Near-IR mediated intracellular uncaging of NO from cell targeted hollow gold nanoparticles

Article abstract of DOI:10.1039/c5cc07989f

We demonstrate modulation of nitric oxide release in solution and in human prostate cancer cells from a thiol functionalized cupferron (TCF) absorbed on hollow gold nanoshells (HGNs) using near-infrared (NIR) light. NO release from the TCF-HGN conjugates

Full text of DOI:10.1039/c5cc07989f

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Cite this: DOI: 10.1039/x0xx00000x
Near-IR mediated intracellular uncaging of NO
from cell targeted hollow gold nanoparticles
Received 00th January 2012,
Accepted 00th January 2012
Elizabeth S. Levy,# Demosthenes P. Morales,# John V. Garcia, Norbert O.
Reich,* and Peter C. Ford*
DOI: 10.1039/x0xx00000x
We demonstrate modulation of nitric oxide release in solution
and in human prostate cancer cells from a thiol functionalized
cupferron (TCF) absorbed on hollow gold nanoshells (HGNs)
using near-infrared (NIR) light. NO release from the TCF-HGN
conjugates occurs through localized surface heating due to NIR
excitation of the surface plasmon. Specific HGN targeting is
achieved through cell surface directed peptides, and excitation
with tissue penetrating NIR light provides unprecedented spatio-
temporal control of NO delivery to biological targets.
Cupferron (CF) was developed over a century ago as an organic
precipitant for copper(II) and iron(III) ions.13,14 More recently, sev-
eral workers have studied cupferron derivatives as precursors that
are triggered by electrochemical or photochemical oxidation reac-
tions to release one equivalent of the caged NO.15-17 Notably, much
earlier thermal stability studies have shown that simply heating CF
leads to decomposition resulting in the release of one equivalent of
NO, although the other products of this redox reaction were not iden-
tified.13 The question is: how does one trigger this NO release in a
controlled fashion? Our premise is that the rapid heating of hollow
gold nanoshells (HGNs) upon excitation of the surface plasmon with
NIR light18-21 will trigger thermochemical NO release from TCF
absorbed on the surface (Scheme 1). Described are the preparation of
such conjugates and photoexcitation studies that validate this strate-
gy for NO uncaging. Furthermore, we show that orthogonal assem-
bly with a targeting peptide22 provides the means to direct these
nanoparticles to a subset of cells with high precision and efficient
uptake. NIR photolysis thus leads to NO delivery with unprecedent-
ed spatio-temporal control.
Nitric oxide (NO) is a bioregulatory small molecule that mediates
a diverse range of activities from blood pressure to cell apoptosis
depending on its intracellular concentration.1 There are many poten-
tial therapeutic applications of NO delivery; however, this presents a
technical challenge owing to NO's diverse and concentration de-
pendent biological responses and associated chemical reactions. For
example, low NO concentrations promote cell survival and even
proliferation, while higher concentrations result in opposite respons-
es.2,3 Thus, it is imperative to exercise precise control over the dos-
age delivered to cellular sites in order to elicit the desired effects and
avoid undesirable ones.
One approach is to use light to trigger NO release ("uncaging")
from an otherwise inactive precursor, thus allowing precise control
of the timing and location of this event. There are now numerous
examples of NO uncaging using donor molecules photoactivated by
UV-to-visible light;4-7 however, strong absorptions of cellular com-
ponents, especially DNA and hemes, interfere with application of
these shorter wavelengths.8 Given that near-infrared light penetrates
more deeply and is relatively benign to tissue, these laboratories9,10
and others11,12 have been interested in designing systems that employ
NIR wavelengths to trigger the photochemical uncaging of various
bioactive small molecules. In the present study, we describe a
somewhat different tack, namely the use of NIR excitation to trigger
the thermochemical release of NO from a thiol functionalized de-
rivative of cupferron, (ammonium N-nitroso(4-mercapto-
methylphenyl)hydroxylamine, TCF).
_______________________________________________________
*Department of Chemistry and Biochemistry, University of California, Santa
Barbara, Santa Barbara, CA, 93106-9510 USA
E-mails: reich@chem.ucsb.edu, ford@chem.ucsb.edu.
# These authors contributed equally.
†Electronic Supplementary Information (ESI) containing detailed descrip-
tions of experimental procedures and ten figures.
