1
RESEARCH POSTER PRESENTATION DESIGN © 2012 www.PosterPresentations.com A large proportion of chemotherapeutic drugs such as paclitaxel (PTX) are water insoluble and thus require the use of specialized delivery vehicles (e.g. micelle, liposome, etc.) for parenteral administration. These nano-sized delivery vehicles are often complex and may be difficult to manufacture, immunogenic, and/or cause toxic side effects. Recently, exosomes (extracellular vesicles ~100nm in size, released by almost all cell types and used for intracellular communication) have begun to be explored for use as drug delivery vehicles for non-native therapeutics such as nucleic acids and low MW drugs such as curcumin[1-3] and doxorubicin[4]. Introduction exoPTX is More Cytotoxic Than Taxol or Paclitaxel In Vitro Sonication provided the greatest loading capacity* (28.29 ± 1.64%) as compared to incubation (12.12 ± 3.67%) and electroporation (12.68 ± 3.53%). The loading capacity of the commercial formulations of paclitaxel, Abraxane and Taxol, are 10% and 1%, respectively. *Loading Capacity calculated by μg exosomal protein exoPTX is more cytotoxic than either Taxol or Paclitaxel alone in vitro, when tested against a Lewis Lung Carcinoma cell line (3LL-M27), as well as MDCK WT and MDCK MDR1 (Pgp+) cell lines. Uptake of exosomes by 3LL-M27 cells is greater than uptake of other commonly used nanocarriers (liposomes or polystyrene (PS) nanoparticles) in vitro . Intranasally administered exosomes and their drug payload co-localize with pulmonary metastases in vivo. Intranasally administered exoPTX exhibits greater therapeutic efficacy as compared to Taxol in a mouse model of pulmonary metastases. Exosomes do not induce any organ toxicity. Summary Exosomes (10 7 particles/10μL x2) or PBS were intranasally administered to healthy C57/BL mice every other day for 12d (6x total injections). Paraffin tissue sections of lung tissue were then stained with H&E and imaged. e xoPTX Exhibits Greater Therapeutic Efficacy than Taxol In Vivo Drug-Loaded Exosomes Co-localize With Pulmonary Metastases In Vivo References 1.Sun, D., et al., A Novel Nanoparticle Drug Delivery System: The Anti-inflammatory Activity of Curcumin Is Enhanced When Encapsulated in Exosomes. Mol Ther, 2010. 18(9): p. 1606-1614. 2.Zhuang, X., et al., Treatment of Brain Inflammatory Diseases by Delivering Exosome Encapsulated Anti- inflammatory Drugs From the Nasal Region to the Brain. Mol Ther, 2011. 19(10): p. 1769-1779. 3.Kalani, A., et al., Curcumin-primed exosomes mitigate endothelial cell dysfunction during hyperhomocysteinemia. Life Sciences, 2014. 107(1–2): p. 1-7. 4.Tian, Y., et al., A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials, 2014. 35(7): p. 2383-2390. Acknowledgements 1 UNC Eshelman School of Pharmacy, Division of Molecular Therapeutics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7362, USA 2 Center for Nanotechnology in Drug Delivery, Division of Molecular Pharmaceutics UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7362, USA 3 Laboratory of Chemical Design of Bionanomaterials, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119899, Russia Myung Soo Kim 1 , Matthew Haney 1 , Yuling Zhao 1 , Richa Gupta 1 , Onyi Okolie 1 , Phi Phua 1 , Shawn Hingtgen 1,2 , Alexander Kabanov 1,2,3 , Elena Batrakova 1,2 Exosome-Encapsulated Water-Insoluble Small Molecule Chemotherapeutics for the Treatment of Pulmonary Metastases PBS Taxol exoPTX 3LL-M27 /DAPI Bar = 50μm This work was supported by the United States National Institutes of Health grant 1RO1 NS057748. We would also like to thank The Center for Nanotechnology in Drug Delivery at UNC for their assistance and for allowing us to use their instrumentation. 