Nanomedicine‐based approaches in Oncologypp gyDr. Shiladitya Sengupta

Harvard  Medical SchoolBrigham and Women’s HospitalDana Farber Cancer InstituteHarvard‐MIT Division of Health Sciences and Technology

Someone diagnosed with cancerSomeone diagnosed with cancer   every second .50 during this talk!

19711971

How doesHow does cancer 

progress?

DNA binders that induce cell death

ClNH2

PtCl

NH2 Cisplatin

Doxorubicin

Cisplatin

Bleomycin

Tubulin inhibitors that prevent the cells from dividing

Vincristine

Taxol

C‐KIT C‐METEGFR VEGFR

Molecular medicine ‐ targeting oncogenic mutations

USD 7000USD 7000 per dose

USD 4500 per dosep

USD 4300 per doseper dose

earlier nowCost of Development       250M       1.5BTime to Market 10 Yrs 15 YrsTime to Market  10 Yrs 15 YrsNCEs ~40 ~30Most Blockbuster Patents expire in next 2 5 yearsMost Blockbuster Patents expire in next 2‐5 years 

3 storiesCancer is more than 

3 storiesjust a dividing cellj g

2D or 3D context matters2D or 3D, context matters

Non‐metastatic Metastatic

Weaver and Bissell

Endothelial cells on 2D or 3DEndothelial cells on 2D or 3D

Endothelial cells in MCF10A

Endothelial cells in MCF7

Endothelial cells in MDAin MCF10A

(normal epithelium)cells in MCF7

(non‐metastatic)cells in MDA(highly‐metastatic)

P*AKT(S473)Green = MDA‐MB‐231Red = P*AKT (S473)Red   P AKT (S473)Blue = DAPI

P*FAK Tyr925Green = MDA‐MB‐231Red = P*FAK (Tyr925)Red   P FAK (Tyr925)

OX

OX

OUronic Acid

OCOO‐OO

OX

NHYO

OH

OX

NHY

OX

2’O position of uronic acid

Glucosamine

2 O position of uronic acid 6’O and 3’O position of glucosamineN‐position of glucosamine can either be sulfatedN‐position of glucosamine can either be sulfated, acetylated, or unsubstituted

Post‐synthetic modification of HSGAGs 

N‐deacetylase/N‐sulfotransferase

OH

O

COOH

O

OH

O

COOH

ON sulfotransferase

OH

NAc

OH

OHO O

OH

NSO4‐2

OH

OHO O

OH

O

O

O

OCOOH

OH

NSO4‐2

OH

OHO O

2‐O Sulfotransferase3‐O Sulfotransferase

OSO4‐2

OSO4‐2

O

OH

OCOOH

3 O Sulfotransferase6‐O Sulfotransferase

OSO4

NSO4‐2

OH

OSO4‐2

O O

Formation of endothelial cells de novo, Vasculogenesis

Day 3PI (Red) vWF (green)

Vasculogenesis

D 7Day 7

Day 10

Characterization of HSGAG disaccharides: Sulfation increases with endothelial differentiation

itsso

rban

ce u

nAb Tri‐sulfate

Knock‐down of NDST1 disrupts neovascularization

zNDST1‐Mo 3.5 pmol

RNA Microarray AnalysisIGF2

RRM2

GRN

GADD45A

IGF2R

MDM2HSPAB

Hsp90

TP53

MDM2 HSPA8

GRN

selenoprotein W 2azgc:110001wu:fb59a01zgc:76878peptidase D

RRM2B

TP73

GADD45A

CCNG1

FOXO3A

PPIDGPI

HSP00AB1

CAV1

MDM2Cav1/3SESN1

HTRA2TP73LC2

TP53

Hsp90IGF2R IGF2

GADD45

FOXO3A

HSP90AB1

24 hours-powu:fb55b11proteolipid protein 1atubulin, alpha 1cytoglobinnuclear receptor subfafatty acid binding prozgc:55364Dr_1689_2_A1_a_atDr_19402_1_A1_atDr_24890_1_S1_atwu:fd20g07fibrinogen alpha chainselenoprotein W, 2a

