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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. 100 nm. Synthesis of the inner NP.

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Synthesis of Nanocells

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  1. Synthesis of Nanocells Titima Songkroh Mahidol University

  2. Synthesis ofNanocells Synthesis of the inner nanoparticles (NP) Conjugation of Doxorubicin to PLGA Conjugation of 5-FU to PLGA Synthesis of the outer nanocores

  3. 100 nm Synthesis of the inner NP • contains the first therapeutic agents(Doxorubicin, 5-FU) encapsulated in a polymeric matrix (PLGA) by conjugation

  4. Conjugation of Doxorubicin/5-FU to PLGA 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

  5. * 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

  6. Dissolve 0.4 g activated PLGA polymer in 3 mL of DMF Add 4 mg Doxorubicin in 4 µL Et3N Stir 24 h at RT under N2 *** Use 1 mg of 5-FU instead of Doxorubicin for making PLGA-5-FU conjugation***

  7. Ppt by adding cold ether Filter and wash ppt with ether and then dry under vacuum Doxorubicin - PLGA, 5-FU - PLGA

  8. **Mechanism of Doxorubicin (DOX) - PLGA**

  9. **Mechanism of 5-FU - PLGA**

  10. * Synthesis & SEM analysis of NP Completely dissolve 50 mg DOX – PLGA in 2.5 mL acetone by stirring 1 h at RT Add MeOH 0.5 mL Emulsify into an aq.soln of PVA (0.5g/25mL) by slow injection with constant homogenization using a tissue homogenizer * This picture was taken from http://www.eberbachlabtools.com/2355.cfm

  11. * ‡ These pictures were taken from * http://biosurplus.com/inventory.asp?PageNum=4 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

  12. * ‡ 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.

  13. 200 nm 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)

  14. 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

  15. * 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 Shake at 65°C for 1 h * This picture was taken fromhttp://www.toshibha.com/products/oven.html

  16. * Resuspend in 1 mL H2O Add NP which containing 250 µg DOX (or 5-FU) Extrude the result suspension through a 200 nm membrane at 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

  17. 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.

  18. Physicochemical release kinetics studies Suspend NC in 1 mL of PBS Seal in a dialysis bag Incubate in 20 mL of PBS buffer at 37°C with gentle shaking This picture was taken from http://web.siumed.edu/ ~bbartholomew/course_material/protein_methods.htm

  19. 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

  20. Outer drug Inner drug Sengupta, S., et al. Temporal targeting of tumour cell and neovasculature with a nano scale delivery system. Nature. (2005) 436, 568-572.

  21. 100 nm 200 nm Summary • Nanocells will provided twotherapeutic agents; anti-angiogenesis (FTY 720) in the outer nanocore and cytotoxic agents (Doxorubicin and 5-FU) in the inner nanoparticle

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