Prospects and challenges of extracellular vesicle-based drug delivery system: considering cell source

Figures & data

Figure 1. Scheme of biogenesis of three types of extracellular vesicles (exosomes, microvesicles, and apoptotic bodies) and component of exosome. Exosomes are cell secreted vesicles of ∼100 nm in size and packed with a variety of cellular components including mRNAs, miRNAs, proteins, enzymes, lipids, carbohydrates, etc. The exosome surface is decorated with various membrane proteins responsible for different pathophysiological functions.

Figure 2. Scheme of the potential of EVs in disease treatment and drug delivery. EVs can be isolated from different ‘factories’ (dendritic cells, mesenchymal stem cells, macrophages, milk, tumor cells, others), loading different cargos (small molecules, nucleic acids, protein, metal nanoparticles), and targeting to precise disease (cardiovascular disease, neurodegenerative disease, osteoporosis, cancer, malignancies, and metastasis).

Table 1. Summary of isolation and purification method of EVs.

Isolation method	Principle of isolation	Characteristic	Grade of isolation efficiency
Ultracentrifugation-based isolation techniques	Density, size, and shape based sequential separations of particulate constituents and solutes	Large sample capacity and yielding of large amounts of exosomes, but high equipment cost, cumbersome, long run time and high speed centrifugation may damage exosomes	Low recovery and high specificity
Size-based isolation techniques	Size difference between exosomes and other particulate constituents	Low equipment cost and fast but shear stress induced deterioration and exosomes loss due to attaching to the filter membranes	Intermediate recovery and intermediate specificity
Immunoaffinity capture- based techniques	Specific interaction between membrane-bound antigens (receptors) of exosomes and immobilized antibodies (ligands)	Suitable for the isolation of specific exosomes with high specificity, but high reagent cost, exosome tags need to be established, low sample capacity and low yields	Low recovery and high specificity
Precipitation	Altering the solubility or dispersibility or exosomes by the use of water-excluding polymers	Easy to use, no need for special equipment, high sample capacity, but low specificity and co-precipitation of other non-exosomal contaminants like proteins and polymeric materials	High recovery and low specificity
Microfluidics-based isolation techniques	A variety of properties of exosomes like immunoaffinity, size, and density	Fast, low cost, portable, easy automation and integration, high portability, but low in sample capacity and no isolation standard	Low recovery and high specificity

Table 2. Summary of drug loading technique of EVs.

Classification	Loading method	Type of cargo	Characteristic	Loading efficiency
Pre-loading method	Transfection	miRNA (Ohno et al., 2013), siRNA (Steinman, 2012), protein (Limoni et al., 2019)	Widely used but uncontrollable in quantity of cargo loading	Low loading efficiency
	Co-incubation	Paclitaxel (Merchant et al., 2017) carboplatin and etoposide (Akao et al., 2011)	Easy to operate but drugs may be cytotoxic to cells	Low loading efficiency
	Activities	miRNA (Pascucci et al., 2014)	Easy to operate but only applicable to specific cells	Low loading efficient
Post-loading method	Co-incubation	Curcumin (Street et al., 2017) hsiRNA (Seo et al., 2018), porphyrins (Lee et al., 2015), catalase (Li et al., 2019c)	A simplest way but uncontrollable in quantity of cargo loading	Low loading efficiency
	Electroporation	SiRNA (Steinman, 2012), TMP (Lee et al., 2015), DOX (Kantoff et al., 2010)	Superior loading of siRNA over chemical transfection but disrupting integrity of exosomes	Medium loading efficiency
	Sonication	PTX (Cheng et al., 2017), catalase (Li et al., 2019c), small RNAs (Sun et al., 2010)	High loading efficiency but not efficient for hydrophobic drugs	High loading efficiency
	Extrusion	Porphyrins (Lee et al., 2015), catalase (Li et al., 2019c)	High drug loading efficiency but potential deformation of membrane	High loading efficiency
	Freeze/thaw cycle	Catalase (Li et al., 2019c), prepare hybrid exosomes (Li et al., 2019d)	Exosomes may aggregate and the drugs loading efficiency is low	Low loading efficiency
	Saponin-assisted loading	Catalase (Li et al., 2019c), hydrophilic molecules (Lee et al., 2015)	High drug loading efficiency but generates pores in exosomes hemolysis/toxicity concerns	High loading efficiency

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