Macrophage-mediated delivery of light activated nitric oxide prodrugs with spatial, temporal and concentration control

Article abstract of DOI:10.1039/c8sc00015h

Nitric oxide (NO) holds great promise as a treatment for cancer hypoxia, if its concentration and localization can be precisely controlled. Here, we report a Trojan Horse strategy to provide the necessary spatial, temporal, and dosage control of such drug-delivery therapies at targeted tissues. Described is a unique package consisting of (1) a manganese-nitrosyl complex, which is a photoactivated NO-releasing moiety (photoNORM), plus Nd³⁺-doped upconverting nanoparticles (Nd-UCNPs) incorporated into (2) biodegradable polymer microparticles that are taken up by (3) bone-marrow derived murine macrophages. Both the photoNORM [Mn(NO)dpaqNO2]BPh₄(dpaqNO2 = 2-[N,N-bis(pyridin-2-yl-methyl)]-amino-N′-5-nitro-quinolin-8-yl-acetamido) and the Nd-UCNPs are activated by tissue-penetrating near-infrared (NIR) light at ～800 nm. Thus, simultaneous therapeutic NO delivery and photoluminescence (PL) imaging can be achieved with a NIR diode laser source. The loaded microparticles are non-toxic to their macrophage hosts in the absence of light. The microparticle-carrying macrophages deeply penetrate into NIH-3T3/4T1 tumor spheroid models, and when the infiltrated spheroids are irradiated with NIR light, NO is released in quantifiable amounts while emission from the Nd-UCNPs provides images of microparticle location. Furthermore, varying the intensity of the NIR excitation allows photochemical control over NO release. Low doses reduce levels of hypoxia inducible factor 1 alpha (HIF-1α) in the tumor cells, while high doses are cytotoxic. The use of macrophages to carry microparticles with a NIR photo-activated theranostic payload into a tumor overcomes challenges often faced with therapeutic administration of NO and offers the potential of multiple treatment strategies with a single system.

Full text of DOI:10.1039/c8sc00015h

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Huang, Y. Iwamoto, K. Ibsen, E. M. Chan, Y. Hitomi, P. C. Ford and S. Mitragotri, Chem. Sci., 2018, DOI:
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Macrophage-Mediated Delivery of Light Activated Nitric Oxide
Prodrugs with Spatial, Temporal and Concentration Control
Received 00th January 20xx,
Accepted 00th January 20xx
Michael A. Evans,†,a,b,c Po-Ju Huang,†,a Yuji Iwamoto,d Kelly N. Ibsen,b Emory M. Chan,e Yutaka
Hitomi,d Peter C. Ford,a,* Samir Mitragotrib,c,
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Nitric oxide (NO) holds great promise as a treatment for cancer hypoxia, if its concentration and localization can be
precisely controlled. Here, we report a “Trojan Horse” strategy to provide the necessary spatial, temporal, and dosage
control of such drug-delivery therapies at targeted tissues. Described is a unique package consisting of 1) a manganese-
nitrosyl complex, which is a photoactived NO-releasing moiety (photoNORM), plus Nd3+-doped upconverting nanoparticles
(Nd-UCNPs) incorporated into 2) biodegradabale polymer microparticles that are taken up by 3) bone-marrow derived
murine macrophages. Both the photoNORM [Mn(NO)dpaqNO2]BPh4 (dpaqNO2 = 2-[N,N-bis(pyridin-2-ylmethyl)]-amino-N′-5-
nitro-quinolin-8-yl-acetamido) and the Nd-UCNPs are activated by tissue-penetrating near-infrared (NIR) light at ~800 nm.
Thus, simultaneous therapeutic NO delivery and photoluminescence (PL) imaging can be achieved with a NIR diode laser
source. The loaded microparticles are non-toxic to their macrophage hosts in the absence of light. The microparticle-
carrying macrophages deeply penetrate into NIH-3T3/4T1 tumor spheroid models, and when the infiltrated spheroids are
irradiated with NIR light, NO is released in quantifiable amounts while emission from the Nd-UCNPs provides images of
microparticle location. Furthermore, varying the intensity of the NIR excitation allows photochemical control over NO
release. Low doses reduce levels of hypoxia inducible factor 1 alpha (HIF-1α) in the tumor cells, while high doses are
cytotoxic. The use of macrophages to carry microparticles with a NIR photo-activated theranostic payload into a tumor
overcomes challenges often faced with therapeutic administration of NO and offers the potential of multiple treatment
strategies with a single system.
