Micron-sized particles

Li Junhua20121113

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.

PC12 cell(differentiated)

Particle

IntroductionCollagen gel culture

1.Make the micron-sized particles

2.Measure the size3.NGF/fluBSA encapsulation

and release study

TOPIC

Methods of making micron-sized particles

• Transport across Oil-water Interface• Reverse Phase Evaporation• Emulsification/gelation method (Agarose microgel

particles)• ...

Preparation of Vesicles

Lipids Suspension(1mM DOPC in Mineral Oil)

Water Phase(PBS)

Stand for 1hr

Vesicle

Centrifuge4,000rpm for 2min. Collect

Vesicle solution(using 18 gauge

of needle)

W/O Emulsions10wt% agarose gel

＋1mM DOPC

in Mineral Oil

DOPC (1,2-Dioleoyl-sn-Glycero-3-phosphocholine)

Transport across oil/water interface

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,phosphatidylglycerol, cholesterol, etc.

Organic solvent(ether) ：diethyl ether, isopropyl ether, Halothane, trifluorotrichloroethane, etc.

Preparation of Agarose microgels (Emulsification/gelation)

1. Make 3% agarose with calcein, heat until 96C2. Make w/o Emulsion (3% agarose in olive oil), stir

(sonicate) at 96C, cool down until around 5C3. Add acetone, stir (vortex) 10 min4. Collect the microgels by filtration (aperture 1µm), acetone washes the filter several times 5. Freeze-dry overnight, dried powder

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—60min2. Add 1 ml of d.water/PBS/DMEM, shake (sonication)3. Examine the particles’ size and the release of

fluorescent BSA by fluorescence spectrophotometer.

Nikon lenses and ruler

Nikon plan fluor 10x lens: 1 pix = 648 nmNikon plan fluor 20x lens: 1 pix = 324 nmNikon plan fluor 40x lens: 1 pix = 164 nmNikon plan fluor 60x lens: 1 pix = 107.5nm

Microgel’s size (40x)

Li:2.5µm

N1:5.7µm

N2:3.2µm

N3:2.5µm

Microgel size: 5.3 µm (20x)

Microgel size: 4.2 µm (20x)

PC12 cell culture in collagen gel (Microgels with fluBSA + NGF)

(20x)

PC12 cell culture in collagen gel (Microgels with NGF + fluBSA)

(40x)

Microgel size: 3.7 µm x 2.5 µm

PC12 cell culture in collagen gel(Microgels with NGF + fluBSA)

(20x)

Fluorescence BSA releases from microgel particles by fluorescence spectrophotometer

Fluorescence BSA releases from microgel particles by fluorescence spectrophotometer

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

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’ PhosphateOH: 3’ hydroxyl

Mechanism of RNA interference pathway

Dicer: ribonuclease proteinRISC: RNA-induced silencing complex

Process of siRNA transfection:

Reagent:Lipofectamine RNAiMaxLipofectamine 2000FuGENE6etc.

Time course of silencing of filamin A gene expression by siRNAs

C. Huang et al. FEBS letters 580 (2006) 1795-1800