0 5 10 15 20 25 30 35 Incubation Electroporation Sonication Loading Capacity (%) Comparison of Drug Loading Methods Exosomes/DAPI 3LL-M27 /DAPI 3LL-M27 with DOX DOX/DAPI Bar = 20μm Drug Cell Line IC50 (ng/mL) exoPTX 3LL-M27 13.57 ± 1.33 Taxol 3LL-M27 23.16 ± 1.88 Paclitaxel 3LL-M27 126.41 ± 31.31 exoPTX MDCK WT 23.33 ± 3.77 Taxol MDCK WT 69.54 ± 11.50 Paclitaxel MDCK WT 428.77 ± 63.37 exoPTX MDCK MDR1 187.5 ± 38.65 Taxol MDCK MDR1 1708.67 ± 299.93 Paclitaxel MDCK MDR1 >10,000 Results Sonication Provides the Greatest Drug Loading 0 100 200 300 400 500 600 700 800 0 10 20 30 40 50 # Particles Millions Time (hrs) Uptake, 3LL-M27, # Particles vs Time (hrs) Exosomes PS Nanoparticles Exosomes are Taken Up More Readily by Lewis Lung Carcinoma Cells than Other Commonly Used Nanocarriers In Vitro Bar: 20 μm 3hrs: 24hrs: Exosomes Liposomes PS Nanoparticles C57/BL mice with pulmonary metastases (3LL-M27) were intranasally administered DiD-labeled doxorubicin- loaded exosomes. After 18d, sections of lung tissue were stained and imaged by confocal microscopy. Exosomes Distribute to Lung Tissue In Vivo x10 x40 Exosomes were labeled with DiD (red) and intranasally administered to healthy BALB/C mice. 4h later, lung sections were stained and examined by confocal microscopy. Future Direction Our exoPTX formulation exhibits greater cytotoxicity in vitro than Taxol, the commercially available formulation of PTX. Furthermore, intranasally administered exosomes travel to sites of pulmonary metastases in vivo and exoPTX exhibits greater therapeutic efficacy than Taxol in vivo. It is worth noting that our exoPTX formulation appeared to mitigate the drug-resistance phenotype afforded by Pgp expression in MDCK MDR1 cells. The exact mechanism behind this phenomenon has yet to be elucidated and warrants further research. Thus, exoPTX holds great promise for use in the treatment of pulmonary metastases and may represent a new platform for the delivery of water-insoluble chemotherapeutics. Conclusions A release study of exoPTX will be conducted in parallel with a stability study to determine the release profile and stability of our exoPTX formulation. The intracellular distribution of PTX when delivered through exoPTX will also be studied. In addition, the apparent ability of exoPTX to circumvent Pgp-mediated resistance will be further investigated, and the cytotoxicity of exoPTX against Pgp+ and/or MRP1+ cell lines will be assessed. Exosomes Do Not Induce Any Organ Toxicity C57/BL mice with pulmonary metastases (red) were intranasally administered exoPTX (10 7 particles/10μL x2) or Taxol (equivalent amount of PTX), or PBS every other day for 12d (6x total injections). After 18d, sections of lung tissue were stained and imaged by confocal microscopy. Exosomes: PBS: Kidney Spleen Liver AFM Image of exoPTX Hypothesis Incorporation of paclitaxel into exosomes (exoPTX) will increase its solubility and therapeutic efficacy against pulmonary metastases. Exosomes & Microvesicles Macrophage Exosome Isolation and Drug Loading Cytotoxicity was assessed using a standard MTT assay. http://www.ozyme.fr/gammes/sbi/images/exoquick_exosomes_proto_hztl505-351.png http://www.sigmaaldrich.com/content/dam/sigma- aldrich/product1/114/cls3289g.jpg/_jcr_content/renditions/large.jpg https://immunology.org/image/17-15_Macrophages_1.jpg Incubate in exosome- free media for 3 days Remove cells and cell debris, take supernatant Exosome Isolation: ExoQuick-TC™ Drug Loading HPLC Nucelosil C18 exoPTX purification (NAP10 column) Incubate exoPTX at 37°C for 30min http://www.iopan.gda.pl/fizyka/biofizyka/images/stories/biooptyka/hplc %20system%20agilent%201200.jpg Data Analysis

