(621e) Laser-Activated Carbon Nanoparticle Cellular Damage and Prevention

The cell membrane is a challenging barrier to
cross because of its structure and composition. Endocytotic
processes can be inefficient, especially for molecules that have therapeutic
value such as siRNA, plasmids, etc. So it is a
challenging task to come up with a method that can address the issue of
intracellular delivery.

Various methods have been employed, like
chemical modification of the drug to induce stronger binding to the cell, using
vectors to ?infect? cells or using a physical force to temporarily disrupt
cells and increase their permeability. The physical methods are sometimes
preferred because they are more broadly applicable and involve less
modification to the drug. Some examples of these methods use ultrasound,
electroporation and lasers for intracellular delivery. The tradeoff associated with
using such methods is that generally the forces that cause the cell to permeablize can also cause a fraction of the cells to
disrupt, fragment and die. So, there is a need to optimize the process and
conditions under which physical methods are operated.

In our previous studies, permeablization
of cells was observed when they were exposed to pulsed laser in the presence of
carbon nanoparticles.¹Photoacoustic pressure
waves², generated as a result of laser-nanoparticle interactions,
are believed to cause temporary membrane disruption, which effectively
increases permeability. Since the carbon nanoparticles serve as strong
laser-absorption sites, the overall absorption capability of the medium
increases. This means that much lower laser fluence
is required to have an effect on the cells compared to laser delivery systems
without nanoparticle absorbers. This kind of drug delivery system is appealing
not only because of its novelty, but also because it has the advantages of low
exposures and better control of the acoustic field, both of which enable better
targeting of drug delivery within the body.

Here we report i) the dependency
of uptake and viability on the time of laser exposure, ii) optimized conditions
to achieve maximum uptake with minimal loss to cellular viability and iii)
potential ways to increase viability of the cells.

A Nd:YAG
nanosecond pulsed laser (Continuum Powerlite III
Plus, Santa Clara, CA) of 1064 nm wavelength was used for the exposure. The
sample holder was made of Pyrex glass (Starna Cuvettes, 37PX-2) since it has high transmittance to
infrared wavelength. Human Prostrate cancer cells (DU145) were used as a model
cell line in this study. Carbon nanoparticles were made by sonicating
carbon black (Black Pearls^® 470, Cabot
Corporation) in DI water, with Tween 80 was
used as a stabilizer, to final carbon concentration of 0.4 g/L. The final
suspension was made by adding calcein (10 µM) and
carbon black (25 mg/L) to cell suspension containing ~1 million
cells/mL in RPMI. Total volume of this
solution exposure to laser was 570 µL.

The laser fluence
and time were varied for the exposure. After exposure the cells were stored in
ice (0 ? 4ºC) for at least 5 min until all the exposures were done. The uptake
and viability analysis was either done visually using a confocal microscope (Zeiss LSM META/NLO 510) or using flow cytometry (FACS LSR II, BD Bioscience, NJ, USA). To quantify
cell fragmentation, we count the number of cell events while keeping the time to
flow through the flow cytometer fixed and then
compare the number of exposed cells to the unexposed controls. For staining the
intact nonviable cells, PI was used. At least three replicates were taken for
each experiment. The data acquired as uptake and viability were
corrected by comparing them with the control (unexposed cells). Paired t-test
was performed to compare between samples. Pluronic
solution was prepared by adding 0.5 g Pluronic F-127
to 50 mL DI water, and then thorough mixing at 37°C,
followed by sonication for 5 min until a clear
solution was formed. The final concentration of the stock was 10 g/L.

When the exposure was set to
18.75 mJ/cm² (Figure 1) and time was varied
from 0, 1, 3, and 7 min, it was observed that uptake of calcein
was observed in 7%, 29%, 61% and 87% of cells, respectively, while viability
remained close to 100%. Moreover, when the fluence
was increased to 25 mJ/cm²,
the uptake initially went up from 55% of cells at 1 min exposure to 70% at 3 min,
and then it fell to 56%. The viability was 87%, 86% and 56% at 1, 3 and 7 min,
respectively. At the lower fluence of
18.75 mJ/cm², the photo-acoustic
pressure field intensity generated as a result of laser carbon interaction should
be lower compared to the 25 mJ/cm² field. We
expect that the cells disrupt less as a result of these waves in the case of
the lower fluence. Even when the time of exposure was
increased, it did not cause increased loss of cell viability. More cells exhibited
uptake of calcein in the presence of the field. In
the case of 25 mJ/cm², exposure initially
at 1 min resulted in about 13% loss of cells with more than half of the cells
with calcein in them. Since here the field was much
stronger, exposure at longer times resulted in death of cells, which caused the
viability to go down, which in turn reduced the uptake of calcein.
The optimal uptake was found to be 87%, where DU145 cells with 25 mg/L carbon nanoparticles were exposed for 7 min at 18.75 mJ/cm² laser fluence.
It should be noted that such efficient uptake with high viability is much
better than uptake reported by many other intracellular delivery methods.

Pluronic surfactants have been shown in
the past to protect against membrane disruption and damage both in-vivo and
in-vitro. Cells were exposed to laser pulses at 44 mJ/cm² for 3 min as a negative control.
This is a very extreme condition where many cells are killed. Figure 2 shows
the effect of adding 5% v/v F-127 Pluronic on the
uptake and viability. It can clearly be observed that when no surfactant is
added to the system, the viability is about 28% with almost all cells calcein positive, which was expected from such extreme
conditions. But when surfactant was added to the system and the same exposure was
carried out, the uptake and viability significantly went up to the value of 85%
and 90%, respectively. This experiment demonstrates that surfactant is able to
save cells from damage.

This study supported the
hypothesis that photoacoutic waves generated by laser
excitation of carbon nanoparticles cause
increased intracellular uptake of molecules into viable cells. The level of
uptake and viability depended on laser exposure parameters. At optimal
condition in this study, 87% of cells exhibited intracellular uptake of the
marker compound, calcein, with cells maintaining almost
100% viability. It was also observed that damage to cells at strong laser
exposures can be controlled using pluronics to further
optimize uptake with reduced loss of cells. Overall this method holds exciting
potential of delivering molecules efficiently into cells with low cytotoxic
effect and can be of interest not only for in-vitro but also future in-vivo
drug delivery into cells.

REFERENCES

1.     
P. Chakravarty, W. Qian, M.A. El-Sayed & M.R. Prausnitz,
Delivery of molecules into cells using carbon nanoparticles activated by
femtosecond laser pulses. Nature Nanotechnology 5, 607?611 (2010)

2.     
H.X. Chen & G. Diebold, Chemical generation of acoustic waves
- A giant photoacoustic effect. Science 270,
963-966 (1995). 

ACKNOWLEDGMENTS

This work was carried out at the Center for
Drug Design, Development and Delivery and the Institute for Bioengineering and
Bioscience at Georgia Institute of Technology with partial financial support
from the U.S. National Institutes of Health.

Figure 1: Photoacoustic effects on intracellular uptake and viability of DU145 cells
as a function of laser exposure conditions, using calcein
as an uptake marker and carbon nanoparticles at 25 mg/L.

Figure2: Effect of adding F-127 Pluronic on the uptake
and viability of DU145 cells. Cells were exposed at 44 mJ/cm² for 3 min.