Synthesis of Nanocells
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Synthesis of Nanocells Titima Songkroh Mahidol University
description
Synthesis of Nanocells. Titima Songkroh Mahidol University. 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. 100 nm. Synthesis of the inner NP. - PowerPoint PPT Presentation
Transcript of Synthesis of Nanocells
Synthesis of Nanocells
Titima Songkroh
Mahidol University
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
Synthesis of the inner NP
100 nm
• contains the first therapeutic agents (Doxorubicin, 5-FU) encapsulated in a polymeric matrix (PLGA) by conjugation
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
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
*
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***
Filter and wash ppt with ether and then dry under vacuum
Ppt by adding cold ether
Doxorubicin - PLGA,5-FU - PLGA
**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
+
**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
+
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
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
‡
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.
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
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
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
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
*
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.
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
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
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
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
