As an exosome service company, we provide customized services to engineer and load exosomes with proteins or acid nucleics based on methods described in the litterature like sonication, extrusion or electoporation. We are also developing our own patented technology to load exosomes by turbuloporation.
Exosome engineering methods
Several studies have reported different strategies to load cargos into exosomes. The cargos that have already been used are drugs, proteins, nucleic acids, or nanomaterials (Fu et al. 2020). Exosomes can load molecules endogenously during their biogenesis or exogenously after isolation of EVs (Elsharkasy et al. 2020). The strategy to load can be direct or indirect. An indirect method means to load the cargo into a donor cell which will release exosomes containing the cargos. Techniques to load indirectly are classically incubation and transfection.
Transfection is a technique to load proteins, or nucleic acids into exosomes. Transfection reagents allow the transduction of specific plasmids into cells that will induce the expression of the desired cargos, that are loaded into ILVs and released as cargo loaded exosomes (Fu et al. 2020). However, this technique shows a low loading efficiency due to the difficulty to control cargo transport intracellularly (Li et al. 2019).
A direct loading is a non-cell-based approach where the molecule is directly load into the exosomes (Han et al. 2021). The different loading technologies can be categorized into two subtypes: the passive and the active loading.
Incubation or passive loading for engineering
The simplest way to load cargos into exosomes is to incubate the cargo with exosomes or with exosome-secreting cells. By concentration gradient, the cargo diffuses into the exosome or cell (Fu et al. 2020). Since exosomes have a lipid bilayer, their hydrophobicity promotes the loading of hydrophobic cargos such as curcumin (Oskouie et al. 2019). For exosome and cargo incubation, the incubation occurs at a determined temperature during a certain time, then unloaded cargos are removed by enzymes, centrifugation, or others purification methods to keep cargo-loaded exosome (Fu et al. 2020). Many studies have already experimented the incubation to load anti-cancer drugs such as paclitaxel with a loading efficiency of 9.2 % (Saari et al. 2015) or doxorubicin with a loading efficiency of 13 % (Goh et al. 2017).
While incubation seems to be the simplest technique and harmless for exosome integrity, the loading efficiency remains low (Fu et al. 2020).
The most successful techniques found in the literature to load cargos directly into exosomes are physical treatments. They induce transitory pores on the membrane of exosomes or a recombination of the membrane which increases the chances for the cargo to enter (Fu et al. 2020).
The use of surfactant reagents such as saponin or triton enable to dissolve the lipids from the exosome membrane. The permeabilization of the membrane helps cargo loading (Fu et al. 2020; Gangadaran and Ahn 2020). A study showed that saponification enables a loading efficiency of porphyrin 11 times higher than a passive loading (incubation) (Fuhrmann et al. 2015). Moreover, in 2017, Goh et al. obtained a 50 % loading efficiency for doxorubicin loading by saponification (Goh et al. 2017). However, some consider that surfactants may alter the integrity of exosomes and the cargos, affecting their potential therapeutical properties (Fu et al. 2020).
Placing exosomes into a hypotonic solution leads to the swelling of exosomes and the creation of pores on their membrane by osmotic pressure. This membrane permeabilization helps to load cargos into exosomes. The loaded exosomes are then placed into an isotonic solution to allow the recovery of their structure (Sutaria et al. 2017). One study explored the loading of porphyrin through hypotonic dialysis and showed a significant loading efficiency compared to incubation (Fuhrmann et al. 2015). Nevertheless, to apply osmotic pressure to exosomes seems to alter the size and charge of exosomes, affecting the cellular uptake of exosomes afterwards (Sutaria et al. 2017).
This method is an alternance of freezing in liquid nitrogen or at -80 °C and thawing (generally at room temperature) in order to disrupt exosome membrane. Commonly, 3 cycles are realized to effectively alter the lipid bilayer and allow the drug to enter (Jin Wang, Chen, and Ho 2021). Several studies have reported a loading efficiency between 10 and 30 % using freeze-thaw cycles (Goh et al. 2017; Izadpanah et al. 2020). Although freeze-thaw treatment seems to be a good technique to load cargos into exosomes, it has been reported that alternating low and high temperatures rapidly, leads to an aggregation of exosomes (Haney et al. 2015).
Sonication is a technique traditionally used to disaggregate exosomes (Nizamudeen et al. 2021). By applying a mechanical shear force in the form of an acoustic wave, exosome membrane is weakened, and this could promote cargo loading (Fu et al. 2020). Thus, researchers investigate this method to load molecules into exosomes. Several studies pointed out loading of catalase, or paclitaxel with loading efficiency of respectively 26.10 %, and 28 % (Haney et al. 2015; M. S. Kim et al. 2016). However recently, Nizamudeen et al. suggested that sonication alters size, membrane integrity and cellular uptake of exosomes (Nizamudeen et al. 2021).
Electroporation is a technique using an electrical field in the form of short high-voltage pulses to generate transitory micro pores at the surface of exosomes (Fu et al. 2020; Joshi, Ortiz, and Zuhorn 2021). A lot of studies experimented electroporation loading of doxorubicin with loading efficiency inferior to 20 % (Schindler et al. 2019; Takenaka et al. 2019). On the other hand, electroporation loading of miRNA shows better yield from 30 to 60 % (Liang et al. 2020; Pomatto et al. 2019). Electroporation is the only method that have been tested to load plasmids into exosomes. Two studies showed opposite results: while Lin et al. obtained 20 % of loaded plasmid (Lin et al. 2020), S. M. Kim et al. obtained only 1.75 % of loading efficiency (S. M. Kim et al. 2017) although the voltage was the same (1,000 V) but the read-out was different. Finally, as electroporation shows very high loading yields the disparity between studies suggests further exploration of the parameters and the methods to assess loading efficiency. Plus, some researchers highlighted the potential of electroporation to induce exosomes and cargos aggregation that should be further investigated (Liang et al. 2020).
Extrusion consists in forcing exosomes to pass through a membrane with small pores to induce deconstruction and reconstruction of the membrane (Fu et al. 2020; Jin Wang, Chen, and Ho 2021). Studies showed high loading efficiency using extrusion to load catalase for example (22.2 %) (Haney et al. 2015). With high potentials of extrusion as a loading technique, it comes with a risk of altering the immune properties of exosomes. Modifying the surface of their membrane could expose them to immune cells in the organism and prevent from cellular uptake (Fu et al. 2020).
Concerning the loading, it is necessary to be particularly precise on the readout method. Actually, many studies in the literature did not distinguish whether the cargo is loaded inside in the EVs, embedded in the membrane or stuck on the surface of the EV. For instance, some studies evaluate the loading efficiency by measuring the amount of cargo before loading, the free cargo after ultracentrifugation and deducing that the difference is encapsulated into the EVs (Liang et al. 2020). Loading efficiency become thereby an estimation and not an accurate measure. We favor methods based on enzymatic digestions at EVerZom in order to obtain a correct assessment of the loading. As an example, we destroy
the free nucleic acids after plasmid-DNA, or mRNA loading by using a DNase or RNase treatment respectively. Only the encapsulated cargos remain protected. We block then the enzyme-activity, extract the loaded
nucleic acid cargos from the EVs and perform a RT-qPCR or qPCR. These quantitative approaches allow us to calculate the real loading capacity (i.e. Number of molecules loaded per vesicle) and loading efficiency (i.e. percentage of cargos loaded on the total amount of cargo to be loaded in the process).