6 somite stageCAV1

BAX

PRDX1

MIF

RP527

FOS

CTH

MMP2

ITGAMS100A9

CLDN4

S100A8C3

PPIATP53

SFRP1

CASP8

PARP2 FOS

pfertilization

6‐somitesangioblasts migrate to midline

20‐somitestubes assemble

28‐somites (24 hpf)onset of angiogenesispleckstrin homology li

Dr_26003_1_A1_atinsulin-like growth fagrowth arrest and DNA-membrane protein, palmDr_19794_1_A1_atmatrix metalloproteinawu:fc49d01Dr_22517_1_S1_atwu:fd50h12wu:fc19g03mesogenin 1wu:fb55b11

PARP2

CASP8MBOAT5

NR2F1

PSMA2

PSMB4

CLU

A2M

HSPA5

LPA

PTPRN

PTPRN2

CTH

FGG

FST

FGA

PI3K1

SFRP4

MMP13angioblasts migrate to midline tubes assemble onset of angiogenesis

wu:fb96a10zgc:92153forkhead box O5murine double minute 2GTP binding protein 1,poly (ADP-ribose) polyDr_7787_1_S1_atcyclin G1caspase 8Dr_15033_1_S1_atwu:fi04f09wu:fc92e10pleckstrin homology-li

20 somite stage

PSMB4LPA

PARP2

CASP8

BAX

FOS

MMP13

MMP2

FSTSFRP1 Symbol Legend

wild‐type NDST1 Mo wild‐type NDST1 Mo wild‐type

heat shock cognate 70-heat shock protein 90-wu:fj64h06v-fos FBJ murine osteozgc:56722zgc:55750wu:fj10e08wu:fc84a08tumor protein p53wu:fb96a10

CCNG1

TP73LMDM2

TP73FOXO3A

IGF2R

GADD45ACytokine

Kinase

Other

Enzyme

Transcription Factor

Nuclear Receptor Acts on

Binds

-3.00

-2.30

-1.70

-1.00

-0.33

0.33

1.00

1.70

2.30

3.00

24 hpf stage

CCNG1IGF2RIGF2 Transmembrane

ReceptorPeptidasePhosphorase

FactorIndirect Interaction

Cross talk between microenvironment and endothelial precursors

shRNA

control NDST1*

pFoxo3A

IGF2 IGF1

Foxo3A

Actin*

NDST1 IGFR1

control shNDST1

PI3K FOXO3

picropodophyllin

LY294002

3

4

*

TIE2 PI3K              FOXO3a

AKT

LY294002

1

2Rn

*

Foxo3A

mTORVEGF

rapamycin

contr

olFox

o3A sh

RNA

0

control shFoxo3A HIF1 Vasculogenesis

Story 2 The story ofStory 2. The story of J h CJohn Cossman

Control (30 h) Combretastatin (30 h) Doxorubicin (30 h)

O CH O CH(A)HO

O

O

CH3

O

OO

O CH3

OH

On PLGA 5050 DL 4A

1. pNC, pyridine, CH2Cl2

2. Doxorubicin, DMF, TEA

(A)(C)

6

8

Doxorubicin (g)Combretastatin (102g)

*

**

*

leas

ed

OH3C

O OH

OH

O

OH

OH

O

O O

O

2

4

6

*

*

#

## #

# #

Tota

l Dru

g R

el

OH3C

OH

NH O

O

O

CH3

O

OO

O CH3

OH

On

O

Doxorubicin - PLGA conjugate

emulsion-solvent evaporation

200 nm

200

0 30 60 90 1200

125 225 325Time (hours)

evaporation

Ultracentrifugation, sizing and phospholipid membrane coating. Combretastatin encapsulated in lipid layer.

