Text S1 for
Improved cellular specificity of plasmonic
nanobubbles versus nanoparticles in heterogeneous
cell systems
Ekaterina Y. Lukianova-Hleb, Xiaoyang Ren, Pamela E. Constantinou,
Brian P. Danysh, Derek L. Shenefelt, Daniel D. Carson, Mary C. Farach-Carson,
Vladimir A. Kulchitsky, Xiangwei Wu, Daniel S. Wagner, Dmitri O. Lapotko
1. Molecular targets and cell models
We studied the six cell models representing four molecular targets (Table 1):
1.
Epidermal growth factor receptor (EGFR) was studied as specific molecular target
in head and neck and lung cancer cell lines.
First, we used multi drug-resistant HN31 squamous carcinoma (target) cells (associated
with head and neck cancers) with high expression level of EGFR [1, 2] and immortalized
normal human oral kerotinocyte NOM9 (non-target) cells. Both cell lines were obtained
from The Johns Hopkins Genetic Resoutces Core Facility. The expression level of EGFR
was verified with western blot method (Figure S1). The EGFR expression was found to
be 2.8 times higher for HN31 target cells compared to non-target NOM9 cells.
The NOM 9 cells were cultured in KGM Complete Medium (Cat# CC-3001, Lonza
Walkersville Inc, Walkersville, MD) in a 37°, 5% CO2 incubator. The malignant cells
were transfected with green fluorescent protein (GFP) to allow identification. The HN31
GFP stable cell line was built by transfecting the EGFP C1 plasmid into HN31 cells. The
selecting marker was G418. The transfected cells were cultured in DMEM High Glucose
medium (Cat# 10013 CV, Mediatech Inc, Manassas, VA) supplemented with MEM
Vitamin Solution (Cat# 11120, Invitrogen Corp., Carlsbad, CA) and MEM NEAA
(Cat#11140, Invitrogen Corp., Carlsbad, CA) and penicillin-streptomycin (Cat# 30002
CT), Mediatech Inc, Manassas, VA) in the same incubator. For co-culture, the cells were
re-suspended in KGM media and counted after trypsinization and neutralization. NOM 9
was mixed with HN31 cell in 100:1 ratio, seeded at a density of 700,000 cells/ml in 15well chambered slide (µ-Slide Angiogenesis, Ibidi LLC, Martinsried, Germany) and
grown 24 hours before NP treatment. The 60 nm hollow gold NSs (2.4*1010 particles/ml)
conjugated with Panitumumab antibody were identically targeted to both types of cells in
vitro by incubating for 24 hour with a co-culture of HN31/NOM9 cells under
physiological conditions (37ºC). After that uncoupled NPs were washed off prior to the
generation of PNBs. The viability of the cells was measured after their incubation with
NP-conjugates. The viability of cells was found to be 98% for HN31 cells and 96% for
NOM 9 cells. In order to determine the role of the targeting vector in cellular specificity
of PNBs, we incubated the co-culture of HN31 and NOM9 cells with bare and
Panitumumab conjugated 60 nm gold NSP as described above. For combinatorial
targeting of cancer surface receptors by the two anti-EGFR body (Panitumumab and
C225) we used the 60 nm gold NSP conjugated to Panitumumab antibody and the same
NSP conjugated to C225 antibody. The cells were cultivated and treated by NSP
conjugates as described above. In the cases of pre-treatment cells by C225 or
Panitumumab antibodies alone, the cells were incubated with antibody (30 min, 4°C @
2µg anitibody/ml), washed off from the uncoupled antibodies and treated by NP
conjugates as described above. The viability of cells was more than 98% in all
combinations of treatment of cells by NP-conjugates and antibodies.