Scheme 1. The surfaces of the hollow gold nanoshells are coated by co-
absorption of the thiolated cupferron TCF and of a thiolated PEG (TPEG or
TPEGRP), which serves as a protective layer and to enhance aqueous solubil-
ity and in the case of TPEGRP provides the cell-targeting peptide. Upon 800
nm laser excitation of the TCF-HGN-TPEG or TCF-HGN-TPEGRP conju-
gates, HGN surface temperatures are rapidly elevated and NO is released.
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Preparation of TCF-HGN conjugates: The hollow gold nanoshells sent,13 one can calculate the surface loading to be ~6 x 104 TCF mol-
(~65 nm diameters, Supporting Information (SI) Fig. S-1)19, 20 and
ecules per nanoparticle (Figure 2, right).
the thiolated cupferron TCF15,16 were synthesized by published
methods as described in the SI. The optical spectrum of the HGNs
HGNs by adding equimolar amounts of TPEG and the non-thiolated
As a control, a nanoparticle suspension was prepared from the
displayed a broad, strong absorption band at λmax ~750 nm with an
extinction coefficient of ~3. x 1010 M-1 cm-1 (Figure 1). This band has
been attributed to the plasmonic transition of these metallic nanopar-
ticles.18,19 Formation of the conjugates between the HGNs and the
cupferron derivative TCF takes advantage of the tendency of thiols
to form self-assembled monolayers on gold surfaces.23 In order to
prevent nanoparticle aggregation and improve aqueous solubility,
this self-assembly was carried out with an equimolar mixture of TCF
and a thiolated polyethylene glycol (TPEG, 5 kDa, purchased from
Nanocs). A 100 µL volume of solution of TCF and TPEG (both 100
µM) in pH 7.4 PBS with 0.01% Tween20 surfactant was mixed with
a 1.0 mL volume of HGNs in 500 µM citrate buffer pH 5.5 (50 pM
HGN concentration). The resulting TCF-HGN-TPEG conjugates
were washed repeatedly by centrifugation for 10 minutes, decanting
the solution from the solid, and then re-suspending in the PBS-
Tween20 solution. This procedure was repeated a minimum of 3
times to remove unbound TCF and TPEG. The visible/NIR spectrum
of the resulting conjugates (Supporting Information Figure S-2) is
qualitatively the same as that found for the HGN particles before
modifying the surfaces. As described below and in the SI, conjugates
with a thiolated polyethylene glycol modified to include the termi-
nally bound targeting peptide RPARAR (TCF-HGN-TPEGRP) and
conjugates that do not present the TCF (HGN-TPEG or HGN-
TPEGRP) were prepared by analogous procedures.
cupferron CF. These were isolated and cleaned in a similar manner,
and then re-suspended in pH 7.4 PBS-Tween20 solution. Analogous
heating at 80 C for 8 h and sampling of the headspace showed that
the NO released was only 10% that released by the TCF-HGN-
TPEG conjugates. The observation that some NO is released by the
control suggests that the preparation of HGN-TPEG ensemble in the
presence of CF retains some of that cupferron, perhaps trapped in the
organic surface coating. However, the much stronger association of
TCF in the TCF-HGN-TPEG conjugates is clearly evident.
o
Figure 2 Right: Nitric oxide release after heating a suspension of the washed
o
and isolated TCF-HGN-TPEG conjugates for 8 h at 80 C. Left: NO release
from comparable solution of conjugates prepared with CF rather than with
TCF. The much stronger signal for TCF (right) can be attributed to the self-
assembly of TCF on the HGN.
At physiological temperatures, both cupferron and thiolated cup-
ferron proved to be quite stable. This was tested by maintaining the
temperature of a 100 µM sample of each in pH 7.4 PBS-Tween20
o
solution for a period of 13 h at 37 C in a closed vessel. The head-
space was then sampled and analyzed, but NO release was not ob-
served in either case. (the NOA detection limit is ~0.5-1 picomoles).
Furthermore, TCF displays very little toxicity as tested with the hu-
man prostate cell line, PPC-1. Cells incubated for 24 h with increas-
ing TCF concentrations showed little effect on cell viability up to 10
µM in the medium according to the PrestoBlue staining assay (Life
Technologies). Higher concentrations did show some modest toxici-
ty (SI Fig. S-3)
400
600
800
1000 1200
Wavelength (nm)
Figure 1. Characteristic NIR absorption spectrum of the hollow gold
nanoshells used to prepare the TCF-HGN-TPEG conjugates. The band cen-
tered at ~750 nm is attributed to surface plasmonic excitation. Inset: TEM of
these HGNs. Scale bar is 100 nm.
