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