synthesis of nanocells titima songkroh mahidol university
TRANSCRIPT
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Synthesis of Nanocells
Titima Songkroh
Mahidol University
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Synthesis of Nanocells
Synthesis of the inner nanoparticles (NP)
• Conjugation of Doxorubicin to PLGA
• Conjugation of 5-FU to PLGA
Synthesis of the outer nanocores
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Synthesis of the inner NP
100 nm
• contains the first therapeutic agents (Doxorubicin, 5-FU) encapsulated in a polymeric matrix (PLGA) by conjugation
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Conjugation of Doxorubicin/5-FU to PLGA
N2
Dissolve PLGA 1.5 g in 15 mL CH2Cl2
Add 14 mg of p-NC and 9.4 mg of pyridine
Stir at 0°C at RT for 3 h under N2
Dilute with CH2Cl2
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Wash with 0.1% HCl and brine solution
Dry on anh.Mg2SO4
Filter
Evaporate
Obtain activated PLGA polymer
* This picture was taken from http://www.rtpumps.com/rtpumps/web/home.nsf/contentview/949D0D491E891025C1256F9000350F4C
*
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Dissolve 0.4 g activated PLGA polymer in 3 mL of DMF
Stir 24 h at RT under N2
Add 4 mg Doxorubicin in 4 µL Et3N
*** Use 1 mg of 5-FU instead of Doxorubicin for making PLGA-5-FU conjugation***
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Filter and wash ppt with ether and then dry under vacuum
Ppt by adding cold ether
Doxorubicin - PLGA,5-FU - PLGA
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**Mechanism of Doxorubicin (DOX) - PLGA**
O Cl
OO2N
OO
O CH3
O
O
O CH3
OH
O
n OH
OO
O CH3
O
O
O CH3
OH
O
n OO
OO2N
O
O
OH
OH O
OH
O
OH
O
O
OH
NH2
O
O
OH
OH O
OH
O
OH
O
O
OH
NH
O
O O
OO
O
CH3
O
O CH3
OH
O
n
..
PLGA
p-nitrophenyl chloroformate (pNC)
Doxorubicin
Doxorubicin-PLGA
+
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**Mechanism of 5-FU - PLGA**
O Cl
OO2N
OO
O CH3
O
O
O CH3
OH
O
n OH
OO
O CH3
O
O
O CH3
OH
O
n OO
OO2N
NH
NHF
O
O
..
:
O
O
OO
O
CH3
O
O CH3
OH
O
n
O
N
NH
OO
F
PLGA
p-nitrophenyl chloroformate (pNC)
5-FU
5-FU - PLGA
+
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Synthesis & SEM analysis of NP
Completely dissolve 50 mg DOX – PLGAin 2.5 mL acetone by stirring 1 h at RT
Add MeOH 0.5 mL
* This picture was taken fromhttp://www.eberbachlabtools.com/2355.cfm
*
Emulsify into an aq.soln of PVA (0.5g/25mL)by slow injection with constant homogenization
using a tissue homogenizer
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Sonicate 1 min
Add to a dilute aq.soln of PVA (0.2g/100 mL)With rapid mixing for 3 h at RT
Ultracentrifuge at 10000, 25000 and 50000 xg
Get the smallest NP size fractions
*
‡ http://www.snupharm.ac.kr/hanbh/about/about_03.asp
These pictures were taken from * http://biosurplus.com/inventory.asp?PageNum=4
‡
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Extrude through a 100 nm membrane using hand-held extruder
Obtain NP for encapsulation within NC
Analyze morphology by SEM at a magnification of 3700X
*
These pictures were taken from * http://www.mudinmind.com/index.asp?cat=173666
‡
‡ from Sengupta, S., et al. Temporal targeting of tumour cell and neovasculature with a nano scale delivery system. Nature. (2005) 436, 568-572.
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Synthesis of the outer nanocores
• coated the inner nanoparticles with a lipid and anti-angiogenesis agent (FTY 720) partitioned in the lipid phase to form a nanocell (NC)
200 nm
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Preparation of Lipid envelope for NC
• Cholesterol (CHOL)
• Egg-phosphatidylcholine (PC)
• Distearoylphosphatidylethanolamine –
polyethylene glycol (mw 2000) (DSPE-PEG)
• Use in ratio of PC:CHOL:DSPE-PEG = 2:1:0.2 molar
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Dissolve 27.5 mg lipid in 2 mL CHCl2
Add 12.5 mg FTY720 in 1 mL CHCl2
Evaporate solvent to create a monolayer lipid/drug film
OH
NH2
OH
Shake at 65°C for 1 h*
* This picture was taken from http://www.toshibha.com/products/oven.html
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Resuspend in 1 mL H2O
Add NP which containing 250 µg DOX (or 5-FU)
Extrude the result suspension through a 200 nm membraneat 65°C using a hand-held extruder to create the lipid vesicles
* This picture was taken from http://www.euroresidentes.com/futuro/nanotecnologia/nanotecnologia_noticias.htm
*
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Determine the average vesicle size by dynamic light scattering
Sengupta, S., et al. Temporal targeting of tumour cell and neovasculature with a nano scale delivery system. Nature. (2005) 436, 568-572.
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Physicochemical release kinetics studies
Suspend NC in 1 mL of PBS
Seal in a dialysis bag
Incubate in 20 mL of PBS bufferat 37°C with gentle shaking
This picture was taken from http://web.siumed.edu/~bbartholomew/course_material/protein_methods.htm
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Take 200 µL aliquots from the incubation medium at predetermined time intervals and stored frozen for analysis
Quantify released drug by RP-HPLC using a C18 column (4.5nm x 150nm, Waters),
MeCN and H2O as gradient eluents,Dexamethasone as an internal control
This picture was taken from http://www.vscht.cz/trp/UTRP-cs/Vyzkum/Vybaveni/HPLC-RP.html
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Sengupta, S., et al. Temporal targeting of tumour cell and neovasculature with a nano scale delivery system. Nature. (2005) 436, 568-572.
Inner drug
Outer drug
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Summary
• Nanocells will provided two therapeutic agents; anti-angiogenesis (FTY 720) in the outer nanocore and cytotoxic agents (Doxorubicin and 5-FU) in the inner nanoparticle
200 nm
100 nm