Bioengineered serum-derived exosomes-nanosystem

for triple negative breast cancer therapy

Amalendu P Ranjan, PhDAssistant Professor

Department of Microbiology, Immunology & Genetics

TCHD 15th Annual Texas Conference on Health Disparities: Women’s Health Disparities

AIAN indicates American Indian/Alaska Native; API, Asian/Pacific Islander; NHB, non‐Hispanic black; NHW,

non‐Hispanic white. 2001 to 2016, United States

Trends in Female Breast Cancer by Race/Ethnicity

Breast cancer statistics, 2019, CA: A Cancer Journal for Clinicians, 69, 6, 438-451.

Incidence rate Mortality rate

Triple Negative Breast Cancer (TNBC) Triple negative breast cancer (TNBC) definition:

Lack of expression of estrogen receptor (-ER)

and progesterone receptor (-PR)

HER2 not overexpressed/amplified (-HER2)

Triple negative breast cancer is the most lethal

form of breast cancer

15-20 % of all breast cancers

The prevalence of TNBC is higher in women of

African American ethnicity

TNBC is heterogeneous and harbors several

molecular alterations

Overall survival in patients with triple-negative breast cancer (TNBC) and non-TNBC

Oncology Letters, 2020, 19: 735-744.

Racial Distribution of TNBC

Nature Reviews Cancer ,2015, 15, 248–254

Current Treatment Option for Breast CancerTNBC

Conventional chemotherapy is only known standard treatment along with surgery and radiation therapy.

Chemotherapy typically includes single agent or combination of Taxanes, Anthracyclines and Oxazophorines.

There is no specific targeted therapy on TNBC. Development of new targeted therapies may be able to treat TNBC.

Bioengineered Serum-derived Exosomes-nanosystem

Membrane protein, integrin, RNA, intracellular protein, metabolites, CAB- cabazitaxel

Isolation of exosome

Isolation and Purification of

exosome membrane

CAB loaded Nanoexos

Exosome

CAB loaded polymeric nanoparticle

Exosomal membrane

Patient serum samples

We will exploit the unique properties and advantages offered by exosomes and drug loaded polymericnanoparticles and create a novel bioengineered serum-derived exosomes-nanosystemCAB: Cabazitaxel

Long body circulations Biocompatibility and safe Cargo protection and encapsulation Biodegradable Enhanced biodistribution Targeted delivery

Properties

Exosomes

Exosome Biogenesis

Cells 2019, 8(4), 307Chuo et al., Journal of Biomedical Science, 2018

Isolation and Characterization of Exosomes from SerumControl: TNBC cells – MDAMB 231 TNBC-Serum samples

Exosomal surface markers: positive markers: TSG 101, CD 81, Hsc70; Negative markers: GM 130, Cyt c, Lamin A/C

CD 81

C E 1 2 3 4 5 6

HSC 70

Lamin A/C

Cyt c

AA CA

CA: Caucasian American, AA: African American

Isolation of exosome

e

Exosome

Patient serum sample

Isolation and Characterization of Exosomes from Pooled Human Serum

TNBC Pool of five different serum samples

Exosomal surface markers: positive markers: TSG 101, CD 81, Hsc70; Negative markers: GM 130, Cyt c, Lamin A/C

Tsg101

Hsc70

GM130

Lamin A/C

CL EL CA AA

CL: Cell lysateEL: Exosomal lysateCA: Caucasian AmericanAA: African American

NTA Measurement

https://systembio.com/shop/exoglow-nta-fluorescent-labeling-kit/

NTA Measurement of Serum ExosomesPolymeric Blank NPS CA-TNBC- Exosomes – light scattering CA-TNBC- Exosomes – Fluorescence

Polymeric Blank NPS AA-TNBC- Exosomes – light scattering AA-TNBC- Exosomes – Fluorescence

