Supplementary Information
Photothermal nanodrugs: potential of TNF-gold nanosphere
conjugates for cancer theranostics
Jingwei Shao1,2, Robert J Griffin3,4, Ekaterina I. Galanzha2, Jin-Woo Kim5,6, Nathan
Koonce3, Jessica Webber3, Thikra Mustafa7, Alexandru Biris7, Dmitry A. Nedosekin2,
Vladimir P. Zharov2,4*
1
College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, Fujian, 350108, China
2
Phillips Classic Laser and Nanomedicine Laboratories, Winthrop P. Rockefeller Cancer Institute,
University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA
3
Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock,
Arkansas 72205, USA
4
Arkansas Nanomedicine Center, University of Arkansas for Medical Sciences, Little Rock, AR, 72205,
USA
5
Bio/Nano Technology Laboratory, Institute for Nanoscience and Engineering, University of Arkansas,
Fayetteville, AR, 72701, USA
6
Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR,
72701, USA
7
Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, AR, 72204,
USA
*Correspondence to ZharovVladimirP@uams.edu
Supplementary Figure Legends
Fig. S1. Optical images of image of Au-TNF nanoparticle clusters during solution drying.
Fig. S2. Atomic force microscopy (AFM) images of Au-TNF nanoparticle clusters during solution
drying. (a) Low resolution of AFM image. (b) Magnified image of part of (a). (c) Magnified image of
part of (b). (d) Magnified image of part of (c). (e) Magnified image of part of (d).
Fig. S3. Histological analysis of Au-TNF in tumor tissues. Silver staining of gold at low
magnification (40x) enhances AuTNF presence and indicated Au-TNF preferential accumulation as
large clusters by black dots around blood vessels in the tumor tissue sections. Biodistribution in tumor
tissue was assessed at 8hrs post IV injection. (Shah et al. 2012, Ref. [30] in the main text).
Fig. S4. Electron micrographs (x 20,000) of biopsies from a patient with inoperable pancreatic
adenocarcinoma: (a) Au-TNF nanoparticles in normal liver, and (b) in tumor tissues. The
distribution of gold nanoparticles indicating as alone (blue arrow) or clusters accumulation (brown
arrow) in tumors, with few particles in healthy tissue (from Cytimmune, Inc. Clinical trial results[1], S.
K. Libutti et al [2], 2007). (c) To confirm the presence of Au-TNF in histological samples and
distinguish them from the biological tissues we performed local X-ray spectroscopy microanalysis on
the particles observed by TEM, the inset .
Fig. S5. Cell viability of 2H11, 4T1-GFP and SCK cells incubation with different concentrations
of Au-TNF for 24 hours. The data represented the means of triplicate measurements. All data are
presented as mean ±SD.
Fig. S6. A schematic of experimental setup for window chamber tumor model integrating: optical
transmission, fluorescence and PA imaging modalities.
Fig. S7. Typical PT signals from cells. (a) Linear PT signal from WBCs; (b) Linear PT signal from
RBCs; (c) Nonlinear PT signal from cancer cell (SCK) with nanoparticles. The time scale: (a) 20 μs/div;
(b) 10 μs/div; (c) 1 μs/div.
Fig. S8. Cell viability after administration of Au-TNF alone and with laser at different Au-TNF
concentrations for 24 h.
Fig. S9. Cell viability after administering 10 µg/mL Au-TNF or AuPEG pre-treated with laser. (a)
2H11 endothelial cells. (b) SCK tumor cells; Nanoparticle samples were left untreated or treated with
different laser wavelengths and energy prior to incubating with cells. Control cell samples (PBS only)
were used to normalize cell viability (100%).
Figure S10. Absorbance of individual and clustered gold NPs. (a) 30-nm and 100-nm NPs spectra
according to Mie Theory. (b) Red shift in clustered NPs. Absorbance efficacy is given for a single NP
in cluster and is normalized on the maximum Kabs for an individual 30-nm particle.
Fig. S11. Theoretical calculations of the temperature increase in 30-nm NPs under 532 and
690-nm laser light. (a) Temperature of 30-nm NP at the end of 10 ns laser pulse as a function of laser
energy fluence. (b) Scheme of possible therapeutic effects of laser heated NP.
Fig. S12. Temperature increase in clustered 30-nm NPs under 690-nm laser. Temperature increases
above the level of explosive water cavitation is for clusters and aligned NPs.
Fig. S13. TEM of Au-TNF nanoparticles exposed to 532 and 690 nm laser light.
Figure S14. Polymer coating around NPs. (a) Au-PEG NPs with no TNF load; (b) Au-TNF NPs not
exposed to laser light (control); (c) Au-TNF NPs exposed to 532-nm laser at laser fluence of 50 mJ/cm2.
(d) average layer thickness for these samples (10, 35, 49 particles, correspondingly); (e) changes in
particle size distribution after laser heating. Laser parameters: 532 nm, 50 mJ/cm2 and 690 nm, 1.0
J/cm2.
