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Advanced Nano-Delivery Systems: Facilitating Tumor Delivery and Mitigating ResistanceSession 5 of the 2018 Drug Design and Delivery Symposium

Mansoor AmijiUniversity Distinguished Professor and Professor of

Pharmaceutical Sciences, Director of the Nanomedicine Education and Research Consortium

(NERC), Northeastern University

Venkat KrishnamurthyAssociate Principal Scientist, Advanced Drug

Delivery Group, AstraZeneca

Dr. Mansoor Amiji’s Academic Website: http://www.northeastern.edu/amijilab

http://www.northeastern.edu/amijilab

• I am interested in the biology of tumor and development of resistance

• I am interested in the nanotechnology solutions

• I am interested in both cancer biology and nanotechnology

• I am new to the field and open to learning

What interests you about this topic?

Audience Challenge QuestionANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

The Hallmarks of Cancer

Ref: D. Hanahan and R.A. Weinberg. Cell; 144: 646-674 (2011).

Normal Cells

Cancer Cells

16

Tumor Hypoxia, Glycolysis and MDR

Ref. O. Trendan, et. al., J. Nat. Cancer. Inst. 99(19): 1441-1454 (2007) Ref. M.G. Vander-Heiden, et al. Science 324 (5930): 1029-1033 (2009)17

Incubation of human breast and ovarianadenocarcinoma cells under normoxic andhypoxic conditions and the expression ofresistance markers (HIF, EGFR, Glut-1, Pgp,HXK2) in cells

Tumor Hypoxia, Glycolysis and MDR – Cont’d

In vivo implantation ofnormoxic and hypoxic cellsin Matrigel® to nude mice

Lara Milane

Funded by NIH/NCI Alliance forNanotechnology in Cancer CNPP,ARRA Supplemental, and NCIR21 Grants.

Ref. L. Milane, et al. Cancer Cell Int. 14 (11): 3 (2011) 18

Western blot analysis of proteinexpression following treatment ofMDA-MB-231 and MDA-MB-435breast cancer and SKOV3 andOVCAR5 ovarian cancer cells undernormoxic and hypoxic conditions(for 3 and 5 days)

Ref. L. Milane, et al. Cancer Cell Int. 14 (11): 3 (2011). 19

Cellular Expression of Resistance Markers

Resistance Markers on Tumor Tissue Established

under Normoxic and Hypoxic Conditions – 100 mm3

MDA-MB-231 estrogen-negative human breast tumor xenograft was established orthotopically in themammary fat pad of female athymic (nu/nu) mice using cells cultured under normoxic and hypoxicconditions. At the time of excision, the tumor size was the same ~100 mm3.


Resistance Markers on Tumor Tissue Established

under Normoxic and Hypoxic Conditions – 500 mm3

MDA-MB-231 estrogen-negative human breast tumor xenograft was established orthotopically in themammary fat pad of female athymic (nu/nu) mice using cells cultured under normoxic and hypoxicconditions. At the time of excision, the tumor size was the same ~500 mm3.


• Programmed cell death

• Mechanism of cellular communication

• Ability of cells to divide

• Secretion of molecules from the cells

Therapeutic resistance in cancer develops due to an increase in the apoptotic threshold of tumor cells. Apoptosis is defined as:


Intracellular Ceramide Modulation as a Strategy to Lower Tumor Apoptotic Threshold

Exogenous Ceramide Co-

Therapy

Lilian van Vlerken

Funded by NIH/NCI through CNPP U01 Grant

23

Multidrug Resistant Human Ovarian and Breast Cancer Models

0

40

80

120

160

0.0001 0.01 1 100

[PTX] (mM)

% c

ell s

urv

ival

0

20

40

60

80

100

0.0001 0.001 0.01 0.1 1 10 100

% c

ell s

urv

ival

a.

b.

Ovari

an

Can

cer

(SK

OV

3)

Bre

ast

Can

cer

(MC

F7)

SK

OV

3T

R

MC

F7

TR

P-glycoprotein

Glucosylceramide

Synthase

Beta-Actin

P-glycoprotein

Glucosylceramide

Synthase

SKOV3TR MCF7TR

a. b.

SK

OV

3T

R

MC

F7

TR

P-glycoprotein

Glucosylceramide

Synthase

Beta-Actin

P-glycoprotein

Glucosylceramide

Synthase

SKOV3TR MCF7TR

a. b.

