Rat Intestine Cells Absorption of Fluorescein
Isothiocyanate–Dextran Induced by Microplasma Treatment
J. Kristof, R. Yokoyama, A.G. Yahaya, M. Blajan, K. Shimizu
Shizuoka University, Nakaku, Johoku, Hamamatsu, 432-8561, Japane-mail: jaroslav.kristof@gmail.com, shimizu@cjr.shizuoka.ac.jp
Introduction. Living cells as drug delivery carriers has gained increasing interest in recent years. Cell-based targeting anddelivery could be used to facilitate a more specific tools for the next-generation drug delivery systems. every cell is surrounded bylipid bilayer membrane. Drug has to pass this barrier of the white blood cells to be incorporated into the cell. This can occur
through the distortion of lipid barrier, by activation of the TRP channels or endocytosis.

Microplasma electrode

Experiment
Endocytosis is process of bringing
substances into the cell throughsurrounding area of the cell membrane
which is pulled inside the cell to form avesicle with the ingested material.
54% of the drug enters into the cellthrough this process after plasma
treatment. The drug delivery efficiency
and cell viability strongly depend on
the plasma source. Drug delivery
efficiency decreases with increase ofmolecular mass of drug.

Microplasma electrode- Dieledctric thickness = 18 μm- Diameter = 3 cm
- Electrode temperature during
discharge – under 45°C

18 μm

FD-150 cell absorption measurements

Excitation: 493 nm
Fluorescence: 518 nm

50000
40000
30000
20000
10000
0control

1.7 mm

2.7 mm

3.7 mm

4.7 mmcell concentration (1/ml)

Molecular weight: 150 kDa

Fluorescence (a.u.)

Fluorescein Isothiocyanate–Dextran(FD-150)

60000

1.00E+08

1.00E+07

1.00E+06

1.00E+051.00E+04
control

1.7 mmdistance

50000
40000
30000
20000
10000(1/ml)

1.00E+07

cell concentration

Fluorescence (a.u.)

4.7 mmNumber of cell decreases 1 order
after plasma treatment and 24 hincubation– effect of distance is negligible

60000

Voltage and current
waveform of
microplasma discharge

3.7 mmdistance

Fluorescence (absorbance of FD-150)
Decreases with treatment distanceMicroplasma treatment of the cells

2.7 mm

1.00E+06

1.00E+05

0control

2.8 kV

3.7 kV

control

4.3 kV

2.8 kV

3.7 kV

4.3 kV

voltagevoltage
Higher voltage of discharge helps toincrease fluorescence (absorbance ofFD-150 into the cells)

Higher voltage of discharge candecrease viability of the cells

Measurementof fluorescence
Transfer toof FD-150
96 well plateby microplate reader

PBS wash 2x

1.00E+07

60000
50000
40000
30000
20000
10000
0controlAdd DMEM
2ml

Trypsin (0.5 ml)
for cell
detachment

1 min

2 min

5 min

treatment time

Treatment time (absorbance of FD-150
into the cells) is very similar for 1 and2 minutes of plasma treatment anddecreased after 5 minutes of plasma
treatment(1/ml)

Incubation for 24 h
37 ℃. 5% CO270000

cell concentration

Add FD-150
to cell culture

Plasma treatment

Fluorescence (a.u.)

Cell treatment procedure

1.00E+06

1.00E+05

1.00E+04
control

1 min

2 min

5 min

treatment time

Length of treatment time does notaffect cell viability significantly
differently

Conclusions■ Applying of air microplasma can increase of drug absorption into intestinal cells■ Increasing of distance from plasma decreases probability of the cells to absorb drug but does not affect cell viability significantly diffrently■ The higher voltage can increase drug uptake but also decrease viability of intestinal cells■ Different treatment time did not affect cell viability differently. Drug uptake was similar for treatment time 1 min or 2 min and decreased in thecase of 5 min of plasma treatment