Fig. S15. PA and optical imaging of mouse vasculature through a window chamber. (a) Control
(healthy) tissues. PA imaging reveals few clusters producing intensive PA signals. (b) Tumor
vasculature. PA imaging reveals both homogeneous accumulation of Au-TNF in the vessels and the
presence of Au-TNF clusters in the vasculature.
Fig. S16. Laser damage of normal vasculature. Control mouse (no tumor) tissues were exposed to
532-nm laser operating at 50 mJ/cm2 energy fluence after injection of Au-TNF. (a) before laser; (b)
after laser irradiation.
Supplementary Document 1.
Laser heating of individual and clustered NPs
Absorbance of individual and clustered NPs.
To estimated absorption efficacy, Kabs, of individual 30-nm NPs we used Mie Theory calculations[3]
(Online Mie Theory Simulator (OMTS), http://nanocomposix.com/support/tools), experimental spectra
of the particles (Fig. 2f) and the data on spectral changes in NP clusters from our previously published
calculations [3]. In general, the calculated spectra matched the experimental well. However, measured
spectra featured slightly higher absorbance in 600-750 nm region, that we believe is associated with the
fact that Au NPs are not ideal spheres (Fig. 2a), and, thus, they absorb light at longer wavelengths.
Figure S10 illustrates changes in absorbance spectra of individual spherical NPs during clustering.
Our calculations, Fig. S10, confirm red shift in absorbance spectra and an increase in absorbance
efficacy of the clustered NPs at longer wavelengths. Absorbance efficacy in the region of 650-750 nm
is higher for NPs in a cluster than for a large spherical particle due to lower symmetry of the cluster.
The calculated spectral shift is usually below 100 nm, thus limiting laser wavelength available for
photothermal therapy to the range of 650-700 nm, where blood has minimal absorbance and clusters of
NPs have rather higher absorbance efficacy.
Calculations of the temperature change in NP core
We calculated temperature level for a NP by the end of a single laser pulse (duration tp = 10 ns, energy
fluence per pulse, F [J/cm2]). We assumed, that a particle is heated uniformly (fast, ~ 1ps,
electron-photon coupling, and high thermal diffusivity of gold, 1.2×10 -4 m2/s [4]). Finally, as laser
pulses were longer than the characteristic heat exchange time (  T ~ r 2 / 4 , where  is thermal
diffusivity, 1.47×10-7 m2/s for water) between NP and medium (0.38 ns for 30-nm NP in water), we will
take into consideration NP cooling by heat transfer to the medium during the laser pulse.
According to [4] the temperature increase, T, at the surface of the particle by the end of a laser pulse
is given as:
T 

FKabs r0 
3kwater
t) ,
1  exp(
2 
4tp kwater 
cNP  NP r 
where 0 and c0 are the density and specific heat capacity of the particle material, kwater is thermal
conductivity (0.596 W m-1 K-1 for water). To simplify the calculations we did not account for the energy
of phase transformations (gold melting, water evaporations and etc).
Individual 30-nm Au-NPs
First, we calculated the increase in the temperature of NP core by 532 and 690 nm lasers, Fig S11a.
Our calculations show that an ideal spherical 30-nm particle cannot efficiently absorb laser light at 690
nm, thus, even at the fluence of 1.0 J/cm2 the temperature of metal core increases to 373 K only (100
C, i.e. no water cavitation). However, the fact that Au-TNF particles are not ideally spherical required
corrections to these calculations. Here, we used experimental spectra to calculate absorption efficacy at
690 nm. Our data shows that even single NPs may be heated up to 755 K, i.e. it is possible to expect
explosive water cavitations and particle fragmentation.
For 532 nm laser light our calculations shows that already at laser fluence of 16 mJ/cm2 it is possible to
expect formation of nanobubbles (nano-cavitation, NP core heated to 100 C). Nanocavitation is
usually safe for the cellular structures as air bubble dimensions are just slightly larger than NPs
themselves and collapse in few hundreds of ns. For laser fluence above 60 mJ/cm 2 much stronger
cavitation is expected, as NP would reach T ~ 650 K, i.e. the temperature of explosive water cavitations
(Pustovalov and Zharov et al. 2008, Ref. [30] in the main text). Strong pressure waves (photoacoustic
effect) would appear in the sample due to blooming of micro-bubbles and may even lead to NP
fragmentation. Further laser fluence thresholds of 170 and 450 mJ/cm2 would correspond to melting
and evaporation of gold nanoparticles, Fig. S11b. In this case direct thermal effects and proteins
denaturation would take place.
Thus, our data shows that heating of individual particles by 690-nm laser may be an effective way
toward heating them up due to the fact that real NPs does not have ideal spherical symmetry. At the
laser fluence levels up to 1 J/cm2 it is possible to expect NP heating up to 700 K and its fragmentation.
