, 1998). However, for cargo loading, interest lies not in the free radicals but the cavitation to damage and tear open the EVs. Theoretically if EVs can be sonicated at a low enough energy transfer level then minimal damage can be applied to the EVs, also encouraging the formation of inlets in the EV structure. This is very similar to the method of loading during electroporation where the concentration gradient encourages the movement of cargo into the EV before the EV reforms. Sonication has been successfully reported to load both nanoparticles and EVs (Haney et al., 2015; Sharma, Yusuf, & Pathak, 2014). Haney et al. loaded EVs via several methods of which sonication was observed to be the most effective (Haney et al., 2015). They

Figure 1-17: diagram showing the action of sonication and how it disrupts vesicles. Energy waves are emitted from the probe in the liquid. This causes cavitation to occur which disrupts the vesicles. Also in the liquid is a high concentration of cargo which should naturally transfer into the vesicles before they repair and reform.

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loaded a large protein called catalase therefore a similar method can be used to load cargo of the same size or smaller.

1.14.3 Cell Penetrating Peptides

Other methods that could be of potential use loading EVs ex vitro include cell penetrating peptides (CPPs) (figure 1-18). CPPs are a family of natural or synthetic proteins which contain domains that allow passage of cargo proteins through the PM. CPPs can be cationic, amphipathic or hydrophobic molecules capable of binding cargoes and transporting them to the inside of cells (Mussbach, Franke, Zoch, Schaefer, & Reissmann, 2011). Cationic peptides rely on their high positive charge to associate with the anionic cell membrane and glycocalix. CPPs can bind either covalently or non-covalently with their cargo. The advantage of using non-covalent bonds over covalent bonds is the lack of change in the cargoes structure or sequence upon binding. Using a non-covalent bond also means that the cargo can theoretically dissociate from the CPP and be released into the cell unchanged. This is particularly useful if the CPP used incorporates itself into the PM. Whilst some CPPs rely on a covalent bond formation with their cargo, the CPPs that will be discussed in this research were used to form non-covalent bonds. This is because the cargo destination is the nucleus. Studies have also shown that depending on the CPP used and method of penetration into the cell, that CPPs can incite the PM repair response therefore preventing any residual damage (Palm-Apergi, Lorents, Padari, Pooga, & Hällbrink, 2009). With many of the older generation CPPs it was found that they struggled with delivery of nucleic acid cargoes to the nucleus. The incorporation of Nuclear Localisation Sequences (NLS) into CPPs has shown promising results with linear DNA (Cartier & Reszka, 2002). A further challenge was also seen in the delivery of nucleic acids in general as they are anionic,

Figure 1-18: diagram showing the process of CPP complex formation and delivery of cargo into the cell or vesicle. The CPP initially interacts with the cargo through ionic bonds due to the difference in charge between the cargo and CPP. The complex then interacts ionically with the membrane of a cell or vesicle but does not bind to a receptor. The CPP, using its structure, charge and hydrophobicity disrupts the membrane through which the cargo is delivered.

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the same as the PM. However, this has been overcome by synthesising highly cationic CPPs such as CADY. There have only been a couple of studies that looked at the toxicity of CPPs. CPP cytotoxicity is caused by perturbation of the PM where the cell does not repair or is not able to repair quickly the disturbance in the PM where the CPP has bound. Saar and colleagues found that MAP and Transportan-10, out of the selection of CPPs that they tested for cytotoxicity, were the most cytotoxic (Saar et al., 2005). However, there are uses for cytotoxic CPPs such as treating cancerous cells. Many CPPs have been synthesised, here I will talk about the three concerned in this research; MPGα, CADY and JBS Nucleoducin.

MPGα is an amphipathic peptide. It is a chimeric protein made from a combination of the fusion sequence of HIV gp41 and the NLS of SV40 T-antigen (M. C. Morris, Vidal, Chaloin, Heitz, & Divita, 1997). As such it is able to deliver oligonucleotides into cells. As mentioned above, MPGα is a non-covalent linking CPP which gives it the advantage of not having to go through a chemical bonding process prior to use. Simeoni and colleagues have shown in their study that the MPGα action of penetration is endocytosis independent. This is because MPGα can deliver siRNA to the nucleus when endosomal pathway inhibitors are co-administered (Simeoni, Morris, Heitz, & Divita, 2003). Thus there is less chance of cargo being degraded or sent to the wrong organelle. Furthermore, upon binding siRNA as a cargo, MPGα changes its structure to a more stable helical form (Konate et al., 2010). MPGα has shown success in embryonic stem cell studies (Zeineddine et al., 2006).

CADY, similar to MPGα, is also an amphipathic molecule. It is able to spontaneously insert into the membrane and undergoes a helical conformation change upon interaction with siRNA cargo. The helical conformation allows the exposure of certain residues to form three domains; tryptophan, charged residues and hydrophobic. It has been found that CADY is one of the most hydrophobic and therefore most stable CPPs (Keller et al., 2013). This means that in an aqueous environment it will readily bind itself to the PM and reduce the time it takes for the CPP to come into initial contact with the cell. CADY is a synthetic peptide made from a combination of PPTG1 peptide, Tryptophan and charged amino acids (Laurence Crombez et al., 2009). Incubation times as short as 30 minutes were seen for CADY uptake with an siRNA cargo where target knockdown was successful (Laurence Crombez

et al., 2009).

JBS-Nucleoducin is a cocktail of CPPs rather than a homogenous population. This allows for coverage of different cargoes and allows multiple cell type delivery. Whilst the composition of CPP cocktails remains a secret with the manufacturer, they are usually either hydrophilic or hydrophobic.

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