Evaluation of the novel nanocellEvaluation of the novel nanocell(in(in vitro/in vivo)vitro/in vivo)

Dr.Ramraj PantheeDr.Ramraj Panthee

Chulabhorn Research InstituteChulabhorn Research Institute

Outline

Introduction Introduction In vitro studiesIn vitro studies In vivo studiesIn vivo studies SummarySummary

IntroductionIntroduction

• Novel nanocellNovel nanocell• Confocal microscopeConfocal microscope• In vitro In vitro

• Cell culture studiesCell culture studies• Co- culture studiesCo- culture studies

• In vivo In vivo • Nude mice tumor studiesNude mice tumor studies

The novel nanocellThe novel nanocellstructurestructure

FTY720FTY720

DoxorubisinDoxorubisin+5FU(D+5F)

PLGAPLGA conjugationconjugation (Both)Both) 200 nm

100 nm

(F)

NanocellNanocell

The nThe novel nanocellovel nanocell Function Function

2 4 1

200 nm

100 nm

3 NanocellNanocell

Tumor cellTumor cellBVs shutdownBVs shutdown

Nc into tumor cellNc into tumor cell

Confocal microscopeConfocal microscopeStructure and functionStructure and function

• Laser rayLaser ray

• 3 dimensional 3 dimensional picture picture

• Computerized Computerized devicedevice

In vitro studiesIn vitro studies controlscontrols

Vehicle is phosphate buffer solution(PBS)

•FTY720

Doxo +5FU

Doxorubicin

•5FU

Dox+FTYDox

v

Cell culture studies

+

Liver cancer cellsLiver cancer cells

Calf serumCalf serum

cultureculture mediummedium

24 hours24 hours5%

Cells + Drugs Viable rateCells + Drugs Viable rateDrugs action on liver cancer cellsDrugs action on liver cancer cells SurvivalSurvival

Cont.Cont. Cell culture studiesCell culture studies

• Plated for 24 hrs,Plated for 24 hrs,• Doxorubicin +5FU Conjugated mixed for Doxorubicin +5FU Conjugated mixed for

48hrs.48hrs.• Evaluation: Trypan blue exclusion method.Evaluation: Trypan blue exclusion method.• Ec 50 calculation curve fittingEc 50 calculation curve fitting CellCell

cultureculture

Trypan blue – cell viabilityTrypan blue – cell viability

EcEc50 50 - 50% test cells effective - 50% test cells effective by drugs

Tumor-endothelium co-Tumor-endothelium co-culture studiesculture studies

• Liver cancer cells with Liver cancer cells with GFP Expre..GFP Expre..

• Endothelial cellsEndothelial cells• MatrigelmatrixMatrigelmatrix• Paraformaldehyde(4Paraformaldehyde(4%%

))• Incubation- propidium Incubation- propidium

IodideIodide• Exposed to different Exposed to different

treatmenttreatment

Liver cancer cell

Endothelial cell

Microscopic studies of cellMicroscopic studies of cell

Control 30 hControl 30 h FTY720FTY720DOXO+5FU

Modified picture from: Nature vol. 436 July 2005 S.sengupta et.al..

Control 12 h Nanocell 12hNanocell 30h

Nanocell in 12h action ,BV Nanocell in 12h action ,BV Nanocell in 30h tumor Nanocell in 30h tumor

Microscopic studies (cont.)Microscopic studies (cont.)

• Yellow stained – Liver cancer cellsYellow stained – Liver cancer cells• Red stained - endothelial cellsRed stained - endothelial cells• Doxorubicin +5FU –Tumor reducedDoxorubicin +5FU –Tumor reduced• FTY720 – Vascular shutdownFTY720 – Vascular shutdown• Nanocell in 12hours little effectNanocell in 12hours little effect• Nanocell in 30hours complete tumor reducedNanocell in 30hours complete tumor reduced

Stereological quantificationStereological quantification of of co-cultureco-culture

• Vascular componentVascular component:

VEHICLE 12hr-no changeVEHICLE 12hr-no change

In 30hrs. Vascular changedIn 30hrs. Vascular changed

Model picture from Nature vol.436 2005

------ 12 h--- -------30 h---------

Veh

icle

Nan

ocel

l

Veh

icle

FT

Y7

20

5Flu

oura

cil +

Dox

o

Stereological quantification of Stereological quantification of co-cultureco-culture

• Tumor componentTumor component:Vehicle 12hrs no changeVehicle 12hrs no change

Doxo+5FU 30hrsDoxo+5FU 30hrs

Model picture from Nature vol.436 2005

Veh

icle

Nan

ocel

l

vehi

cle

FT

Y72

0

Dox

o+5F

Tumor component

Physicochemical release Physicochemical release kinetic of drugskinetic of drugs

• Doxorubicin + 5FU– slow releaseDoxorubicin + 5FU– slow release• FTY720 —rapid releaseFTY720 —rapid release

FTY720FTY720Doxorubicin+5FUDoxorubicin+5FU

Model picture from Nature vol. 436 2005

In vivo nanocell studiesIn vivo nanocell studiesNude mouseNude mouse

Nude mouseNude mouse

1. High sensitive to antigen2. Well manifestation of tumor

In vivo nanocells (cont.)In vivo nanocells (cont.) in vivo tumor studies in vivo tumor studies

• GFP expressionGFP expression• Liver cancer cellsLiver cancer cells• Implanted male nude miceImplanted male nude mice• Tumor vol. 50mmTumor vol. 50mm33

• Treatment started with 100Treatment started with 100µ l ivµ l iv• FTY720 andFTY720 and• doxorbicin+5FUdoxorbicin+5FU• Animals were killed in periods Animals were killed in periods • Necropsies tumor - histopathologyNecropsies tumor - histopathology

In vivo NanocellIn vivo NanocellNude mouse tumorNude mouse tumor

Liver cancer cells injected into sub -Liver cancer cells injected into sub -cutaneous tissuecutaneous tissue

Tumor 50mmTumor 50mm33 in size in size

Tumor studiesTumor studies

Size of tumor with different controlsSize of tumor with different controls

Modified picture from Nature vol.436,july 2005 S. sengupta et.al.

D+5F+F

F

5FN

D+FV

Effects of treatmentEffects of treatment

• SurvivalSurvival• ToxicityToxicity

Traditional chemotherapy Traditional chemotherapy 30days survive30days survive

Nanocell combined 65 Nanocell combined 65 days survivedays survive

Less toxicLess toxic

WBC count show - normalWBC count show - normal

Without any treatment20days Without any treatment20days survivesurvive Source-Nature:436:2005 S.Sengupta et.al

Tissue distribution studiesTissue distribution studies

• NC + Fluorescence dyeNC + Fluorescence dye• Injected – miceInjected – mice• Mice were killed – 5, 10, 24 hrs. after injectionMice were killed – 5, 10, 24 hrs. after injection• Organs collection –liver, lungs, spleen, tumor and Organs collection –liver, lungs, spleen, tumor and

serum after necropsedserum after necropsed• Wt. – organs and fluorescence extractedWt. – organs and fluorescence extracted

Purpose - Distribution of drugs into the tissues Result – Nanocells were detected within 5 hoursResult – Nanocells were detected within 5 hours

SummarySummaryAA novel nanocell therapy

Slow release Target specific

Less toxic Cytotoxic Lifespan