C: Caucasian American, AA: African American

Particle Size and Zeta Potential

AA-TNBC

CA-TNBC0

20

40

60

80

100

0

5

10

15

Part

icle

Siz

e (n

m)

Con

cent

ratio

n *1

010

(par

ticle

s/m

l)

Particle Size Concentration AA-TNBC CA-TNBC

-8

-6

-4

-2

0

Zeta

Pot

entia

l (m

V)

In vivo Biodistribution of Exosomes in Nude Mice

15 min 45 min 1 hr

1.5 hrs 2 hrs 3hrs

6 hrs 24 hrs 48 hrs

15 min 45 min 1 hr

1.5 hrs 2 hrs 3hrs

6 hrs 24 hrs 48 hrs

CA: Caucasian American, AA: African American

AA TNBC patient serum exosomes CA TNBC patient serum exosomes

A. Representative bio

illuminance image of tumor

B. Representative fluorescence

image of IR dye tagged

exosomes from serum

samples,

C. Serum exosome distribution

in tumor of different mice.

AA

CACA

AA AA

CA

Biodistribution of serum exosomes in tumor bearing athymic nude mice

AA-African American and CA – Caucasian American

A B C

Formulation of Cabazitaxel Loaded PLGA Nanoparticles

Cabazitaxel+PLGA polymer dissolved

in acetone

Drop wise

NanoprecipitationCAB-PLGA

nanoparticles

Particle size (nm): 87.83±1.5

PDI: 0.113±0.014

Zeta potential (mV): -45.5±2.60

Drug Loading and Encapsulation Efficiency

0

1 0

2 0

3 0

4 0

5 0

8 .5

9 .0

9 .5

1 0 .0E

nc

ap

su

lati

on

Eff

icie

nc

y (

%)

Dru

g lo

ad

ing

(%)

E n c a p s u la t io n e ff ic ie n c y

D ru g lo a d in g

Bioengineered serum-derived exosome coated PLGA nanoparticles

Schematic of exosome coated PLGA NP preparation by Co-extrusion methods

Physical extrusion

Exosome

Bio-Exo-NPs

CAB-PLGA-NPs

Physiochemical Characterization of Bio-Exo-NPs

B-PLGA-NP

CAB-PLGA-NP

AA-EXO

AA-EXO-CAB-PLGA-N

P

CA-EXO

CA-EXO-CAB-PLGA-N

P0

50

100

150

0.0

0.1

0.2

0.3

0.4

Part

icle

Siz

e (n

m)

PDI

Particle Size PDI

CAB-PLGA-NP

AA-EXO

AA-EXO-CAB-PLGA-N

P

CA-EXO

CA-EXO-CAB-PLGA-N

P-60

-40

-20

0

Zeta

Pot

entia

l (m

V)

In vivo Tumor Efficacy of Bio-Exo-NP in the Tumor Mouse Model In progress

Tumor cells Engraftment

Patient serum samples Exosomes

+Extrusion

CAB loaded NPsBio-Exo-NPs

Tumor burden mice

IV injection

Conclusions/Take Home Messages Triple negative breast cancer (TNBC) is a molecularly heterogeneous disease whose

incidence is disproportionately higher in African American (AA) women compared to European American (EA) women.

TNBC is an aggressive subtype of breast cancer, with nearly no targeted therapies available today.

Exosomes are membrane bound extracellular vesicles generally have a natural high targeting potential make them attractive for use in targeted drug delivery.

Exosome from distinct racial distinct patient serum samples shows disparate localization in mouse model

Further, these exosome shows specific tumor targeted localization due to diverse signature molecules on surface.

We have successfully bioengineered the exosome clocked PLGA nanoparticles loaded with cabazitaxel for investigating the targeted chemotherapy of TNBC.

AcknowledgmentsRohan Joshi, PhD studentPriyanka Desai, PhD studentJana Lampe, PhD studentJianmei Wang, Pharmaceutical Analysis Core Lab, College of Pharmacy

This work supported in part by:

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