the progression of specific disease states.3-6 In this context, we and
others have developed photo-activated NO releasing moieties
(photoNORMs), since triggering with light can provide precise
Introduction
Nitric oxide (NO) has exhibited significant potential as a cancer
therapy, but its effects are highly concentration- and location-
dependent.1 Furthermore, NO has a relatively short lifetime in
physiological media and induces significant side effects when
delivered systemically.2 Targeting strategies must be a key feature
for the delivery of any drug, and this is especially true for a
bioregulator such as NO. Photochemical uncaging allows one to
define the location, timing and dosage of such drug delivery, thus
this technique has value as an investigative tool and for addressing
spatial and temporal control of NO delivery.7-10 However, this
methodology is limited by the strong wavelength dependence of
light transmission through tissue.11,12 Ultraviolet and shorter visible
light are much less tissue penetrating than are longer red or
(ideally) near-infrared (NIR) wavelengths. The ability of NIR light to
transmit deeply into tissue has inspired various approaches to
designing photoNORM systems including the engineering of
molecular compounds that are photoactive at longer
wavelengths13,14 and the use of antennas for multi-photon
sensitization of NO release with NIR light.15-18 However, delivering
these conjugates to the desired physiological targets continues to
be a challenge.
It is difficult to deliver the desired drug payload specifically to
hypoxic areas of tumors via simple diffusion from the blood stream,
owing to poorly developed vascular structures.19-21 Instead it would
be particularly valuable to utilize the inherent biological
mechanisms to facilitate such delivery. Tumor hypoxia generates
inflammatory signals that recruit monocytes from the blood via
chemotaxis, that once inside the tumor differentiate into
macrophages.22 This behavior has stimulated interest in recruiting
macrophages as carriers23-28 to localize drugs in tumors at levels
difficult to attain with conventional delivery methods.
* Corresponding authors: PCF: ford@chem.ucsb.edu; SM: Mitragotri@g.harvard.edu
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in acetonitrile solution does indeed lead to NO release as measured
using the Sievers Nitric Oxide Analyzer (NOA) with a quantum yield
of 0.18. (ESI Figure S1).
The Nd-UCNPs were high quality core/shell upconverting
nanoparticles (Figure 1, bottom) synthesized using the robotic
Workstation for Automated Nanocrystal Discovery and Analysis
(WANDA) of the Molecular Foundry at Lawrence Berkeley
National Laboratory.31–33 The host material for the 10 nm
diameter cores was NaY0.8Gd0.2F4. (The Gd3+ adduct favors the
hexagonal structure that typically gives higher upconversion
efficiency).34-36,§ The host material was also doped with the
lanthanide ions Yb3+ (30%), Nd (1.0%) and Tm (0.5%). The 2 nm
thick shell was composed of NaGdF4 doped with Nd3+ (20%). The
shell minimizes surface quenching effects and improves the
luminescence efficiency of UCNPs. The Nd3+ sensitizers in these
Scheme 1. Illustration of macrophage cellular Trojan Horse strategy to carry
polymer based microparticles containing a photochemical precursor of NO
(photoNORM) plus upconverting nanoparticles (UCNPs) into a tumor. NIR
activation of the photoNORM/UCNP combination releases NO to mediate
the tumor environment in order to facilitate various types of therapy as well
NaYF4:Yb/Gd/Nd/Tm(30/20/1/0.5%)@NaGdF4:Nd(20%)
nano-
as providing the opportunity for photoluminescence imaging
.
particles (Nd-UCNPs) absorb NIR wavelengths near 800 nm
allowing these materials to be excited at wavelengths where the
The present study demonstrates the utility of macrophages as
carriers of micron-sized, poly(lactic-co-glycolic acid) (PLGA)
microparticles in which one can incorporate a theranostic payload.
The encapsulated payload here includes a photoNORM that can be
triggered for NO release by NIR light together with Nd3+ doped
upconverting nanoparticles (Nd-UCNPs) to provide imaging
capabilities.29,30 The BALB/c bone marrow derived macrophages
(BMMs) were shown to undergo phagocytosis of these
microcarriers. Such macrophages allow far deeper penetration into
large NIH-3T3/4T1 co-cultured tumor spheroids (Scheme 1) than do
the microparticles alone. Thus, as previously described by Clare and
coworkers,23 such macrophages have the potential to act as
“cellular Trojan Horses” to carry a therapeutic payload into tumors.
It is shown here that, once carried inside the spheroid by a BMM,
the photoNORM in the microparticles can be activated with NIR
light to release NO. At high light intensity, NO concentrations
sufficient to cause direct tumor cell cytotoxicity are generated,
while at low light intensity, the NO released leads to a significant
drop in the expression of hypoxia inducible factor HIF-1α in the
tumor microenvironment.
Results and Discussion:
NIR active photoNORM and Nd-UCNPs in microparticles.
