integrated fluorescence intensity
Assessment of use as Photosensitizers through Quantum Yield and Binding Studies of Novel Nanoparticles Attached with
Fluorosensitive Dyes
Nick Jenkins1, Ragini Jetty2, Michael Daniele2, Stephen Foulger2
1
Department of Physics, Furman University, Greenville, SC 29613
2
School of Materials Science and Engineering, Clemson University, Clemson, SC 29634
Introduction: Photo Dynamic Therapy (PDT) is a relatively
fluorescent intensity peak via quenching. Additional
new method of cancer treatment. It requires a photosensitizer
absorbance and PL readings were taken at peak absorbance
that is excited via a light source to an excited triplet state
wavelengths for three more dyes which were then used in
which then quenches out releasing to a ground state in the
click reactions with each other and examined through NMR to
oxygen present. This process releases a singlet oxygen which
confirm the success of the click. The clicks were carried out
is highly toxic and eliminates the cancerous tissue. A similar
by stirring for 24 hours the “A” dye, “B or C” dyes, Sodium
method can be used for imaging1 and drug detection. Previous
Ascorbate, and Copper Sulfate all in Methanol in a 1:1.5:10:2
studies4 have shown that low Quantum Yield and non-specific
mol ratio. An extraction was performed with DCM, products
binding and quenching are some of the main difficulties that
were washed with water. The organic layer was separated out
must be overcome to make an effective fluoroprobe for
using Sodium Sulfate through gravity filtration. Lastly, the
imaging or elimination of cancerous tissue. In order to access
dye was recrystalized in Diethyl Ether.
the novel nanoparticles and dye’s efficiency as
Results:
photosensitizers they were tested for fluorescent quantum
1.8e+6
yield (QY) via a 5-point method. Also, the Indocyanine Green
HITCIl_abs vs HITCI_int
04p23s1_abs vs 04p23s1_int
Dye (ICG) which has been examined previously for its use in
HITCI fit
04p23s1 fit
2
1.6e+6
fluoroprobes that was attached to the particles was tested for
3
changes in binding properties and thermal stability with
1.4e+6
Bovine Serum Albumin (BSA) and the Human counterpart
(HSA), proteins which make up the majority of their
respective blood plasmas. Various dyes were tested for
1.2e+6
absorbance and PL intensity both before and after a series of
click reactions with each other were performed. After
1.0e+6
extracting the product from the click, Nuclear Magnetic
Resonance (NMR) was performed to compare the projected
8.0e+5
results of the click with the actual results to confirm the
success of the click reaction. 6.0e+5
0.008
0.010
0.012
0.014
0.016
absorbance
Determination of slopes resulting from integrated peak areas,
directly proportional to QYs. Lower slope indicates lower QY.
QYs averaged for this example was 12% for the marker and
9% for the particles.
newBSA-ICG (PBS)
7000
0microl ICG
20microl ICG
40microl ICG
80microl ICG
120microl ICG
200microl ICG
280microl ICG
440microl ICG
6000
5000
fluoresence
Experimental: The quantum yield measurements were found
through the comparative method by first taking the absorbance
of HITCI as a marker though DLS ( Perkin Elmer
UV/VIS/NIR Spectrometer Lambda 900) at 5 points between
0.75 and 1.25 relative absorbance and then taking the
photoluminescent (PL) intensity on a fluoroscope (Jobin Yvon
Spex, model: ASC-UV) when excited at the peak wavelength
of absorbance for each concentration. This process was
repeated with 5 concentrations of both single and double azide
modified particles attached to Propyl Acetate (PA) as well as
the single azide modified without the PA group. These were
done at the same concentrations as the HITCI for comparative
evaluation of the QY. Taking a ratio of the integrated area
under the PL peak to the integrated area under the absorbance
peak gives a factor which is directly proportional to the QY.
This value was used to then determine the QYs of the particles
and marker through comparative analysis. The definitive data
for the ICG binding studies was carried out on a (Jobin Yvon
Horiba Fluorolog) as it allowed to excite the sample closer to
the accurate wavelength than the former fluoroscopes (Jobin
Yvon Spex, model: ASC-UV) allowed on the initial runs of
this procedure. In both 1.4 microMolar 98% pure BSA was
used and 1 mM ICG in MeOH was titrated in 20 microLiter
increments into an initial 2.9 mL of BSA with PL being taken
after each titration. This same procedure was carried out with
the same concentrations and amounts using HSA. In another
experiment, particles themselves without ICG were titrated
into the same amount and concentration of BSA using the
same methods. The total amount titrated in was half as much
as the ICG to account for its increased ability to lower the
4000
3000
2000
1000
0
200
250
300
350
400
450
500
550
wavelength (nm)
Quenching of the fluorescent peak for BSA through addition
of ICG added in 20microl increments.
but much smaller than the peak at which it was excited. The
opposite can be seen when exciting about 15nm lower at the
left peak which then has the dominant PL signature while the
right peak can be seen offset from it. Many solvents were tried
to attempt to recrystalize the product from the click reactions.
