Transcript Seminar 20121113

Micron-sized particles
Li Junhua
20121113
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 premixed 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.
Introduction
Collagen gel culture
Particle
PC12 cell
(differentiated)
TOPIC
1. Make the micron-sized particles
2. Measure the size
3. NGF/fluBSA encapsulation and release study
Methods of making micron-sized particles
• Transport across Oil-water Interface
• Reverse Phase Evaporation
• Emulsification/gelation method (Agarose microgel
particles)
• ...
Preparation of Vesicles
DOPC (1,2-Dioleoyl-sn-Glycero-3-phosphocholine)
Lipids Suspension
(1mM DOPC in Mineral Oil)
W/O Emulsions
10wt% agarose gel
＋
1mM DOPC
in Mineral Oil
Stand for
1hr
Water Phase
(PBS)
Centrifuge
4,000rpm
for 2min.
Collect
Vesicle solution
(using 18 gauge
of needle)
Vesicle
Transport across oil/water interface
Preparation of Vesicles: Reverse Phase Evaporation (REV) Method
Lipids：
Phosphatidylcholine,phosphatidylglyc
erol, cholesterol, etc.
Organic solvent(ether)：
diethyl ether, isopropyl ether,
Halothane, trifluorotrichloroethane,
etc.
Advantage
・High concentration vesicle solution
Disadvantage
・Leaving the small amounts of ether in the solvents
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
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.
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
Microgel’s size (40x)
N1:
5.7µm
N2:
3.2µm
N3:
2.5µm
Li:
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
1200
1000
800
DMEM+fluBSA+NGF
600
DMEM+fluBSA
400
water+fluBSA+NGF
water+fluBSA
200
0
10min
30min
50min
18h
Fluorescence BSA releases from microgel
particles by fluorescence spectrophotometer
300
274
276
250
200
150
2d+
4d
100
91
89
54
50
16
17
23
25
18
0
d.water
d.water+fluBSA
DMEM
DMEM+fluBSA
fluBSA
fluBSA releases from microgels in
d.water, PBS or DMEM buffer
350
320
310
293
300
277
260
250
235
220
216
200
30min
150
3d
100
63
55
50
1615
0
2121
2019
2019
1615
2325
2525
2424
2521
5854
5552
siRNA : small interfering RNA
short interfering RNA
silencing RNA
P: 5’ Phosphate
OH: 3’ hydroxyl
• 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.
Mechanism of RNA
interference pathway
Dicer: ribonuclease protein
RISC: RNA-induced silencing complex
Process of siRNA
transfection:
Reagent:
Lipofectamine RNAiMax
Lipofectamine 2000
FuGENE6
etc.
Time course of silencing of filamin A gene expression by siRNAs
C. Huang et al. FEBS letters 580 (2006) 1795-1800