The cell-mediated delivery platform described here consisted
of murine BMMs loaded with polymer-based microcarriers into
which were incorporated a NIR sensitive photoNORM and NIR
active bioimaging Nd-UCNPs. The photoNORM was prepared by
metathesis of the water-soluble salt [Mn(dpaqNO2)(NO)]ClO4
(dpaqNO2
= 2-[N,N-bis(pyridine-2-ylmethyl)]-amino-N′-5-nitroquin-
olin-8-yl-acetamido)13 with sodium tetraphenylborate to give the
corresponding, hydrophobic photoNORM [Mn(dpaqNO2)(NO)]BPh4
(I). The hydrophobicity was needed to minimize leakage of I from
–
the polymer microcarrier into the medium. The BPh4 salt was
further purified by recrystallization to obtain black needles. Figure 1
(top) shows the spectrum of I in acetonitrile. Although the λmax of
the longest wavelength band is ~650 nm, this absorbance extends
to the NIR region (ε: 20.2 M-1cm-1 at 794 nm) and overlaps with the
output from a 794 nm diode laser used as a continuous wave (CW)
excitation source in the present study. Direct 794 nm photolysis of I
Figure 1. Top: Absorption spectrum of [Mn(dpaqNO2)(NO)]BPh4 (I) in
acetonitrile. The red spike at the right is the spectrum of the 794 nm diode
2 | J. Name., 2012, 00, 1-3
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laser. Bottom: TEM image of the core-shell the nano-particles
NaYF4:Yb/Gd/Nd/Tm(30/20/1/0.5%)@NaGdF4:Nd(20%). Inset: Upconversion
emission spectrum of Nd-UCNPs under excitation by an 800 nm CW laser.
released from these microcarriers in response to the different
excitation laser intensities. Notably, both plots appear roughly
linear over this narrow range, consistent with a single photon
transmittance through water is most efficient. Energy absorbed by excitation mechanism for NO release. Additionally, the efficiencies
Nd3+ ions in the shell migrates via resonant transfer to Nd3+ in the of NO
core and energy transfer to Yb3+ ions.36 The energy from multiple
photons is transferred sequentially from the excited Yb3+ dopants to
Tm3+ emitters, thereby producing upconverted photoluminescence
(PL) bands in the UV and visible range. The transmission electron
microcopy (TEM) and PL spectra of these Nd-UCNPs are shown in
Figure 1 (bottom).
The polymer-based microcarriers were prepared from PLGA
dissolved in dichloromethane (DCM) by a micro-emulsion technique
as described previously37,38 and in the Experimental Methods
section (see ESI). The procedure simultaneously encapsulated the
photoNORM I and/or Nd-UCNPs into spherical PLGA particles with
ca. 1-micron diameter (0.35-2 µm) (ESI Figures S2 & S3). The
average loading of the manganese photoNORM was 4.36 ± 0.66
wt% as determined by inductively coupled plasma atomic emission
spectroscopy (ICP-AES). The spherical micro-particles have the
optimal shape to facilitate phagocytosis.39 The PLGA was acid
terminated and the resulting surface carboxylates were modified
Figure 2. Plots of the NOA detected NO released vs laser intensity (W/cm2)
for the CW 794 nm photolysis of PBS suspensions of the microcarriers PLGA-
with immunoglobulin G (IgG) by amide coupling in 0.1 M pH 5.5 2-
(N-morpholino)ethanesulfonic acid (MES) buffer to increase the
efficiency of phagocytosis. A bicinchoninic acid (BCA) protein assay
indicated there is ca. 44 µg of IgG per mg of PLGA particles.
1 (black) and PLGA-2 (red).
release for PLGA-1 and PLGA-2 are 1.63 pmole W-1 cm2 s–1 and 2.06
pmole W-1 cm2 s–1 respectively. When differences in loading are
taken into account, this represents a lower NO release from the Nd-
UCNP loaded microparticles, although this experiment is somewhat
qualitative given that these are suspensions of the microparticles.
Nonetheless, under these experimental conditions, the Nd-UCNPs
have value primarily for imaging purposes.
NO release from photoNORM loaded polymer microparticles.
Previous studies in these laboratories16,40 have shown that
UCNPs can serve as photosensitizers that absorb NIR photons and
emit visible light to trigger NO release from photoNORMs that were
not NIR sensitive. This process requires relatively high intensity
irradiation to effect the multi-photon upconversion mechanism of
UCNPs and energy transfer from UCNPs to photoNORMs. In the
present case, the Mn photoNORM I is photosensitive toward NO
release via direct single-photon excitation with 794 nm light, and
this allows one to overcome the scattering constraints that may be
problematic for multiphoton excitation in deeper tissue. However,
since the extinction coefficient for I at this wavelength is low (20.2
M-1cm-1), it was of interest to see whether the visible light
generated by upconversion from the Nd-UCNP would enhance NO
release owing to the significantly higher extinction coefficients of I
in the visible spectral region.