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RESEARCH POSTER PRESENTATION DESIGN © 2012

www.PosterPresentations.com

A large proportion of chemotherapeutic drugs such as paclitaxel (PTX) are

water insoluble and thus require the use of specialized delivery vehicles (e.g.

micelle, liposome, etc.) for parenteral administration. These nano-sized

delivery vehicles are often complex and may be difficult to manufacture,

immunogenic, and/or cause toxic side effects. Recently, exosomes

(extracellular vesicles ~100nm in size, released by almost all cell types and

used for intracellular communication) have begun to be explored for use as

drug delivery vehicles for non-native therapeutics such as nucleic acids and

low MW drugs such as curcumin[1-3] and doxorubicin[4].

Introduction

exoPTX is More Cytotoxic Than Taxol or Paclitaxel In Vitro

• Sonication provided the greatest loading capacity* (28.29 ± 1.64%) as compared to incubation (12.12 ±

3.67%) and electroporation (12.68 ± 3.53%). The loading capacity of the commercial formulations of

paclitaxel, Abraxane and Taxol, are 10% and 1%, respectively. *Loading Capacity calculated by µg exosomal protein

• exoPTX is more cytotoxic than either Taxol or Paclitaxel alone in vitro, when tested against a Lewis Lung

Carcinoma cell line (3LL-M27), as well as MDCK WT and MDCK MDR1 (Pgp+) cell lines.

• Uptake of exosomes by 3LL-M27 cells is greater than uptake of other commonly used nanocarriers

(liposomes or polystyrene (PS) nanoparticles) in vitro .

• Intranasally administered exosomes and their drug payload co-localize with pulmonary metastases in vivo.

• Intranasally administered exoPTX exhibits greater therapeutic efficacy as compared to Taxol in a mouse

model of pulmonary metastases.

• Exosomes do not induce any organ toxicity.

Summary

Exosomes (107 particles/10µL x2) or PBS were intranasally administered to healthy C57/BL mice every other

day for 12d (6x total injections). Paraffin tissue sections of lung tissue were then stained with H&E and

imaged.

exoPTX Exhibits Greater Therapeutic Efficacy than Taxol In Vivo

Drug-Loaded Exosomes Co-localize With Pulmonary Metastases In Vivo

References1.Sun, D., et al., A Novel Nanoparticle Drug Delivery System: The Anti-inflammatory Activity of Curcumin Is

Enhanced When Encapsulated in Exosomes. Mol Ther, 2010. 18(9): p. 1606-1614.

2.Zhuang, X., et al., Treatment of Brain Inflammatory Diseases by Delivering Exosome Encapsulated Anti-

inflammatory Drugs From the Nasal Region to the Brain. Mol Ther, 2011. 19(10): p. 1769-1779.

3.Kalani, A., et al., Curcumin-primed exosomes mitigate endothelial cell dysfunction during

hyperhomocysteinemia. Life Sciences, 2014. 107(1–2): p. 1-7.

4.Tian, Y., et al., A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for

targeted tumor therapy. Biomaterials, 2014. 35(7): p. 2383-2390.

Acknowledgements

1UNC Eshelman School of Pharmacy, Division of Molecular Therapeutics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7362, USA2Center for Nanotechnology in Drug Delivery, Division of Molecular Pharmaceutics UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7362, USA

3Laboratory of Chemical Design of Bionanomaterials, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119899, Russia

Myung Soo Kim1, Matthew Haney1, Yuling Zhao1, Richa Gupta1, Onyi Okolie1, Phi Phua1, Shawn Hingtgen1,2, Alexander Kabanov1,2,3, Elena Batrakova1,2

Exosome-Encapsulated Water-Insoluble Small Molecule Chemotherapeutics for the Treatment of Pulmonary Metastases

PBSTaxolexoPTX

3LL-M27 /DAPIBar = 50µm

This work was supported by the United States National Institutes of Health grant 1RO1 NS057748. We would

also like to thank The Center for Nanotechnology in Drug Delivery at UNC for their assistance and for allowing

us to use their instrumentation.

