Extracellular vesicles-based drug delivery system for cancer treatment

Figures & data

Table 1. Therapeutic potential of EVs derived from various donor cells

Donor Cell	Cargo	Purpose	References
Prokaryotes
Gram-positive bacteria
Bacillus anthracis	Naturally toxin-carrying EVs	Vaccination	(Rivera et al., 2010)
Streptococcus pneumoniae	Naturally toxin-carrying EVs	Vaccination	(Olaya-Abril et al., 2014)
Gram-negative bacteria
Neisseria meningitides	Naturally toxin-carrying EVs	Vaccination	(van der Pol et al., 2015)
Escherichia coli	Naturally toxin-carrying EVs	Vaccination	(van der Pol et al., 2015)
Eukaryotes
Protists
Dictyostelium discoideum	Hypericin	Anti-tumour	(Lavialle et al., 2009)
Animals
1. RBC	1a. Protoporphyrin IX	1a. Photodynamic therapy	(L. Y. Wang et al., 2013)
	1b. Ultrasmall superparamagnetic iron oxide	1b. MRI	(Chang et al., 2010)
2. Human milk	2. Naturally immune relevant proteins-carrying EVs	2. Immune responses	(Admyre et al., 2007; Alsaweed et al., 2016)
3. Bovine milk	3a. Withaferin A, bilberry-derived anthocyanidins, curcumin, paclitaxel, and docetaxel	3a. Cancer therapy	(Munagala et al., 2016)
	3b. Naturally bioactive TGF-β-containing EVs	3b. Immunoregulator	(Pieters et al., 2015)
4. Mesenchymal cells	4a. Paclitaxel	4a. Anti-tumour	(Pascucci et al., 2014)
	4b. Renilla luciferase (Rluc)-expressing EVs	4b. Anti-tumour	(Kalimuthu et al., 2016)
5. Tumour cells	5a. Topotecan	5a. Anti-tumour immunity	(Kitai et al., 2017)
	5b. Unmodified TC-EVs	5b. Anti-tumour	(Smyth et al., 2015)
	5c. Doxorubicin, methotrexate, hydroxyl camptothecin, and cisplatin	5c. Anti-tumour	(Tang et al., 2012)
6. Dendritic cells	6a. Doxorubicin-loaded EVs carrying lamp2b fused to αv integrin-specific iRGD peptide	6a. Targeted tumour therapy	(Tian et al., 2014)
	6b. Unmodified autologous DC-EVs	6b. Vaccination of metastatic melanoma patients	(Escudier et al., 2005)
Plants
1. Grapefruit	1. Methotrexate	1. Targeted drug delivery to intestinal macrophages	(B. Wang et al., 2014)
2. Grape extract	2. Unmodified grape-EVs	2. Prevent oral mucositis associated with chemoradiation treatment of head and neck cancer	https://clinicaltirals.govct2/show/NCT0166849

Figure 1. Endogenous and exogenous loading strategies to obtain drug-loaded EVs.

Figure 2. Generation of SPIONs containing EVs (Yuana Y, unpublished results). A. Exogenous loading were performed by growing cells in medium containing SPION and stimulating by sonoporation. B. EVs were harvested from conditioned medium containing SPIONs. C. Negative control EVs from cells grown without SPIONs and stimulated by sonoporation. Scale bars are 100 nm.

Figure 3. CFSE-containing cells stimulated with calcium ionophore (CaI) released EVs with CFSE (Yuana Y, unpublished results). A. 0.5 µM CFDA-SE were used to label cell using diffusion method. The CFDA-SE was cleaved by intracellular esterase enzymes to form an amine-reactive product, CFSE which can be detected by fluorescent microscopy. B. Negative control image (non-labelled cells). C. CFSE-labelled cells treated with 10 µM CaI have comparable CFSE signal to untreated CFSE-labelled cells as detected by flow cytometry. D. Detection of CD9-positive EVs using flow cytometry captured by CD9-antibody (AB) conjugated beads and stained with Alexa Fluor 647-tagged anti-CD9 AB. E. Detection of CD63-positive EVs using flow cytometry captured by CD63-antibody (AB) conjugated beads and stained with Alexa Fluor 647-tagged anti-CD63 AB. EVs were stained positive for CD9 or CD63 and CFSE as shown in the region Q2 (green rectangle) in D and E. E. Negative control (EVs captured with beads and unstained).

Table 2. Tissue distribution of EVs derived from different cell lines in mice model

Donor cells	Isolation Method	Loading method	Labeling method	Injection site	Duration of tracking	Tissue distribution	References
B16BL6	Sequential centrifugation and filtration through 0.22 µm filter	Endo Exo	Glu-LA PKH26	I.v.	10–240 min 10 min	Liver, spleen, lung Liver, spleen	(Imai et al., 2015)
4T1, MCF-7, PC3	Sequential centrifugation followed by sucrose density gradient ultracentrifugation	Exo	DiR	I.v. I.t.	1–24 h	Liver, spleen Tumour	(Smyth et al., 2015)
HEK293T, N2a	Ultracentrifugation Ultraspin column 100 kDa followed by Size-exclusion liquid chromatography fractionation	Exo	DiR	I.v.	24 h	Liver, spleen, lung	(Nordin et al., 2015)
HEK293T	Sequential centrifugation and filtration through 0.22 µm filter	Endo	GlucB	I.v.	30–360 min	Spleen, liver, kidneys, lung	(C. P. Lai et al., 2014)
B16BL6	Sequential centrifugation and filtration through 0.22 µm filter	Exo	Iodine-125-labeled biotin	I.v.	1 min-4 h	Liver	(Morishita et al., 2015)
Raw 264.7	Extrusion through 10, 5, and 1 μm-sized polycarbonate membrane filters followed by iodixanol-density gradient ultracentrifugation	Exo	99mTc-HMPAO	I.v.	0.5, 1, 3, and 5 h	Liver, spleen	(Hwang Do et al., 2015)
RBC	Sequential centrifugation followed by filtration through a 600-nm polycarbonate filter	Exo	99mTc-HMPAO	I.v.	45 min	Liver, spleen	(Varga et al., 2016)
Endothelial cells	Sequential centrifugation	Endo Exo	8-nm anionic citrate-coated maghemite nanoparticles CFDA-SE	I.v.	5 min	Liver, spleen, lung	(Al Faraj et al., 2012)

Note. Endo: endogenous, Exo: exogenous, I.v.: intravenous, I.t.: intratumoural, min: minute, and h: hour

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