Excitation of TCF-HGN conjugates: NIR irradiation at a wave-
length corresponding to excitation of the surface plasmon of the
thiolated cupferron-hollow gold nanoparticle conjugates leads to NO
release. A solution of the TCF-HGN-TPEG conjugates (48 pM in
100 µL volume of the pH 7.4 PBS-Tween20 solution) in a sealed
quartz cuvette was subjected to 800 nm excitation from the high
intensity pulses from a Ti:sapphire ultrafast pulsed laser. The result
was controlled NO release. Irradiation for 30 s at 1 KHz (140 fs
pulses, 5 mm beam diameter, Gaussian profile pulses) at an average
power of 5 W cm-2 generated 240 pmoles of NO as detected by sam-
pling the gaseous headspace and measuring the NO content using the
NOA. This quantity corresponds to 80% conversion of the total TCF
present, according to the above data on the loading. Similarly, 800
nm photolysis with the same laser system but at the lower average
laser powers 0.9, 1.5 and 2.4 W cm-2 generated 42, 126 and 156
The loading of the thiolated cupferron on the HGN conjugates was
evaluated by heating a 100 µL volume of the nanoparticle suspen-
sion at 80 °C for 8 h in a closed vessel having a port fitted with a
syringe cap. A gas-tight syringe was then used to withdraw a sample
of the gaseous headspace and transfer this to a GE Siever's Nitric
Oxide Analyzer (NOA). The amount of NO thus detected by the
NOA indicated that 300 pmoles of NO were released from the solu-
tion containing the TCF-HGN-TPEG conjugate. Further heating did
not result in the release of additional NO, so it was concluded that all
the TCF had undergone thermal decomposition. The HGN concen-
tration in the 100 µL solution was calculated to be 48 pM from the
optical density. Thus, by drawing on the earlier observation that one
mole of NO is generated thermally for each mole of cupferron pre-
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pmoles of NO, respectively, from analogous solutions of the TCF- made it difficult to detect NO production in the PPC-1 cells, those
HGN-TPEG conjugates (Figure 3).
experiments were conducted using the 22Rv1 cells.
As described in the SI, the 22Rv1 cells were first treated with the
nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (L-
NAME). One set of cells was then incubated with the TCF-HGN-
TPEGRP conjugates for 2 h, while a second set was incubated with
the HGN-TPEGRP analog without the caged NO TCF, and repeated-
ly washed with buffer to remove non-internalized nanoparticles be-
fore use. Both sets of cells were incubated with DAF-2 DA (see SI).
Imaging was accomplished using a laser scanning confocal two-
photon microscope (Olympus Fluoview 1000 MPE) equipped with a
tunable fs pulsed laser, as well as a 473 nm CW laser. The continu-
um emission of the hollow gold nanoshells resulting from 800 nm
excitation demonstrates nanoparticle internalization for both experi-
ments. Figure 4 (left panels) shows the gold particles within the
outlines of the cells. Before NIR excitation, virtually no fluorescence
was seen from either set of cells when probed using single photon
continuous irradiation at 473 nm (Figure 4, center panels). However,
upon excitation at 800 nm by rastering the fs pulsed laser 5X
through the two sets of cells, the 22RV1 cells treated with the TCF-
HGN-TPEGRP conjugates clearly showed fluorescence, indicating
NO generation as detected by the fluorophore formation with the
DAF-2 DA (Figure 4, top right). In contrast, cells treated similarly
with the HGN-TPEGRP conjugate (without the NO donor) did not
exhibit any change in fluorescence before or after irradiation with
800 nm pulsed laser light (Figure 4, bottom right).
Figure 3. NOA signal demonstrating the release of NO from TCF-HGN-
TPEG conjugates upon excitation with a 800 nm pulsed laser operating at 1
KHz. Signals are for different excitation powers 0.9 W cm-2 (A) 1.5 W cm-2
(B), 2.4 W cm-2 (C) and 5 W cm-2 (D). (NOA signal: 1 mV = ~ 13.3 pmoles).
Signal D corresponds to release of ~80% of the NO available based on the
TCF loading of the TCF-HGN-TPEG conjugates (see SI Figure S-4)
As previously reported,23 the high peak intensities of the pulsed
ultrafast laser system are exceptionally effective in the rapid heating
of gold nanoparticles with NIR light. However, it is notable that NO
release could also be effected by excitation with a continuous wave
(CW) NIR laser operating at 800 nm, although the process was con-
siderably less efficient. In that case, it was necessary to focus the
CW beam to a diameter of 0.9 mm where the intensity was 157 W
cm-2. Irradiation of a comparable solution of the TCF-HGN-TPEG
conjugates at this intensity for 60 s gave 41 pmol NO, while irradia-
tion at 78.5 W cm-2 gave 9.4 pmol NO (SI Figure S-5). This behav-
ior is consistent with non-linear absorption of light by the HGN sur-
face plasmon leading to localized heating and consequently to ther-
mochemical NO release from the TCF. Notably, there were only
modest changes in the bulk solution temperatures with excitation
from either the pulsed or continuous laser (SI Figure S-6).