0

5

10

15

20

25

30

35

Incubation Electroporation Sonication

Load

ing

Cap

acit

y (%

)

Comparison of Drug Loading Methods

Exosomes/DAPI 3LL-M27 /DAPI 3LL-M27 with DOXDOX/DAPI

Bar = 20µm

Drug Cell Line IC50 (ng/mL)

exoPTX 3LL-M27 13.57 ± 1.33

Taxol 3LL-M27 23.16 ± 1.88

Paclitaxel 3LL-M27 126.41 ± 31.31

exoPTX MDCK WT 23.33 ± 3.77

Taxol MDCK WT 69.54 ± 11.50

Paclitaxel MDCK WT 428.77 ± 63.37

exoPTX MDCK MDR1 187.5 ± 38.65

Taxol MDCK MDR1 1708.67 ± 299.93

Paclitaxel MDCK MDR1 >10,000

ResultsSonication Provides the Greatest Drug Loading

0

100

200

300

400

500

600

700

800

0 10 20 30 40 50

# Pa

rtic

les

Mill

ion

s

Time (hrs)

Uptake, 3LL-M27, # Particles vs Time (hrs)

Exosomes PS Nanoparticles

Exosomes are Taken Up More Readily by Lewis Lung Carcinoma Cells than Other Commonly Used Nanocarriers In Vitro

Bar: 20 µm

3hrs:

24hrs:

Exosomes Liposomes PS Nanoparticles

C57/BL mice with pulmonary metastases (3LL-M27) were intranasally administered DiD-labeled doxorubicin-

loaded exosomes. After 18d, sections of lung tissue were stained and imaged by confocal microscopy.

Exosomes Distribute to Lung Tissue In Vivox10 x40

Exosomes were labeled with DiD (red) and intranasally administered to healthy BALB/C mice. 4h later, lung

sections were stained and examined by confocal microscopy.

Future Direction

Our exoPTX formulation exhibits greater cytotoxicity in vitro than Taxol, the commercially available

formulation of PTX. Furthermore, intranasally administered exosomes travel to sites of pulmonary metastases

in vivo and exoPTX exhibits greater therapeutic efficacy than Taxol in vivo. It is worth noting that our exoPTX

formulation appeared to mitigate the drug-resistance phenotype afforded by Pgp expression in MDCK MDR1

cells. The exact mechanism behind this phenomenon has yet to be elucidated and warrants further research.

Thus, exoPTX holds great promise for use in the treatment of pulmonary metastases and may represent a new

platform for the delivery of water-insoluble chemotherapeutics.

Conclusions

A release study of exoPTX will be conducted in parallel with a stability study to determine the release profile

and stability of our exoPTX formulation. The intracellular distribution of PTX when delivered through exoPTX

will also be studied. In addition, the apparent ability of exoPTX to circumvent Pgp-mediated resistance will

be further investigated, and the cytotoxicity of exoPTX against Pgp+ and/or MRP1+ cell lines will be assessed.Exosomes Do Not Induce Any Organ Toxicity

C57/BL mice with pulmonary metastases (red) were intranasally administered exoPTX (107 particles/10µL x2)

or Taxol (equivalent amount of PTX), or PBS every other day for 12d (6x total injections). After 18d, sections

of lung tissue were stained and imaged by confocal microscopy.

Exosomes:

PBS:

Kidney Spleen Liver

AFM Image of exoPTX

HypothesisIncorporation of paclitaxel into exosomes (exoPTX) will increase its solubility and therapeutic efficacy against

pulmonary metastases.

Exosomes & Microvesicles

Macrophage

Exosome Isolation and Drug Loading

Cytotoxicity was assessed using a standard MTT assay.

http://www.ozyme.fr/gammes/sbi/images/exoquick_exosomes_proto_hztl505-351.pnghttp://www.sigmaaldrich.com/content/dam/sigma-aldrich/product1/114/cls3289g.jpg/_jcr_content/renditions/large.jpg

https://immunology.org/image/17-15_Macrophages_1.jpg

Incubate in exosome-

free media for 3 days

Remove cells and cell

debris, take supernatant

Exosome Isolation: ExoQuick-TC™

Drug Loading

HPLC

Nucelosil C18

exoPTX purification

(NAP10 column)

Incubate exoPTX

at 37°C for 30min

http://www.iopan.gda.pl/fizyka/biofizyka/images/stories/biooptyka/hplc%20system%20agilent%201200.jpg

Data Analysis