Date post: | 14-Dec-2015 |
Category: |
Documents |
Upload: | alvin-sarsfield |
View: | 213 times |
Download: | 0 times |
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