Multidrug resistant (mdr-1 positive)

Wild-type (drug sensitive)

Ref. L. van Vlerken, et al., The AAPS Journal, 12(2): 171-180 (2010). 24

Effect of Temporal Spacing of Paclitaxel and

C6-Ceramide on In Vitro Resistant Cell-Kill Efficacy

0

20

40

60

80

100

1 uM PTX PTX + CER sol'n

PTX + CER @t=6hrs

CER + PTX @t=6hrs

% c

ell s

urv

ival

P<0.001

P<0.05

0

20

40

60

80

100

% c

ell s

urv

ival

P<0.05

P<0.001SKOV3TR

Resistant Ovarian Cancer

Cells

MCF7TR

Resistant Breast Cancer

Cells

Paclitaxel

C6-Ceramide

Ref. L. van Vlerken, et al.,The AAPS Journal, 12(2):171-180 (2010). 25

Polymer Blend Nanoparticles for Temporal-Controlled Delivery

5 mm500 nm

PLGA

Location of ceramide

PbAE

Location of paclitaxel

PEO surface

modification

Atomic composition (%) High resolution C1

peak area (%)

C N O C-H /

C-C

C-O/ C-N C=O

100% PBAE 73.6

0.7

5.7 0.5 16.6 0.1 81.4

0.2

11.1

0.2

7.5 0.0

100% PLGA 58.5 0.1 0.0 0.0 43.6 6.2 29.3

0.2

36.6

0.0

34.2

0.2

70% PLGA/ 30% PBAE 61.5

0.3

0.8 0.3 37.8 0.6 32.6

0.7

34.8

0.1

12.8

9.2

PTX

CER

0

20

40

60

80

100

120

0 5 10 15 20 25 30

Time (hours)

% d

rug

re

lea

se

d (cu

mu

lative

)

a.

b.

c.

d.


Tumor Growth Suppression with Combination Therapy using Polymer Blend Nanoparticles

0

50

100

150

200

250

300

% t

um

or

vo

lum

e c

ha

ng

e (fr

om

in

itia

l)

control

PTX + CER NP

PTX + CER

PTX NP

PTX

0

50

100

150

200

250

0 5 10 15 20 25

time (days)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

fin

al tu

mo

r w

eig

ht

(g)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

fin

al tu

mo

r w

eig

ht

(g)

a. b.

c. d.

*

*

**

**

#

#

##

*

^

SKOV3TR

MCF7TR


PTX

control

PTX + CER

PTX NP

PTX + CER NP

MCF7TRSKOV3TR

Augmentation of In Vivo Apoptotic Activity in Tumor Tissues with Combination Therapy

Ref. L. van Vlerken, et al., The AAPS Journal, 12(2): 171-180 (2010).

Periphery Core Periphery Core

28

• Carbon dioxide and water

• Lactic acid

• Ethanol

• Polymer of glucose

Tumor cells metabolize glucose by a process of aerobic glycolysis. In this process, glucose is converted into:


30

EGFR-Targeted Polymeric Nanoparticles for

Combination Paclitaxel/Lonidamine Therapy

Ref. L. Milane, et al. Mol. Pharm., 8(1): 185-203 (2011).31

Uptake and Intracellular Localization of Control

and EGFR-Targeted Nanoparticles in MDA-MB-231 Cells

15 min 30 min

T

T

C

N

N

C

MDA-MB-231-

WT

MDA-MB-231-

HYP

MDA-MB-231-

WT

MDA-MB-231-

HYP

L. Milane, et al. Mol. Pharm., 8(1): 185-203 (2011). 32

In Vitro Efficacy of Combination Paclitaxel/Lonidamine

Administered in EGFR-Targeted Nanoparticles

PTX dose = 1 mMLON dose = 10 mM

Ref. L. Milane, et al. Mol. Pharm., 8(1): 185-203 (2011). 33

The mean weight and volumes of tumors upon treatment with different formulations on day 28 drug post-administration. The dose of paclitaxel (PTX) was 20 mg/kg and lonidamine (LON) was 80 mg/kg administered in MDA-MB-231 human breast cancer-bearing female athymic (nu/nu) mice.

Efficacy of Combination Paclitaxel/Lonidamine Administered

in EGFR-Targeted Nanoparticles to Tumor-Bearing Mice

Ref. L. Milane, et al. PLoS ONE, 6(9): e24075. (2011).

