1. Synthesis of Nanocells Titima Songkroh Mahidol University

2. 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

3.  Synthesis of the inner NP 100 nm 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 CH 2 Cl 2 Add 14 mg of p-NC and 9.4 mg of pyridine Stir at 0°C at RT for 3 h under N 2 Dilute with CH 2 Cl 2

5. Wash with 0.1% HCl and brine solution Dry on anh.Mg 2 SO 4 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 Stir 24 h at RT under N 2 Add 4 mg Doxorubicin in 4 µL Et 3 N *** Use 1 mg of 5-FU instead of Doxorubicin for making PLGA-5-FU conjugation***

7. Filter and wash ppt with ether and then dry under vacuum Ppt by adding cold ether 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 * This picture was taken from http://www.eberbachlabtools.com/2355.cfm * Emulsify into an aq.sol n of PVA (0.5g/25mL) by slow injection with constant homogenization using a tissue homogenizer

11. Sonicate 1 min Add to a dilute aq.sol n 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 ‡

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.

14.  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

15. 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

16. Dissolve 27.5 mg lipid in 2 mL CHCl 2 Add 12.5 mg FTY720 in 1 mL CHCl 2 Evaporate solvent to create a monolayer lipid/drug film Shake at 65°C for 1 h * * This picture was taken from http://www.toshibha.com/products/oven.html

17. Resuspend in 1 mL H 2 O 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 *

18. 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.

19. 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

20. 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 H 2 O 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

21. 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

22. 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