1. Micron-sized particles Li Junhua 20121113

2. Abstract Micron-sized agarose particles were prepared using emulsification/ gelation method as a reservoir for protein or peptide drugs. The size of agarose microgels were measured by microscope and the ruler. In this study, nerve growth factor (NGF) aqueous solution with fluorescence BSA were pre- mixed to the microgels for encapsulation. The 3D collagen gel culture of differentiated PC12 cells was employed to examine the effects of released NGF from agarose microgels on those axons growth. To understand the released amount of proteins from microgel particles in different solution, fluorescence BSA in d.water, PBS and DMEM supernatant was measured by fluorescence spectrophotometer in different time intervals, respectively. Results suggested that after 3days incubation, encapsulated microgels in DMEM culture buffer released more protein (13.6%) than in d.water (2%) or in PBS (3.8%). More experiments are needed to confirm the results.

3. PC12 cell (differentiated) Particle Introduction Collagen gel culture

4. 1.Make the micron-sized particles 2.Measure the size 3.NGF/fluBSA encapsulation and release study TOPIC

5. Methods of making micron-sized particles Transport across Oil-water Interface Reverse Phase Evaporation Emulsification/gelation method (Agarose microgel particles)...

6. Preparation of Vesicles Lipids Suspension (1mM DOPC in Mineral Oil) Water Phase (PBS) Stand for 1hr Vesicle Centrifuge 4,000rpm for 2min. Collect Vesicle solution (using 18 gauge of needle) W/O Emulsions 10wt% agarose gel ＋ 1mM DOPC in Mineral Oil DOPC (1,2-Dioleoyl-sn-Glycero-3-phosphocholine)

7. Transport across oil/water interface

8. Preparation of Vesicles: Reverse Phase Evaporation (REV) Method Advantage ・ High concentration vesicle solution Disadvantage ・ Leaving the small amounts of ether in the solvents Lipids ： Phosphatidylcholine,phosphatidylglyc erol, cholesterol, etc. Organic solvent(ether) ： diethyl ether, isopropyl ether, Halothane, trifluorotrichloroethane, etc.

9. Preparation of Agarose microgels (Emulsification/gelation) 1. Make 3% agarose with calcein, heat until 96C 2.Make w/o Emulsion (3% agarose in olive oil), stir (sonicate) at 96C, cool down until around 5C 3.Add acetone, stir (vortex) 10 min 4.Collect the microgels by filtration (aperture 1µm), acetone washes the filter several times 5. Freeze-dry overnight, dried powder

10. NGF + fluBSA Encapsulation and Release study 1. 20 µl of NGF + fluBSA aqueous solution 2 mg of freeze-dried microgels Store at R.T. for 30—60min 2.Add 1 ml of d.water/PBS/DMEM, shake (sonication) 3.Examine the particles’ size and the release of fluorescent BSA by fluorescence spectrophotometer.

11. Nikon lenses and ruler Nikon plan fluor 10x lens: 1 pix = 648 nm Nikon plan fluor 20x lens: 1 pix = 324 nm Nikon plan fluor 40x lens: 1 pix = 164 nm Nikon plan fluor 60x lens: 1 pix = 107.5nm

12. Microgel’s size (40x) Li: 2.5µm N1: 5.7µm N2: 3.2µm N3: 2.5µm

13. Microgel size: 5.3 µm (20x)

14. Microgel size: 4.2 µm (20x)

15. PC12 cell culture in collagen gel ( Microgels with fluBSA + NGF) (20x)

16. PC12 cell culture in collagen gel ( Microgels with NGF + fluBSA) (40x) Microgel size: 3.7 µm x 2.5 µm

17. PC12 cell culture in collagen gel ( Microgels with NGF + fluBSA) (20x)

18. Fluorescence BSA releases from microgel particles by fluorescence spectrophotometer

20. fluBSA releases from microgels in d.water, PBS or DMEM buffer

21. siRNA : small interfering RNA short interfering RNA silencing RNA Double-stranded molecules, 20-25 nucleotides in length The main role of siRNA is involved in RNA interference (RNAi) pathway, to knock down the target gene expression An important tool for drug development and biomedical research of genes of unknown function. P: 5’ Phosphate OH: 3’ hydroxyl

22. Mechanism of RNA interference pathway Dicer: ribonuclease protein RISC: RNA-induced silencing complex

23. Process of siRNA transfection: Reagent: Lipofectamine RNAiMax Lipofectamine 2000 FuGENE6 etc.

24. Time course of silencing of filamin A gene expression by siRNAs C. Huang et al. FEBS letters 580 (2006) 1795-1800