Microparticle uptake and compatibility.
The goal of these experiments was to determine the amount
of loaded PLGA microparticles that can undergo phagocytosis into
the murine bone marrow macrophages that had been prepared as
described in the Experimental section (see ESI). It has been
previously shown that rigid spheres, 1-3 microns in diameter, are
readily taken up by this mechanism.27,41,42 As noted above, said
microparticles were also surface modified with a covalently bound
layer of IgG to enhance uptake (ESI Figure S4). An alamarBlue® cell
viability assay was used to determine whether the microparticles
proved toxic to the BMM cells. Figure 3 shows that these particles
displayed no significant acute toxicity for concentrations as high as
100 μg/mL for incubation periods of 24 and 48 h with BMMs.
Moderate toxicity was observed at higher concentrations. (Notably,
a recent review of lanthanide-UCNP toxicity has stated that, while
there are numerous reports of negligible or low toxicity, “there is a
paucity of knowledge concerning primary and secondary toxicity
In order to test this possibility, microparticles of two different
compositions were prepared. One contained both I and Nd-UCNPs
(PLGA-1), the other contained only the manganese photoNORM I
(PLGA-2). Data obtained from dynamic light scattering (DLS) showed
these two particle groups to be similar in average size (~1 micron,
ESI Figure S2 Top), while ICP-AES analysis showed the former to
have somewhat higher loading of I (4.76 wt% vs 3.79 wt%
respectively). Particles of both types (0.5 mg) were separately
suspended in 2.5 mL pH 7.4 phosphate buffered saline (PBS)
solution and were irradiated with a 794 nm diode laser while
stirring and purging with medical grade air. The purge gas was
analyzed for NO using the NOA for 1.0 s irradiation times at
different intensities (in W/cm2). The NOA signals recorded are
shown in ESI Figure S3 while Figure 2 plots the quantity of NO
effects on the environment and humans.”)43
For the present
study, the microparticle concentration of 100 µg/mL was selected
for further incubations. Analysis of BMMs containing NO-donor
loaded particles with ICP-AES determined an uptake of 0.667 µg
manganese per 1 x 106 cells. This translates into 263 µg particles or
12.1 nano-equivalents of NO as photoNORM I in 106 cells.
Intracellular NO release.
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BMMs loaded with microparticles containing both I and Nd-
UCNPs (BMMp+) were then tested to verify internal release of NO.
The reporter was 4-amino-5-methylamino-2',7'-difluorofluorescein
diacetate (DAF-FM), which reacts to form a fluorescent compound
when exposed to intracellular NO. DAF-FM was incubated with the
Journal Name
co-cultured breast tumor spheroids imaged by confocal microscopy. Blue is
4',6-diamidino-2-phenylindole (DAPI) and red represents macrophages
stained with CellTrackerTM deep red. Green spots in (c) and (d) are Cy-3-IgG
labeled microparticles. (b) Spheroids stained with a HypoxyprobeTM Red549
to label hypoxia (cyan). (c) Spheroid incubated with microparticles-laden
BMMs. (d) Spheroid incubated only with IgG modified PLGA microparticles.
BMMp+ macrophages and
microparticles. Prior to DAF-FM incubation, both sets were treated
with L-N-nitroarginine methyl ester (L-NAME), nitric oxide
a control set of BMMs without
analysis showed migration of both the native BMM cells and the
BMMp+ cells across the endothelium layers, although under these
conditions, fewer of the BMMp+ cells ((10.4 ± 0.8) x 103) migrated
relative to the BMM control cells ((17.1 ± 2.4) x 103). Thus,
phagocytosis of microparticles did reduce chemotaxis as reported
previously,27 although in the latter case an even sharper drop in
chemotaxis was observed. However, there are various strategies
that can be used to enhance overall macrophage recruitment and
therapeutic efficacy, if needed.28,46,47
a
synthase inhibitor, to reduce background from biological NO
production.44 Upon 794 nm laser exposure at 13.0 W/cm2 for 90 s,
the BMMp+ set clearly produced a visible fluorescence response
while the BMM set without particles did not (ESI Figure S5).
Microparticle effects on macrophage chemotaxis.