Heating of clustered Au-NPs
Next, we estimated temperature of the clustered NPs using two approximations: a) several NPs are
arranged into a straight line b) N NPs form a 3D cluster. According to our previous calculations [3] the
normalized absorption efficacy depends on the configuration of the cluster and the distance between
particles. For example, 4 Au-NPs arranged into a line produce spectral shift to NIR range with a
maximum at 720 nm (1 nm distance between particles), Fig. S10b. Thus, the absorption efficacy for
such cluster at 690 nm is 68 time higher than for a single NP. Larger gap between particles (thicker
coating) may minimize spectral shift and cluster absorbance in NIR, however the absorbance still
would be higher than for individual particles. For a large cluster composed of many particles
absorbance maxima shifts only to ~ 600 nm due to higher spherical symmetry of the construct. An
average absorption efficacy for a cluster having more than 20 NPs at 690 nm is ~ 7 times higher than
that of a single 30-nm particle. Still, the cumulative thermal effect of the cluster is much higher, as all
the NPs of the cluster produce heat. Figure S12 illustrates temperature changes in NPs of the clusters
by the end of 690-nm laser pulse.
Thus, clustering produces red shift in spectra of the nanoparticles increasing the probability of cell
damage or TNF release under NIR laser light. Clustering of NPs into a large aggregate with a certain
spherical symmetry increases PT contrast 4-7 times. Thus, it is hard to expect melting or explosion of
such clusters under 690-nm laser. Still, formation of microbubbles around each NP in such cluster may
be expected at laser fluence of only 0.45 J/cm2. Moreover, cumulative heat release from clusters may
cause certain direct thermal effects in tissues as clusters produces many times more heat than a single
NP. Finally, we should note that smaller clusters not having spherical symmetry and consisting of 4-8
NPs may have an important role as they would have much higher absorbance compared to individual
particles, Fig. S12, in optimal for PT therapy NIR range.
Supplementary Document 2.
TNF release by Au-TNF NPs under the laser light
We used transmission electron microscopy (TEM) to analyze the effects of laser heating on TNF load
of Au-TNF nanoparticles. Based on the calculations of the metal core temperature (Supplementary
Document 1) we have selected the following laser irradiation conditions: 532 nm laser at 50 (ANSII
level of safety for visible range), 125, and 250 mJ/cm2 and 690 nm at 1.0 J/cm2. All the samples of Au
NPs were prepared fresh before the experiment and were exposed to 600 laser pulses (60 s exposure at
10 Hz) in accordance with the methodology developed for irradiation of the cells.
The exposure of Au NPs to the laser light resulted in a number of different effects: particle
fragmentation, melting and complete stripping of polymer layer from the NP surface (Fig. S11b, S13).
We have observed fragmentation of the nanoparticles taking place at a small extent already at 50
mJ/cm2 and destroying almost all the NPs at laser fluence of 125 and 250 mJ/cm2 (Fig. S13). Higher
laser energy also resulted in particle melting leading to formation of ideal spherical particles of smaller
diameter in solution. We believe that the main damage mechanism in this case is the explosion of NPs,
not surface evaporation of gold atoms expected only at much higher laser fluence. We also observed
NP fragmentation with the use of 690-nm laser light at fluence of 1.0 J/cm2. This data well correlates
with the results of theoretical calculations (Supplementary Document 1) and confirms that
nonsymmetrical shape of the NPs have an important impact on sample absorbance in NIR range.
Second, we analyzed the polymer layer around the Au NPs, Fig. S14. To enhance the contrast of
polymer layer around the Au core we used 2% osmium tetraoxide stain conventionally used in
biological matrixes. We calculated average thickness of the layer for samples having only PEG
(Au-PEG), and for samples having both PEG and TNF load (Au-TNF). We observed a significant
difference between layer thickness for Au-PEG and Au-TNF NPs. We believe that increased thickness
of the layer should be related to TNF load as other NPs parameters are the same. Moreover, after
exposure of these NPs to laser light we observed a decrease in layer thickness to the level close to that
of Au-PEG particles. Thus, we can hypothesize that laser light causes unloading of the NPs from the
PEG coating into the surrounding medium. NPs themselves and PEG coating remain mostly intact. We
also detected no polymer coating around NPs fragmented or at those melted at higher laser energy.
Thus, TEM imaging proved that laser heating of NPs unload TNF from the polymer coating, while
leaving PEG layer intact, i.e. not affecting the biostability of the NPs. The fragmentation of the NPs
under high energy laser light may be one of the possible mechanisms of tissue damage with NP
fragments delivering kinetic energy, and potentially toxic fragments deep into tissues.
References:
[1] http://www.cytimmune.com/go.cfm?do=Page.View&pid=31.
[2] Libutti S., Paciotti G., Myer L., et al., Preliminary results of a phase I clinical trial of CYT-6091: a
pegylated colloidal gold-TNF nanomedicine, in J Clin Oncol (ASCO Meeting Abstracts). 2007. p. 3603.
[3] Khlebtsov B., Zharov V., Melnikov A., et al. Optical amplification of photothermal therapy with gold
nanoparticles and nanoclusters. Nanotechnology, 2006, 17(20): 5167.
[4] Pustovalov V.K. Theoretical study of heating of spherical nanoparticle in media by short laser
pulses. Chemical Physics, 2005, 308(1–2): 103-108.