(B)

100

150

Dia

met

er (n

m)

H3CO

H3CO

OCH3OH

anop

article

nano

cell

50

D

OCH3 nan

Normal blood vessels Tumor blood vessels

Vehicle L[C] L[C]Vehicle

Ve(A

)vWFTUNEL/HIF1a

NC[D] L[CD] L[CD]NC[D]ehicle

L[

NC[D]+L[C] NCNC[D]+L[C] NC

NC (ld)

[C]

NC

Lewis lung carcinoma B16/F10 melanoma10000 B16/F10 melanoma5500 Lewis lung carcinoma

C[D

]N

C[

2000

2500

5000

7500

10000

olum

e m

m3

30003500400045005000

olum

e m

m3

[D]+L[C

]

500

1000

1500

Tum

or V

o

5001000150020002500

Tum

or V

o NC

9 11 13 15 170

Veh L[C] L[CD]NC[D]NC[D]+L[C] NC NC( ld)

Days9 11 13 15 17

0

Days

L[CD

]

Coming back to JohnComing back to John

Some results from Phase 1, currently Phase 2

Story 3. The future: design ofy gnanomedicine based on SAR

Understanding SAR to design a nanomedicine

DNA Cl

aquationCl Cl OH

bindingq

Cisplatin

Carboplatin Oxaliplatin

Understanding SAR to design a nanomedicine

DMF, H2O

Dry DMF,  DBU

pH<7

umbe

r %

10

2030

pH>7

OO

n

OHN O

O H

O HH O

O H

HNO

nO O

OHHO

Nu

1             10          100         1000      10000Size (d.nm)

010

‐1611.54

‐2210

P t

OO

H 3N N H 3

PtHNO

H3N NH3 O OH

OHOH

‐

ppm‐500 ‐1500 ‐2500

Lung CancerCisplatin(3mg/kg)

PIMA‐GA‐CisPt(3mg/kg)

PIMA‐GA‐CisPt(1.25 mg/kg)

Cisplatin(1.25 mg/kg) Vehicle 

PIMA‐GA‐CisPt [1.25mg/kg] PIMA‐GA‐CisPt [1.25mg/kg]

Ovarian Cancerg

PIMA GA CisPt [1.25mg/kg]PIMA‐GA‐CisPt [3 mg/kg]CisPt [1.25 mg/kg]CisPt [3mg/kg]Vehicle

PIMA‐GA‐CisPt [3 mg/kg]CisPt [1.25 mg/kg]CisPt [3mg/kg]Vehicle

(C)

Vehicle Control PIMA-GA-CisPtCi Pt [3 /k ] PIMA-GA-CisPt 225Vehicle Control [1.25 mg/kg]

Kidn

ey

CisPt [3 mg/kg] [3 mg/kg]

75

100

125

150

175

200

225

***mg

Tum

or

vehic

leCis

(3mg/k

g)Cis

(1.25

mg/kg)

PIMA-G

A-Cis(

1.25)

PIMA-G

A-Cis(

3)

0

25

50

Translating it to the clinics(a) (b)

( ) tmen

t

ControlCisplatin(3mg/kg)

Cisplatin‐NP(3mg/kg)

(a)

LLC 4T1 CP20(a) (b) (c)

Pretreat

ent

Post treatm

e

(b)

‐1621.5

(f)

100010 100

Cisplatin (3 mg/kg)(c)

Cisplatin (1 mg/kg) Cisplatin (3 mg/kg)Cisplatin (1 mg/kg)

Cisplatin NP  (3 mg/kg)Cisplatin NP (1 mg/kg) Cisplatin NP  (3 mg/kg)Cisplatin NP (1 mg/kg)

What drives cancer? SO4

Metastasis

Abhimanyu ParaskarDoD Postdoctoral Scholar

l l

Funding Support

Cisplatin‐nanoparticle

Sudipta basuCharles A King Postdoctoral Scholar

NIHDoDCoulter FoundationAmerican Heart AssociationMary Kay Ash FoundationCharles A King Postdoctoral Scholar

Nano‐biotechnology

f h

y yCIHRCharles A King Trust

Rania HarfoucheCIHR Postdoctoral ScholarVasculogenesis

Yamicia ConnorNIH‐MD,PhD ScholarTumor Microenvironment

Stephanie PiecewiczHarvard MIT PhDHarvard‐MIT PhDVasculogenesis

Shape affects outcome