The ability of macrophages to target tissue such as tumors is
governed by their ability to undergo chemotaxis towards sights of
inflammation.22 Thus, it is crucial that the macrophages retain this
function after drug loading. This question was probed using a
transendothelial assay (ESI Figure S6). First, monolayers of bEnd.3
blood brain barrier (BBB) endothelial cells were grown on
FluoroBlokTM transwell inserts inside of the wells of a 24-well plate
as model endothelium barriers. Such monolayers are very tight
compared to those from other endothelian cells, thereby making
migration through the barrier challenging.45
After monolayer confluency, media containing 1 x 105 BMMp+
cells, media containing a comparable number of BMMs without
particles, and media alone were added to the tops of separate
transwells and the monocyte chemoattractant protein-1 (MCP-1)
was added below. After 24 h, the inserts were stained with
NucBlue® allowing for quantification of chemotaxis by counting the
nuclei of cells that had migrated to the bottom of the insert using a
fluorescence microscope (corrected for the signal from wells with a
confluent monolayer to which only media had been added). This
Tumor spheroid penetration
In order to model a 3D tumor environment, tumor spheroids
were prepared from co-cultured murine NIH/3T3 fibroblast: 4T1
breast cancer cells with a 5:1 cell seeding ratio similar to that used
previously.48 Incorporation of fibroblasts into tumor spheroids
induces formation of a tumor stroma which enhances spheroid
compactness and increases the expression of pro-inflammatory
cytokines used in leukocyte chemotaxis.49,50 With this seeding ratio,
the hanging drop technique produced spheroids with an average
diameter of 962 ± 60 µm (~1.35 x 105 cells/spheroid) after 10 days
of incubation with the microparticle loaded macrophages
introduced on day 7. These spheroids are considerably larger than
those produced with 4T1 cells alone (ESI Figure S7) as well as those
NIH/3T3: 4T1 spheroids grown without the loaded BMM cells (ESI
Figure S8), the latter a likely result of macrophage supported tumor
growth.50 Pimonidazole (PIMO) staining of the spheroids revealed
hypoxia throughout the spheroid interior. Considering that hypoxia
normally occurs 100-150 µm into a tumor,51 this is not surprising.
Macrophages labeled with CelltrackerTM deep red were used to
monitor the chemotactic penetration of the BMMp+ cells into
spheroids. Images of tumor spheroid center slices indicated deep
penetration (Figure 3b), although the macrophages did not reach
the center but instead formed a visible ring around the central
section. Since oxygen is required for macrophage chemotaxis, this
behavior may reflect a central necrotic region with high hypoxia.
Flow cytometry of spheroids dissociated with AccumaxTM
demonstrated a macrophage cell composition of 0.53 ± 0.4% or ~
715 ± 540 macrophages per spheroid (ESI Figure S9). Assuming that
the BMMp+ retained their payload at the concentration shown by
ICP, these macrophages brought an estimated 8.7 ± 6.6 pico-
equivalents of the photoNORM into the spheroid. The value of the
macrophages as "Trojan Horses" was demonstrated using particles
labeled with fluorescent IgG. Spheroid slices (Figure 3c)
demonstrated that macrophages with internalized fluorescent
particles carried their payload into the spheroids (Figure 3c) but
that IgG modified microparticles alone were unable to penetrate
more than a few cell layers when incubated with the spheroids
(Figure 3d).
(a)
(b)
(c)
(d)
Nd-UCNP imaging.
The Nd-UCNPs introduced to the polymer-based microparticles
provide an imaging agent that allows one to track the location of
Figure 3. (a) Viability of BMMs with various particle incubation
concentrations after 24 and 48 h. (b), (c) and (d) are slices of 4T1/NIH-3T3
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these prodrug carriers and their macrophage hosts using tissue- concentrations in excess of 1 µM were generated in the spheroids,
penetrating NIR excitation wavelengths. In addition, since their
a
concentration exceeding that needed to induce p53
chemotactic capacities allow macrophages to hone in on
phosphorylation and/or nitrosative stress mediated apoptosis.1
Medical
Air
(a)
(b)
3min
No Purging
4.5min
No Purging
NO
NOA
Purging
6min
Purging
No Purging
Purging
4.5min
No Purging
Control
Purging
Purging
4T1
Cancer Spheroid
6min
No Purging
794 nm excitation
Figure 4. Z-stack two-photon confocal images (dimensions = 508.4 µm x
508.4 µm) of live spheroids containing BMMs (a) with blank particles and (b)
with UCNP and NO-donor loaded particles. Spheroids were stained with
calcein AM (green). Particles were identified via emission (red) from
embedded UCNPs with laser excitation at 810 nm and a pulse energy of 37.5
nJ. The wavelength range for detection of UCNP emission was 420–460 nm.
(c)
(d)
inflammation sites, the PL from UCNPs provides
a potential
mechanism to detect hidden metastatic sites. Since I is photo-
activated by the same NIR wavelength, the combination of this
photoNORM and Nd-UCNP in these macrophage-carried
microparticles would provide theranostic capability.
