Evaluation of the novel nanocell(in vitro/in vivo)Dr.Ramraj PantheeChulabhorn Research Institute

OutlineIntroduction In vitro studiesIn vivo studiesSummary

IntroductionNovel nanocellConfocal microscope In vitro Cell culture studies Co- culture studiesIn vivo Nude mice tumor studies

The novel nanocellstructureFTY720Doxorubisin+5FU(D+5F)PLGA conjugation (Both)(F) Nanocell

The novel nanocell Function24 13 NanocellTumor cellBVs shutdownNc into tumor cell

Confocal microscopeStructure and function Laser ray 3 dimensional picture Computerized device

In vitro studies controls

Vehicle is phosphate buffer solution(PBS)FTY720

Doxo +5FUDoxorubicin5FU

Dox+FTYDoxv

Cell culture studies+Liver cancer cellsCalf serum culture medium24 hours5%Cells + Drugs Viable rateDrugs action on liver cancer cells Survival

Cont. Cell culture studies

Plated for 24 hrs,Doxorubicin +5FU Conjugated mixed for 48hrs.Evaluation: Trypan blue exclusion method. Ec 50 calculation curve fittingCellcultureTrypan blue cell viabilityEc50 - 50% test cells effectiveby drugs

Tumor-endothelium co-culture studiesLiver cancer cells with GFP Expre..Endothelial cellsMatrigelmatrixParaformaldehyde(4%)Incubation- propidium IodideExposed to different treatmentLiver cancer cellEndothelial cell

Microscopic studies of cellControl 30 hFTY720DOXO+5FU Modified picture from: Nature vol. 436 July 2005 S.sengupta et.al.. Control 12 hNanocell 12hNanocell 30hNanocell in 12h action ,BV Nanocell in 30h tumor

Microscopic studies (cont.)Yellow stained Liver cancer cellsRed stained - endothelial cellsDoxorubicin +5FU Tumor reducedFTY720 Vascular shutdownNanocell in 12hours little effectNanocell in 30hours complete tumor reduced

Stereological quantification of co-culture

Vascular component:VEHICLE 12hr-no changeIn 30hrs. Vascular changed Model picture from Nature vol.436 2005------ 12 h--- -------30 h---------VehicleNanocellVehicle

FTY7205Fluouracil +Doxo

Stereological quantification of co-cultureTumor component:Vehicle 12hrs no change

Doxo+5FU 30hrsModel picture from Nature vol.436 2005VehicleNanocellvehicleFTY720Doxo+5FTumor component

Physicochemical release kinetic of drugsDoxorubicin + 5FU slow releaseFTY720 rapid releaseFTY720Doxorubicin+5FU Model picture from Nature vol. 436 2005

In vivo nanocell studiesNude mouse

Nude mouseHigh sensitive to antigenWell manifestation of tumor

In vivo nanocells (cont.) in vivo tumor studiesGFP expressionLiver cancer cellsImplanted male nude miceTumor vol. 50mm3Treatment started with 100 l ivFTY720 anddoxorbicin+5FUAnimals were killed in periods Necropsies tumor - histopathology

In vivo NanocellNude mouse tumorLiver cancer cells injected into sub -cutaneous tissue Tumor 50mm3 in size

Tumor studies

Size of tumor with different controls Modified picture from Nature vol.436,july 2005 S. sengupta et.al.D+5F+FF5FND+FV

Effects of treatment

SurvivalToxicityTraditional chemotherapy 30days surviveNanocell combined 65 days surviveLess toxicWBC count show - normal Without any treatment20days survive Source-Nature:436:2005 S.Sengupta et.al

Tissue distribution studiesNC + Fluorescence dyeInjected miceMice were killed 5, 10, 24 hrs. after injectionOrgans collection liver, lungs, spleen, tumor and serum after necropsedWt. organs and fluorescence extracted

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

SummaryA novel nanocell therapy Slow release Target specific Less toxic Cytotoxic Lifespan