evaluation of the novel nanocell (in vitro/in vivo) dr.ramraj panthee chulabhorn research institute
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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
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Outline
Introduction Introduction In vitro studiesIn vitro studies In vivo studiesIn vivo studies SummarySummary
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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
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The novel nanocellThe novel nanocellstructurestructure
FTY720FTY720
DoxorubisinDoxorubisin+5FU(D+5F)
PLGAPLGA conjugationconjugation (Both)Both) 200 nm
100 nm
(F)
NanocellNanocell
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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
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Confocal microscopeConfocal microscopeStructure and functionStructure and function
• Laser rayLaser ray
• 3 dimensional 3 dimensional picture picture
• Computerized Computerized devicedevice
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In vitro studiesIn vitro studies controlscontrols
Vehicle is phosphate buffer solution(PBS)
•FTY720
Doxo +5FU
Doxorubicin
•5FU
Dox+FTYDox
v
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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
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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
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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
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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
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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
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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
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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
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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
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In vivo nanocell studiesIn vivo nanocell studiesNude mouseNude mouse
Nude mouseNude mouse
1. High sensitive to antigen2. Well manifestation of tumor
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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
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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
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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
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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
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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
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SummarySummaryAA novel nanocell therapy
Slow release Target specific
Less toxic Cytotoxic Lifespan