Second, we used the EGFR-positive A549 (target) cells [3-5] (associated with lung
carcinoma) and EGFR-negative normal cells, fibroblasts (non-target cells) (cell lines
were obtained from the American Type Culture Collection). The cells were grown in
RPMI-1640 media for 24-30 hours at 37º C in a CO2 incubator in growth chambers (LabTek II, Thermo Fisher Scientific, Pittsburgh, PA) to 70% confluence. For targeting we
used the 60 nm gold 60 nm NSP conjugated with anti-EGFR antibody C225 (BioAssay
Works LLC, Ijamsville, MD). The both types of cells were identically incubated with
NSP-C225 conjugates (concentration 6*1010 particles/ml) for 30 minutes at 37C in the
CO2 incubator. Following incubation, the cells were washed twice by a serum and phenol
red free RPMI-1640 media to remove free NPs. Cell viability was measured after NP
treatment and was 98% for A549 cells and 98% for fibroblasts and not differed from
viability of intact A549 and fibroblast cells (98% and 98%, respectively).
2.
Mucin molecule MUC1 was studied as molecular target in HES cells, a derivative
of WISH/HeLa cells (kindly provided by Dr D. Kniss, Ohio State University, Columbus,
OH). The HES cells with high level of MUC1 expression [6-8] and HS-5 cells with low
expression of MUC1 were used as target and non-target cell, respectively. Cells were
identically targeted with the gold 60 nm NSP conjugated with 214D4 by BioAssay Works
LLC (Ijamsville, MD). HES cells were grown in high glucose DMEM (Life
Technologies, Grand Island, NY) supplemented with 5% (vol/vol) FBS (Life
Technologies, Grand Island, NY) and 1 mm sodium pyruvate (Sigma-Aldrich, St. Louis,
MO). HS-5 cells were grown in low glucose DMEM (Life Technologies, Grand Island,
NY) supplemented with 10% (vol/vol) FBS. Cells were seeded in growth chambers (LabTek II, Thermo Fisher Scientific, Pittsburgh, PA) and cultured for 24-48 h before NP
treatment. The NSP-214D4 conjugated were re-suspended in phenol red free DMEM
media before concentration 2.4*1010 particles/ml. Cell were incubated with the NPcontaining media for 1 h at 37°C in CO2 incubator. After that the cells were washed with
fresh DMEM media to remove free NPs. Viability of NP-treated HES and HS5 cells was
found to be 90% and 85%, respectively.
3.
Prostate Specific Membrane Antigen (PSMA) was studied as a molecular target
in C4-2B prostate cancer cells (were obtained from Dr. Leland Chung (MD Anderson
Cancer Center, Houston, TX) with high PSMA enzymatic activity [9]. As non-target we
used HS5 (human bone stromal cells; were obtained from American Type Culture
Collection) with low level of PSMA expression [10]. Both cell lines were grown as
described earlier [11]. Both cell cultures were identically targeted with gold NPs using
one or two specific cancer surface markers: PSMA that is primarily expressed in prostatic
tissues as well as in bone metastases [10, 12] and EGFR that is expressed in prostate
cancer and malignancies [13]. Target (C4-2B) and non-target (HS-5) cells were
maintained as described previously [14]. Cells were grown to 80% confluence,
trypsinized, washed 1 time with PBS, and re-suspended in serum-free low glucose
DMEM (Life Technologies, Grand Island, NY). Both cell populations were identically
treated (30 minutes at 37 °C in CO2 incubator) with the 60 nm gold spheres conjugated
by BioAssay Work LLC (Ijamsville, MD) to PSMA antibodies [15]. After that the cells
were washed three times with media, and re-suspended in fresh media before PNBs
generation. For combinatorial targeting of two cancer surface receptors we used the 60
nm gold spheres conjugated to PSMA antibodies and the 110 nm silica-gold NSs
conjugated to C225 (anti-EGFR antibody) by BioAssay Work LLC (Ijamsville, MD).
Cell viability was measured by Trypan Blue staining of intact and NP-treated cells and
was found to be 97% and 96% for NP-treated C4-2B and HS5 cells, respectively.
4.
Membrane receptor CD3 was studied as molecular target in the two cell models.
CD3-positive J32 [16] (target) and CD3-negative JRT3-T3.5 (non-target) clones of Jurkat
T-cell leukemia cell lines were purchased from the American Type Culture Collection.