Figure 5. (a) The NIH/3T3:4T1 tumor spheroids in 1 mL Hank’s buffered salt
solution (HBSS) under 794 nm laser irradiation. (Photo by Ping-Lin Yang)
Inset (up): a modified 1mL cuvette for photolysis experiment of cancer
spheroids. Inset (down): the scheme represents that the spheroids can be
placed at the corner of the cuvette and exposed to NIR laser excitation. (b)
Up: NO released from five spheroids infiltrated with bone marrow
macrophages loaded with PLGA microparticles containing I and Nd-UCNPs
under 794 nm laser irradiation with 13.1 W/cm2. Down: The control
experiment of six spheroids loaded with PLGA microparticles. (c) Plot of NO
detected vs irradiation time. (d) The viability of spheroids after 6, 12, or 18
min of laser exposure in 6-min increments. Viability was measured using a
PNPP assay 24 h after spheroid treatment. * = p ≤ 0.05 (students t-test).
Figure 4 shows 3D images of spheroids incubated with BMMp+
or with BMMp– and recorded with a confocal microscope using a
808 nm laser source. The spheroids were labeled with calcein AM to
provide visualization of their periphery. PL Signals were noted from
the spheroids treated with BMMp+ cells while those treated with
BMMp– cells failed to produce
a signal. These luminescent
emissions from the Nd-UCNPs provide a proof-of-principle with
regard to using UCNPs to track the macrophages, but it is clear that
either more efficient emitters or (given that the nonlinear
relationship between UCNP PL and excitation intensities) a more
intense laser source may be needed for in vivo diagnostic
applications. On-going studies will address this issue.
The effect of generating such high localized NO concentrations
was examined by evaluating cell viability after 24 h using a p-
nitrophenyl phosphate (PNPP) assay.51 Figure 5d illustrates the
results of irradiating spheroids containing BMMp+ or BMMp– cells
with 794 nm light for 1 to 3 six-minute periods at 13.1 W/cm2. In
general, the data in Figure 5D consistently show reduced cell
viability for irradiated spheroids infiltrated with BMMp+ cells
relative to those loaded with BMMP– cells, the most convincing
example being the 26% reduction for spheroids containing
photoNORM loaded macrophages after 12 min irradiation
compared to 6.6% reduction for similarly treated BMMp– loaded
spheroids. However, the experimental uncertainties are large and
barely statistically significant owing no doubt to unavoidable
variability in spheroid loadings and the difficulty in preparing,
photolyzing and analyzing a statistically large number of loaded
spheroids. Notably, the spheroids used in this study were formed
from 4T1 breast cancer cells, which are p53 deficient,52 so the
observed damage can largely be attributed to nitrosative stress
induced apoptosis. Cancers with p53 pathways are likely to be more
sensitive to NO delivery at these concentrations.
Photoactivated NO release inside spheroids.
The next question was whether NO released by NIR excitation
of these BMMp+ infiltrated spheroids can be detected externally
using the NOA, which to the best of our knowledge, would be
unprecedented. Five spheroids incubated with BMMp+ or BMMs
with PLGA-only particles (BMMp–) were positioned in the corner of
a custom designed cuvette (Figure 5a containing 1 mL of Hank’s
Balanced Salt Solution (HBSS) solution. In typical NOA analysis, the
solution is entrained with the carrier gas and stirred to facilitate
transfer of NO to the detector. However, such conditions would
likely cause spheroid disassembly and cell lysis. Instead, the
spheroids were blanketed with unstirred HBSS solution and
irradiated with the 794 nm diode laser operating at 13.1 W/cm2 for
specified time periods (Figure 5a). Subsequent gentle bubbling with
medical-grade air above the spheroids released NO from the
solution without disrupting the spheroids. The procedure could be
repeated several times with each sample (Figure 5b & 5c). The NO
released photolysis from the five BMMp+ infiltrated spheroids after
the 6 min (total) was 13.6 pmol (~2.7 pmol per spheroid (avg)). In
contrast, spheroids treated with BMMp– cells gave no measurable
NOA response. Based on volumes of ~1 μL, NO steady state
Low NO concentrations have been shown to produce
beneficial shifts in tumor microenvironment through reduction of
factors such as P-glycoprotein and HIF-1α that are implicated in
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increased resistance to chemo- and radio- therapy.53-55 HIF-1α is a
transcriptional factor upregulated in hypoxia that controls the
expression of genes correlated with tumor cell survival, metastasis,
and angiogenesis.56 The effect of generating lower NO
summary of flow cytometry analysis for the control (i) and the NO release
study (ii). (i) represents spheroids with BMMp— with and without 735 nm
LED irradiation.