Targeting with peptides: Cellular internalization of the nanoparti-
cles was facilitated by Neuropilin-1 receptor mediated endocytosis
through coupling the C-end Rule (CendR) peptide, RPARPAR,24 to
the end of a 5-kDa thiolated polyethylene glycol (see SI). This pep-
tide derivatized thiolated PEG (TPEGRP) was then used to prepare
TCF-HGN-TPEGRP conjugates in the manner used to prepare the
TCF-HGN-TPEG analogs. Quantification of the peptide was done
by comparison of rhodamine dye label on the sequence against a
standard calibration curve. This gave the estimate of ~3000 peptide
per particle after KCN etching (data not shown). Microscopy analy-
sis of HGN internalization confirms that nanoparticles are readily
internalized within 2 h only for cells that overexpress the Neuropilin-
1 receptor, such as PPC-1 prostate cells. The HeLa cell line, lacking
Neuropilin-1 receptor did not do so (SI Fig. S-7 and S-8).
Figure 4. Cell-specific targeting of NO release with NIR-mediated spatio-
temporal control. The 22Rv1 cells were treated with TCF-HGN-TPEGRP
conjugates and the NO was detected using DAF-2 DA fluorescent reporter.
First column: incorporation of HGN is visualized by continuum emission
(orange) localized within cells during irradiation. Scale bar is 100 µm. Top
row: 22Rv1 cells treated with TCF-HGN-TPEGRP exhibit increased fluores-
cence from the reporter (green) upon laser irradiation with 800 nm pulsed
laser light indicating generation of NO. Bottom row: Cells treated with HGN-
TPEGRP do not increase in fluorescence indicating that NO was not generat-
ed. (See also SI Fig. S-9.)
Notably, irradiation of the TCF-HGN-TPEGRP conjugates inter-
nalized in cells showed little alteration in cellular viability at 2.4 W
cm-2 excitation power but modest decreases (20-40%) in viability at
5 W cm-2 (SI Fig. S-10).
The release of NO from the TCF-HGN-TPEGRP conjugates inside
the cells was tested by using the diaminofluorescein diacetate fluo-
rescent reporter (DAF-2 DA). A control analog without TCF was
generated by omitting the addition of TCF (HGN-TPEGRP). Both
HGN-TPEGRP and TCF-HGN-TPEGRP readily underwent endocy-
tosis with two different prostate cancer cell lines is seen in the mi-
croscopy images shown in SI Figure S-7 and S-8 for the PPC-1 cells
and in Figure 4 for the 22Rv1 cells, which are also positive for Neu-
ropilin-1 overexpression.25 Since background fluorescence problems
In summary, we have demonstrated that using NIR light to excite
the surface plasmon of hollow gold nanoshells is a viable strategy
for the controlled release of NO in solution and in human prostate
cancer cells. While others have described gold nanoparticle plat-
forms for NO delivery,26,27 the present report is the first to utilize
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NIR light with such systems. This NO uncaging can be attributed to
rapid heating of the thiolated cupferron bound to the HGN surfaces
of the TCF-HGN-TPEG conjugates upon NIR irradiation with a
pulsed laser at frequencies resonant with the surface plasmon. The
quantities of NO released in the small solution volumes irradiated
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26. N. Nakatsubo, H. Kojima, K. Kikuchi, H. Nagoshi, Y. Hirata, D.
This work was supported by NSF Chemistry Division grants (CHE-
1058794 and CHE-1405062) to PCF, a National Institutes of Health
grant (R01 EB012637) to NOR and by a fellowship to ESL from the
UCSB IRES-ECCI Program (NSF grant OISE-1065581). ). Two
photon microscopy was available through NIH (1 S10 OD010610-
01A1) and the NRI-MCDB Microscopy Facility. We thank E. Ru-
oslahti for cell lines. We thank A. Mikailovsky for his help with the
ultrafast laser experiments, M. Raven for discussions pertaining to
the two photon microscope and A. E Pierri for many helpful discus-
sions.
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Conflict of interest statement.
None of the authors have a financial conflict of interest to declare.
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