JRT3-T3.5 do not express CD3 [17]. Both cell populations were identically treated with
the 60 nm gold spheres conjugated with anti-CD3 monoclonal antibody OKT3 (BioAssay
Works LLC, Ijamsville, MD). Cells were re-suspended in a serum free RPMI-1640
medium (Invitrogen, Grand Island, NY) containing OKT3-conjugated NPs at a
concentration 1.2*1011 particles/ml and incubated with the NP-containing media for 45
minutes at 37°C in CO2 incubator. Following incubation, the cells were washed to remove
free NPs and finally suspended in fresh RPMI-1640 medium supplemented with 10%
FBS (Atlanta Biologicals, Lawrenceville, GA), 2 mM l -glutamine, 100 units/mL
penicillin, and 100 mg/mL streptomycin (Life Technologies, Inc., Grand Island, NY) and
placed in growth chambers (Lab-Tek II, Thermo Fisher Scientific, Pittsburgh, PA). Then
the cells were treated with laser pulses. The cell viability was tested immediately after
treatment of cells by NP conjugates. The viability of J32 and JRT3-T3.5 cells was 96%
and 98%, respectively.
In another CD3 cell model we used human T-cells. PBMC (peripheral blood
mononuclear cells) were isolated from buffy coats (obtained from the Gulf Coast Blood
Center, Houston TX), by ficoll gradient. CD3-positive T cells (target) were enriched from
PBMC using a Pan T cell Isolation Kit II (Miltenyi Biotec, Inc., Auburn, CA), and CD3negative cells (non-target) were enriched by depletion of CD3-positive cells from PBMC
using CD3 microbeads (Miltenyi Biotec, Inc., Auburn, CA) according to the
manufacturer’s instructions. The expression levels of CD3 in target and non-target cells
were obtained with flow cytometry (Gallios Flow Cytometer from Beckman Coulter)
(Figure S2) and showed significant difference: the expression of CD3 in target cells was
found to be 25 times higher than in non-target cells. CD3-positive (target) and CD3negative (non-target) cells were incubated at 5*106 cells/ml with the 60 nm gold NSP
conjugated (1.2*1011 particles/ml) by BioAssay Works LLC (Ijamsville, MD) with
target-specific antibody OKT3 for 30 min at 37ºC, 5% CO2, washed three times with
media, and re-suspended in phenol red-free RPMI with 10% FBS for PNB treatment. Cell
viability was tested in 72h after NP-treatment and was 98% for CD3+ cells and 97% for
CD3- cells.
2. Plasmonic nanobubble generation
Optical generation and detection of the PNBs was performed with a photothermal laser
microscope that we developed previously [18, 19]. In our work we employed a pulse of
length 70ps with wavelength 532 nm or 778 nm (PL-2250, Ekspla, Vilnius, Lithuania) or
500 ps with wavelength 532 nm (STH-01, Standa Ltd, Vilnius, Lithuania) and/or 787 nm
(custom made dye laser). Laser beam diameter was 15µm for PNB generation around
individual NP clusters and in individual cells and 260 µm for PNB generation in
monolayers of co-culture of HN31/NOM9 cells in scanning mode with a covering area of
several hundred cells. The laser pulse fluence (10-90 mJ/cm2) was experimentally
determined for each pair of the target and non-target cells to exceed the PNB generation
threshold in target cells and to be below PNB generation threshold for non-target cells.
The fluence of each laser pulse was measured by registering its image and measuring of
the beam diameter (at the level of 1/e2 relative to the maximal intensity in the center of
the beam) at the sample plane with the imaging device (Luka, Andor Technology,
Belfast, Northern Ireland) and by measuring of the pulse energy using a pulse energy
meter (Ophir Optronics, Ltd., Jerusalem, Israel). This scheme provided direct and precise
measurements of the incident optical fluence at the cell plane for each excitation pulse
(Figure S3). Operation of all hardware was controlled by PC through custom software
modules developed using the LabView platform.