concentrations on HIF-1α levels in the tumor spheroids was Summary
examined by using LED excitation (0.58 mW/cm2 at 735 nm). After
These studies demonstrate that the timing and dosage of NO
8.5 h exposure, the spheroids were dissociated into a single cell
suspension. Cells were then fixed and labeled with anti-HIF-1α and
by a fluorescent secondary antibody. Analysis with flow cytrometry
(Figure 6) revealed that the low intensity excitation reduced
proportion of cells expressing HIF-1α from 96.7 ± 0.5 % to 77.7 ± 8.7
% in irradiated spheroids containing BMMp+ macrophages while
spheroids with infiltrated with BMMp– macrophages showed little
change in HIF-1α level with or without light. These data agree with
previous studies demonstrating that low concentrations of NO
destabilize HIF-1α in hypoxia due to the inhibition of cytochrome c
oxidase,54 a critical component of mitochondrial respiration. In a
second trial, HIF-1α expression in spheroids containing BMMp– and
BMMp+ macrophages was shown by flow cytometry to be 90.3 ±
5.1% and 62.2 ± 2.5%, respectively, after LED excitation for 7 h (ESI
Figure S10), thus confirming the impact on this proliferative factor
upon delivering a low concentration of NO.
release from photoNORM loaded microparticles can be controlled
inside tumor models by triggering with tissue penetrating NIR light.
Bone marrow derived macrophages serve as Trojan Horse carriers
for the polymer-based microparticles incorporating both the
photoNORM (I) and Nd3+-doped upconverting nanoparticles for
therapeutic and imaging applications. In this manner, the carrier
macrophages load large quantities of a photoactivated therapeutic
agent without significant effects on viability. The loaded BMMp+
cells maintain the chemotactic ability to traverse an in vitro brain
blood barrier transendothelial model and to penetrate tumor
spheroids with their photoactive cargos. Such penetration does not
occur with the microparticles alone. However, while the tumor
spheroids provide a valuable proof-of-principle, in vitro model to
demonstrate that macrophages can infiltrate and deliver a payload
to such tissues, the next stage will be to demonstrate such targeting
in vivo. In this context we are encouraged by a recent study57 where
blood monocytes loaded with nano-sized polymeric micelles
containing paclitaxel (PTX) were used to treat metastatic breast
cancer in mice. Such PTX delivery was significantly more efficient
than using free PTX or PTX loaded nanoparticles alone.
Both I and the Nd-UCNPs are excited with NIR light at ~800 nm,
the ideal wavelength for tissue transmission. Thus, the Nd-UCNP
emission is used to visualize BMMp+ cells infiltrated into spheroids
while NIR photoactivation of I released NO. We demonstrate two
types of potentially therapeutic effects by using different light
intensities to irradiate the BMMp+ cell infiltrated tumor spheroids.
The micromolar NO concentration generated by high intensity NIR
laser excitation leads to nitrosative stress and cell mortality. In
contrast, lower concentrations of NO served to modify the tumor
microenvironment by destabilizing HIF-1α. Although not explored in
the current study, it should be noted that targeted NO release in
the hypoxic regions of tumors should enhance the effectiveness of
cancer chemotherapy55 and radiotherapy.58,59 In these contexts,
macrophage-mediated delivery of photoNORMs combined with NIR
excitation represents a fresh approach that allows one to target
tumors or other diseased tissues characterized by inflammation.
This strategy will facilitate the spatially and temporally controlled
release of NO or of other caged compounds for precise therapeutic
applications.
Experimental Section
Materials.
All cell lines (4T1, NIH-3T3, and bEnd.3) were purchased from
ATCC. BALB/C mice (6-8 weeks old) were purchased from Charles
River. Sodium tetraphenylborate, sodium trifluoroacetate, sodium
oleate, ammonium fluoride, lanthanide chlorides (99.9+%), oleic
acid (OA) (90%), 1-octadecene (ODE) (90%), poly(vinyl) alcohol
(PVA, Mw: 13000-23000), 4-morpholineethanesulfonic acid (MES,
low moisture content, 99+%), immunoglobulin G (IgG from mouse
Figure 6. Top: Flow cytometry analysis of dissociated tumor spheroids
stained for HIF-1α under different treatments. Gated cells correlate with a
positive signal from staining. (i) and (ii) three runs of analysis for spheroids
loaded with PLGA microparticles incorporating I and Nd-UCNPs with and
without 735 nm LED exposure at 0.58 mW/cm2 for 8.5 h. (iii) The
comparison of (i) and (ii) for each run. Red and black lines represent
particles with and without light exposure respectively. Bottom: The
serum),
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
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hydrochloride (EDC.HCl, commercial grade), TritonTM X-100 The polymer solution was slowly added into 200 mL of 1 wt%
(BioXtra), Tween® 20 (BioXtra), agarose (BioReagent), sodium polyvinyl alcohol (PVA) aqueous solution contained in a 250 mL
hydroxide (NaOH, ACS reagent grade), sodium acetate half-spherical container while the ultrasonic homogenizer was
(RegentPlus®), purified human plasma fibronectin (1 mg/mL), and turned to 350 watts for 30 s. A dark brown colloidal solution formed
Perfecta3D® 96-well hanging drop plates were purchased from immediately. The flask containing the colloidal suspension was fully
Sigma-Aldrich. 10X PBS (OmniPur® liquid concentrate) was covered with aluminum foil and stirred overnight to evaporate the
purchased from EMD Millipore. Poly(lactic-co-glycolic acid) (5050 volatile organic solvents. The milky solution was centrifuged to
DLG 8A, acid terminated) was purchased from Lakeshore collect a solid, which was washed with 18 megohm pure water to
Biomaterials.
