Targeting intracellular

bacteria by PDT
 Uptake of MB by THP1

Aim- To study the uptake of MB by THP1 cells with and without

surfactant.

Materials and Methods

THP1 cells at a concentration of 1 million/mL was added with increasing

concentration of MB (10, 25, 50 and 100 uM).

The cells were incubated for 30 minutes at 370C.

These cells were collected by centrifugation and the concentration of the

supernatant.
Result

The association of MB with THP1 cells increased with increasgin

concentration (A) and sensitization time (B).
There was no significant increase in the uptake of MB when
applied with membrane disrupting triton X100.
The uptake of MB was studied at increasing concentration of MB.
The uptake of MB was about 20% of the applied concentration.
There was obvious difference in the uptake even when MB was
applied with Triton X100 above the critical micellar
concentration of the surfactant.
MTT assay and trypan blue exclusion study to assess the
viability of THP1 cells subjected to increasing sensitization
time.
 Uptake of MB by THP1

Aim- To study the uptake of MB by THP1 cells with and without

surfactant.

Materials and Methods

THP1 cells at a concentration of 0.72X106/mL was added with 50 uM

concentration of MB.

The cells were incubated for increasing time intervals of 1, 5, 10 and 20

minutes at 370C.

These cells were collected by centrifugation and were subjected to 2% SDS

The absorbance of the MB in the supernatant solution was taken and was
calculated for amount of MB taken by THP1 cells.
 6 5E+12

uM of MB
5 NO of MB/Cell
4E+12

NO: of MB/Cell
3E+12
uM of MB

2E+12
2

1E+12
1

0 0
1 min 5 min 10 min 20 min
Duration of sensitization

Increasing the duration of sensitization increased the MB uptake by

THP1 cells
 Targeting internalized bacteria using PDT

• Aim- to kill the bacteria internalized by monocyte (THP1) using PDT approach
• Materials and Methods
• E. faecalis biofilm was grown under nutrient deprived (10% AC in PBS) for 3 days, in 6 well
plates. After the incubation, biofilm was washed three times using PBS. THP1 cells at a
concentration of 2X106/mL was added on to the biofilm. It was incubated for 4 hours in a CO2
incubator. After the incubation time HAP buffer supplemented with antibiotics, penicillin and
gentamicin was added. Incubated for another 6 hours and the monocytes were collected by
centrifugation. These cells were sensitized using 50µM MB in PBS (1mL). The cell
suspension taken in 24 well multiwellplate was irradiated with 664nm for different time
intervals viz, 0, 5 minutes, 10 minutes and 20 minutes. After the specified irradiation time,
samples were withdrawn for 1. CFU counting 2. Trypan blue exclusion assay.
• CFU counting
• 10µL of cell suspension withdrawn was mixed with 10µL of 0.1% tritonx100. After 3 minutes
the resulting mix was added with 980 µL of BHI medium and serially diluted to 102 and 104.
100 µL was taken and plated on BHI agar. The plates were counted and the CFU was plotted
against the irradiation time.
• Trypan blue exclusion assay
• The viability of monocyte with and without the PDT treatment was analyzed using trypan blue
exclusion assay. The cells stained with trypan blue was taken in a hematocytometer and
observed using light microscope.
 6

The log number of bacteria after the PDT 5

treatment. 20 minutes of irradiation

4
resulted in 100% bacterial death.
3

0
0 Min 5 Min 10 Min 20 Min

120

100

80

% Viability
The proportion of viable cells before and 60
after irradiation. The irradiation dose that
result in 100% bacterial kill, produce 70- 40

80% cell death.

20

0
0 Min 5 Min 10 Min 20 Min
 6

Log number of bacteria

The log number of bacteria after the PDT 4

treatment. 20 minutes of irradiation resulted

3
in 100% bacterial death.
2

0
Control 5 mins 10 mins 20 mins

90

80

70

60
The proportion of viable cells before and after % cell survival 50
irradiation. The irradiation dose that result in 40
100% bacterial kill, produce 70% cell death. 30

20

10

0
Control 5 mins 10 mins 20 mins
Influence of sensitization time on the inactivation of intracellular bacteria

Aim- To study the effect of sensitization period on the photoinactivation of E. faecalis internalized
by THP1 cells.

Materials and Methods

THP1 cells at a concentration of 1 million/mL was added to biofilm of E. faecalis (grown under

nutrient deprived condition- 1 day old)

The cells were incubated for 4 hours in CO2 incubator at 370C.