OrthotopicTumor

34

• Genes that regulate the efflux pump (e.g., mdr-1)

• Genes that regulate cellular apoptosis (e.g., survivin)

• Genes that regulate cell division (e.g., mad-2)

• Genes that regulate aerobic glycolysis (e.g., PKM-2)

• None of the above

What genes are responsible for the development of therapeutic resistance in tumors? (Multiple correct answers may exist)


Overcoming MDR by RNAi/Drug Therapy

Inhibiting ATP-dependent drug effluxtransporters (e.g., mdr-1 and mrp-1 genes)

Circumventing anti-apoptotic mechanisms(e.g., survivin and Bcl-2 expression)

Inhibition of aerobic glycolysis (e.g., PKM-2, HK, and LDH-A genes)

Affecting genes that regulate cell-cyclecheckpoints (e.g., mad-2)

Ref. M. Susa, et al., Pharm. Res., 28(2): 260-272 (2011).

Funded by NCI Alliance in Nanotechnology, Platform Partnership Grant 36

Combinatorial-Designed Nano-Assemblies

Shanthi Ganesh

Arun IyerTarg

etin

g

Mo

du

le

Fluorescence

Radioactive

Imag

ing

Mo

du

leP

aylo

ad E

nca

psu

lati

on

M

od

ule

Peptide

Payl

oads

Pol-Thiol

Pol-Lipid C2-4

Pol-Lipid C6-10

Pol-Lipid C12-18

Pol-PEG

Amit Singh

37

Hyaluronic Acid Derivatives

Hyaluronic acid (HA) is a natural, biocompatible, andbiodegradable polymer

Long history of safe use in clinical applications (e.g.,for visco-supplementation therapy in arthritis)

Intrinsic targeting to CD44 receptors over-expressedon tumor cells (e.g., cancer stem cells) andmacrophages

Modular HA nanoparticle platform synthesized usingdifferent functional substitutions (EDC coupling or“click” chemical cojugation)

Combinatorial library of formed nanoparticles byself-assembly of the constituents at specific weightratios of each (i.e., LEGO blocks)

1

3

4

2

5

Small Molecule Drug

TargetedSelf-Assembling

Nano-system

HA-PEG HA-Thiol HA-PEG-Peptide

EGFR Peptide

OR

siRNA/miRNA Duplexes

OR

38

Evolution of HA Nano-Assemblies for siRNA Delivery

siRNADELIVERY

HA-PEI

HA-PLL

HA-TMPTA, HA-spermine, HA-

Pipirazine

HA-butyl amine, HA-hexylamine, HA-octyamine, HA-diamino

hexane, HA-diaminooctane, HA-

stearylamine

HA-butanoic acid, HA-hexanoicacid, HA-octanoic acid, HA-stearic

acid, HA-oleic acid

Nit

roge

n c

on

ten

t

Po

siti

ve c

har

ge

39

Characterization of HA-PEI/HA-PEG Nanoparticles

Gel retardation showing 100% siRNA

encapsulation

HA-PEI/PEG/siRNA +PAA

HA

(-ve charge) PEI

(+ve charge)+

HA-PEI/siRNA (nanoparticle)

PEI modified HAsiRNA (-ve charge)

Nanoparticle Size = 50 nmSurface charge = -6.5 mV

HA-PEI/siRNA nanoparticles are formed by complexing HA-PEI with PLK-1 silencing siRNA in deionized distilled waterat 1:54 (siRNA:polymer) weight ratio.

Transmission electron

microscopy showing

core-shell nanoparticle

structure

Ref. S. Ganesh, et al., Biomaterials. 34(13): 3489-3502 (2013).40

Evaluation of Combination mdr-1 Silencing and

Paclitaxel Co-Therapy in Resistant Ovarian Cancer

SKOV-3TR SKOV-30.0

0.5

1.0

1.5

2.0CD44Pgp

Re

lati

ve

E

xp

res

sio

n

Pgp

β-actin

CD44

SKOV-3TR SKOV-3

Encapsulation of mdr-1 siRNA in HA-PEI/HA-PEG nanoparticles for CD44 targeted delivery in resistant SKOV3TR human ovarian adenocarcioma model