3. Imaging and metrics for NPs and PNBs
To image and quantify the uptake of gold NPs by individual cells we imaged and
measured optical scattering by gold NPs in individual cells. As a rule we used laser
confocal microscopy (LSM 710, Carl Zeiss Inc, Germany) to obtain the stack of several
images per each cell (in case of A549 cells the NP scattering was imaged and quantified
with regular inverted microscope). In each population (sample) 30-50 cells were analyzed
and the population-average image pixel amplitudes I mean were calculated for each cell
sample:
𝐼𝑚𝑒𝑎𝑛 =
𝐾
∑𝑀
𝑖=1(∑𝑗=1((𝐼𝑠𝑐𝑖,𝑗 −𝐼0 )−(𝐼𝑠𝑐1𝑖,𝑗 −𝐼01 ))
𝑀
(1)
where Isc – mean scattering pixel amplitude of individual NP-treated cell as measured in
specific region of interest (ROI) with its shape and area being close for those of
individual cell, Isc1 - mean scattering pixel amplitude of individual intact cell (obtained
under identical settings), I0, I01 – background image amplitude obtained from
nanoparticle- and cell-free space in the NP-treated and intact samples, respectively, K – a
number of slices in stack obtained for each cell, M – number of analyzed cells.
We used previously developed methods and experimental set-up [18, 19] to image, detect
and quantify the NP clusters and PNBs in water and individual cells through their optical
scattering images and time-responses detected in parallel with two probe lasers. We used
the two optical metrics of PNB (Figure S3): optical scattering time-resolved image pixel
amplitude and duration of optical scattering time response (measured independently and
simultaneously with optical scattering image). First, time-resolved optical scattering was
used for imaging of PNBs. Excellent optical scattering properties of a PNB [20] were
used for its imaging in water and cells with the second, pulsed probe beam (576 nm, 70
ps, 0.1 mJ/cm2). This beam was directed at the sample under high angle of incidence and
with 10 ns delay relative to the excitation laser pulse. Thus only the light scattered by
transient PNB was collected by the microscope objective (20x) and detected by the
imaging device, a CCD camera (Luka, Andor Technology, Belfast, Northern Ireland) and
custom-designed programs developed with LabView platform. For objects whose
dimensions are smaller than a wavelength, the size of their image does not represent their
actual size. If the image is formed by the light scattered by the object, the maximal
brightness of the scattered light correlates to the size of the object. We used this wellknown rule for the quantitative control of the relative size of the gold NP clusters. This
allowed us to choose and use in one experiment the NP clusters of similar size. The
maximum pixel amplitude that corresponded to the brightest PNB, I, was measured for
each individual cell (or NP cluster) and the background signal I0 was measured for a cellfree (or NP-free) space. The PNB scattering pixel amplitude S was calculated:
𝑁
𝑆=
∑𝑛=1(𝐼−𝐼0 )
𝑁
(2)
where N- number of cells or NP clusters (for studies of NP clusters in water).
Second, the PNB-specific optical scattering response (Figure 3c) was obtained with
additional continuous probe laser (Figure S3). The shape of the time-response signal was
PNB-specific, while the duration of the PNB-specific response characterized the PNB
lifetime. The maximal diameter of the PNB is proportional to the PNB lifetime [20-24].
The second PNB metric was independently obtained with another, continuous, probe
laser (Figure S3). The PNB-induced scattering of a part of the probe beam decreased its
axial amplitude, resulting in a dip-shaped output signal of the photodetector that
monitored the probe beam (Figure 3c). Thus we registered the time response of the probe
laser radiation to the transient scattering effect of the PNB. This mode provided the
monitoring of PNB growth and collapse, and delivered the PNB lifetime that
characterizes its maximal diameter [19-24]. The lifetime was measured as the duration of
the PNB-specific signal at the amplitude level being a half of the maximum of the PNB
response. In addition, we measured the probability of the PNB generation at specific
fluence of the excitation laser pulse. This allowed us to determine the PNB generation
threshold fluence as the excitation laser fluence that provides PNBs generation
probability of 0.5.
All three described above metrics were used for the relative comparison of the two
samples, target and non-target cells, in each cell model.
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