HAP buffer was added and was incubated for 6 hours to kill extracellular bacteria

THP1 cells with internalized bacteria was washed and were subjected to PDT using MB (50 uM).

Increasing sensitization period of 0min 1 min, 5 mins, 10 mins, 15 mins, 20 mins and 25 mins

were used.

100uL of cells were subjected to 1 minute of irradiation (1.8J) and were assayed for CFU (bacteria

survival) and cell viability (monocyte survival)

 Result

There was a linear

decrease in the THP1
viability as the
sensitization time
increased. THP1

However, increasing the

E. faecalis
sensitization time
beyond 1 minute had no
effect on the viability of
bacterial cells

Work done on 24-11-2007

120

THP1
100 E. faecalis

80

60

40

20

0
0 Min 1 Min 5 Min 10 Min 15 Min 20 Min 25 Min
 Percentage Kill
100
120

20
40
60
80

0
0J
/c
m
2
1.
8J
/c
m
2
9J
/c
m
2
18
J/c
m
2
27

Fluence
J/c
m
2
36
J/c
m
2
45
J/c
m
THP1

2
E. faecalis
Effect of PDT on the inflammatory potential of THP1
Aim- To study the effect of PDT on the inflammatory potential of THP1
cells.

Materials and Methods

THP1 cells at a concentration of 0.66X106/mL was added with 50 uM
concentration of MB.

The cells were incubated for increasing time intervals of 1, 5, 10 and 20

minutes at 370C.

These cells were collected by centrifugation and were washed with PBS

Washed cells were added with RPMI media and 100uL taken in 96 wells
plate was subjected to irradiation for 1 minute

These cells were left in CO2 incubator for 2 days and were assayed for
nitrate using Total NO/Nitrite/Nitrate assay (R&D Systems Parameters
Kit).

Absorbance read on plate well reader at 540nm with correction wavelength

at 664nm
 0.1
Irradiated
0.08 Non-Irradiated

0.06

Absorbance 0.04

0.02

0
Control I Min 5 Min 10 min 20 min
-0.02

-0.04

-0.06

-0.08
Duration of sensitization

No enhancement in the inflammatory potential was evident as

indicated by the nitrate level in the cell culture medium.
 Effect of PDT on DNA of THP1-comet assay
Aim- To study the effect of PDT on integrity of DNA of THP1 cells
subjected different duration of sensitization.

Materials and Methods

THP1 cells at a concentration of 0.66X106/mL was added with 50 uM
concentration of MB.

The cells were incubated for increasing time intervals of 1, 5, 10 and 20

minutes at 370C.

These cells were collected by centrifugation and were washed with PBS

Washed cells were added with RPMI media and 100uL taken in 96 wells
plate was subjected to irradiation for 1 minute

These cells were collected by centrifugation and were analyzed for DNA
damage by comet assay
Effect of PDT on the DNA damage of THP1-comet assay

Control 1minute 5 minutes

10 minutes 20 minutes
 Effect of PDT on the DNA damage of THP1-comet assay

Control H2O2

Irradiation alone Sensitization alone PDT

 MTT assay on THP1 subjected to PDT after
different duration of sensitization
Aim- To study the effect of PDT on viability of THP1 cells subjected different
duration of sensitization.

Materials and Methods

THP1 cells at a concentration of 0.66X106/mL was added with 50 uM concentration
of MB in water or in PF4 containing emulsified H2O2 and perfluoro-
decahydronaphthalene.

The cells were incubated for increasing time intervals of 1, 5, 10 and 20 minutes at
370C.

These cells were collected by centrifugation and were washed with PBS

Washed cells were added with RPMI media and 100uL taken in 96 wells plate was
subjected to irradiation for 1 minute

These cells were collected by centrifugation and were assayed for mitochondrial
activity using MTT assay (1mg/mL MTT (100uL), 3 hours of incubation,
precipitate dissolved in DMSO and absorbance measured at 570nm with 630 as
the reference wavelength).
 100
PDT-MB
90

% Reduction in viability of THP1

PDT-PF4
80
70
60
50
40
30
20
10
0
1 min 5 min 10 min 20 min
Duration of sensitization

PDT conducted using PF4 showed higher cell death when

compared to PDT with 50uM of MB.
There was no significant effect on viability upon increasing
the duration of sensitization. However, the mean value of
percentage cell loss increased gradually along the
sensitization time when PDT was conducted using MB in
aqueous media.