Zhenfeng Duan, MGH

CD44 Pgp

SKO

V3 T

RSK

OV

3

Sensitive TumorPlatinum Refractory Tumor

CD

44 E

xpre

ssio

n

40X

CD44 expression in human ovarian tumor samples E

HA shell for tumor targeting

PEI for condensing mdr1 siRNA

PEG corona for prolonging circulation

mdr-1 siRNA payload

Ref. X. Yang, et al., Scientific Reports, 5(8509): 1-9 (2015).41

0 . 0

0 . 4

0 . 8

1 . 2

S K O V - 3 T R c e l l o n l y

H A - P E I / H A - P E G / n o n - s p e c i f i c s i R N A

L i p o f e c t a m i n e / M D R 1 s i R N A 9 0 n M

H A - P E I / H A - P E G / M D R 1 s i R N A 4 5 n M

H A - P E I / H A - P E G / M D R 1 s i R N A 9 0 n M

H A - P E I / H A - P E G / M D R 1 s i R N A 1 8 0 n M

Re

lativ

e M

DR

1 E

xp

re

ss

ion

Relative mdr-1 gene silencing after 24 h by qPCR

60%

1: SKOV-3TR cell only

2: HA-PEI/HA-PEG/non-specific siRNA 45nM



5: MDR1 siRNA alone 45nM



8: Lipofectamine /MDR1 90nM

9: HA-PEI/HA-PEG/MDR1 siRNA 45nM



Pgp

β-actin

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

0.0

0.2

0.4

0.6

0.8

Rela

tive P

gp

Exp

ressio

n

Relative P-gp expression after 24 h

Down-regulation of mdr-1 in SKOV3TR Cells using

siRNA-Encapsulation in HA-PEI/HA-PEG Nanoparticles


Evaluation of P-gp and CD44 Expression in Tumor Tissues

upon Administration of mdr-1 siRNA in HA Nanoparticles

Ref. X. Yang, et al., Scientific Reports, 5(8509): 1-9 (2015).

1: saline + paclitaxel

2: MDR1 siRNA + paclitaxel

3: HA-PEI/HA-PEG/none specific siRNA + paclitaxel

4: HA-PEI/HA-PEG/MDR1 siRNA + paclitaxel

CD44

β-actin

Pgp

1 2 3 4

1 2 3 40.0

0.2

0.4

0.6

0.8

1.0 CD44Pgp

Re

lati

ve

E

xp

res

sio

n

CD44 Pgp

1

2

3

4

mdr-1 siRNA dose = 0.5 mg/kg x 3 doses

43

5 8 1 1 1 4 1 7 2 0 2 3 2 6 2 9 3 2 3 5

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

s a l i n e + p a c l i t a x e l

M D R 1 s i R N A + p a c l i t a x e l

H A - P E I / H A - P E G / n o n - s p e c i f i c s i R N A + p a c l i t a x e l

T i m e a f t e r i n j e c t i o n ( d a y s )

H A - P E I / H A - P E G / M D R 1 s i R N A + p a c l i t a x e l

Re

lativ

e T

um

or

Vo

lum

e

1 2 3 5 8 12 15 33

Group 1 - 4:

paclitaxel (20mg/kg)

Group 4: HA-PEI/HA-PEG/mdr1 siRNA

Group 3: HA-PEI/HA-PEG/non-specific siRNA

Group 2: MDR1 siRNA alone

Group 1: saline

Tumors collection

In Vivo Anti-tumor Efficacy of Combination mdr-1

siRNA/Paclitaxel Therapy in SKOV3TR Xenograft Model

Saline + paclitaxel

mdr-1 siRNA alone

+ paclitaxel

HA-PEI/HA-PEG/

Non-pecific siRNA

+ paclitaxel

HA-PEI/HA-PEG/

mdr-1 siRNA

+ paclitaxel


Summary

Poor drug delivery efficiency and the various soluble and insoluble micro-environmental factors areimportant in tumor drug resistance development

Multifunctional polymeric nano-systems offer a versatile platform for multi-pronged tumor-targeteddelivery of therapeutics and modulation of tumor drug resistance

First, we have investigated modulation of intracellular ceramide, an apoptotic second messenger, todecrease the elevated tumor apoptotic threshold in MDR

Our second strategy to modulate tumor MDR is by affecting the aerobic glucose metabolism oftumors through inhibition of hexokinase-2 with lonidamine delivered along with paclitaxel in EGFR-targeted nanoparticles

Lastly, combination of gene silencing by RNA interference and cytotoxic chemotherapy delivered inhyaluronic acid-based nano-assemblies offers an important approach for overcoming MDR in tumor

In all of the above, our strategy focuses on the use of safe materials and scalable manufacturingprocesses that could allow for rapid translation of these experimental approaches in clinically-viabletherapeutic options for cancer patients

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• Preclinical (including Discovery)• Bioanalytical• Clinical Pharmacology• Manufacturing & Bioprocessing• Formulation & Quality

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