Finally, the product was first purified by running through an
improvised column created with a pipette and then
recrystalized in diethyl ether in an ice bath. The NMR spectra
for the predicted and actual product of the click reaction dyes
differed in the intensity of many of the peaks, but the locations
were primarily correct. The best evidence to support a
successful click was strong and accurate signals from the
protons on the bridge connecting the two dyes.
YB317 (MeOH)
1.4
5microg/ml
6.67microg/ml
1.2
1.0
absorbance
0.8
0.6
0.4
0.2
0.0
-0.2
0
200
400
600
800
1000
1200
wavelength (nm)
Absorbance peak for the dye that was the common factor in
both click reactions. Shows doublet peak centered around
525nm.
YB317 (MeOH)
1e+6
5microg/ml
6.67 microg/ml
Photoluminescence
8e+5
PL for the same dye, excited
at its higher absorbancy peak,
easily distinguished as around
525 nm.
6e+5
4e+5
2e+5
0
450
500
550
600
650
700
750
wavelength (nm)
Discussion:
The data obtained from the QY measurements is problematic
as the slopes of the marker (HITCI) and the particles are not
even, which is necessary in comparative QY analysis. This
was seen through all QY testing done on each of the different
particles. HITCI was chosen because it emits in the near
infrared (725nm) , but a different marker will need to be
chosen and the tests rerun to obtain accurate QY values.
However, the preliminary value of the ICG attached particles
put them on an order of magnitude 100 times greater than the
particles without, at least giving an indication of positive
correlation of ICG clicked on and QY increasing. The ICG
binding showed that as it approaches 14% ICG to total
solution of ICG in BSA that the fluorescence peak is quenched
almost completely out and the aggregate peak dominates
strongly. Similarly, the particles into the BSA showed the
same strong quenching approaching 9%.
In the absorbency for the YB317 particles a double peak
appears centered around 525nm. When exciting at the higher
peak you obtain the PL shown, where the left peak is present
Conclusion: Quantum yield measurements for each of the
particles tested not sure of accuracy because of choice of
HITCI as a NIR marker. However, preliminary results show a
100 fold increase with ICG attached than without. This
indicates promise for future testing with different markers and
particle-dye combinations.
ICG binding results show consistency to a high degree with
current literature1. Quenching was achieved at approximately
14% pure ICG and 9% with the particles by themselves. This
shows quantitatively the binding relationship between the ICG
or particles and BSA.
The click reactions have been carried out and NMR data
collected that strongly indicates affirmation of the reaction
occurring. Data collection for the combined product and the
comparison to pre-reaction data for the individual dyes is
underway.
References: 1. Altinoğlu, E. Russin, T.J. Kaiser, J.M. Barth,
B.M. Eklund, P.C. Kester, M. Adair, J.H. Near-Infrared
Emitting Fluorophore-Doped Calcium Phosphate
Nanoparticles for In Vivo Imaging of Human Breast Cancer;
ACS Nano. 2008 Oct 28;2(10):1984-6.
2. Berezin, M.Y. Guo, K. Akers, W. Livingston, J. Solomon,
M. Lee, H. Liang, K. Agee, A. Achilefu, S. Rational
Approach To Select Small Peptide Molecular Probes Labeled
with Fluorescent Cyanine Dyes for in Vivo Optical Imaging
Biochemistry. 2011 Apr 5;50(13):2691-700. Epub 2011 Mar
8.
3. Michnik, A. Thermal Stability of Bovine Serum Albumin
DSC study. Journal of Thermal Analysis and Calorimetry.
2003. Volume 71, Number 2, 509-519.
4. Rungta, P. Bandera, Y. Roeder, R. Li, Y. Baldwin, W.
Sharma, D. Sehorn, M. Luzinov, I. Foulger, S. Selective
Imaging and Killing of Cancer Cells with Protein-Activated
Near-Infrared Fluorescing Nanoparticles. Macromolecular
Bioscience. 2011. Volume 11, Issue 7, pages 927-937, July 7,
2011.
Acknowledgements: The authors would like to acknowledge
funding from the National Science Foundation, Award
Number: 1062873, for the REU Site: Interfaces and Surfaces:
Exploring and Experiencing Science (I SEE Science). The
lead author acknowledges the aid of the R. Jetty and M.
Daniele for their training and assistance and S. Foulger for his
guidance. Thanks go to B. Baker for his initial advising and
M. Kennedy for her consistent interest and support in leading
the REU program that made this possible.