4-Amino-5-methylamino-2',7'-difluorofluorescein remove the PVA. Following particle purification, the brown pellet
diacetate (DAF-FM-2DA, Molecular Probes®) was purchased from was re-suspended in 45 mL pure water and then the 1-micron
Life Technologies. N-hydroxysuccinimide (NHS), fetal bovine serum particles were separated by different centrifuge speed (4000 rpm to
(FBS), Dulbecco’s Modified Eagle Medium high glucose (DMEM), remove particles < 500 nm and 300 rpm to remove particles size > 3
Dulbecco’s Modified Eagle Medium: Nutrient mixture F12 μm). The resulting particles were dried under vacuum and re-
GlutaMAXTM and high glucose (DMEM/F12), sodium pyruvate suspended in 0.1
M pH 5.5 MES buffer solution with the
solution (100 mM), penicillin streptomycin 10,000 U/mL (P/S), concentration of 1 mg/mL. EDC.HCl (70.94 μmol/1 mg particles) and
trypsin/EDTA (0.25%), Hank’s Balanced Salt Solution without NHS (106.87 μmol/1 mg particles) were added into the colloidal
calcium and magnesium (HBSS) Dulbecco’s Phosphate Buffered MES solution, which was then sonicated at room temperature for
Saline without calcium and magnesium (DPBS) NucBlue®, 30 min. Subsequently, 10 μL IgG solution (11.21 mg/mL) per 1 mg
CellTrackerTM Deep Red, goat serum, FITC labeled Goat-anti-rabbit, microparticles was added and the mixture stirred overnight. The
Rabbit anti-HIF-1α, p-nitrophenyl phosphate (PNPP) substrate IgG modified particles were collected by centrifugation and washed
tablets, calcein AM, micro bicinchoninic acid assay kit, and with 18 megohm pure water at least three times. IgG concentration
alamarBlue® was purchased from Thermo Fisher Scientific. All TC was determined with
a micro BCA assay according to the
and Non-TC treated plasticware for cell culture, and FluoroblokTM manufacturer’s instructions. After incubation, particles were
transwell insersts were purchased from Corning. BambankerTM cell removed from solution with centrifugation to avoid light scattering
freezing media was purchased from Bulldog Bio. AccumaxTM was during absorbance measurements.
purchased from Innovative Cell Technologies. L-N-nitroarginine
Initially, a beaker was utilized during the emulsion process,
methyl ester hydrochloride salt (L-NAME) was purchased from however; better yields were obtained using a half-spherical glass
Caymen Chemical. Murine monocyte colony stimulating factor 1 flash, since there are no corners in the latter and the sonic energy is
(MCSF-1) and murine monocyte chemoattractant protein 1 (MCP-1) distributed equally preventing settling and allowing more of the
were purchased from Peprotech. Hypoxyprobe-Red549 Kit PLGA to form particles of the correct size.
containing pimonidazole HCl and mouse DylightTM 549-Mab was
Bone marrow macrophage preparation and other cell cultures.
purchased from Hypoxyprobe. Immunoglobulin G labeled with
Bone marrow was harvested from 6-8 week old BALB/c mice in
Cyanine 3.5 (Cy3.5-IgG) was purchased from Jackson Immuno.
accordance with previously published methods.60 Experiments were
The [Mn(dpaqNO2)(NO)]BPh4 salt (I) was prepared under reduced
performed in compliance with all United States federal and
lighting by anion metathesis of the perchlorate analog that had
been synthesized as reported.13 [Mn(dpaqNO2)(NO)]ClO4, (119.3 mg,
California state regulations governing the humane care and use of
laboratory animals, including the USDA Animal Welfare Act
0.195 mmol) was dissolved in
3
mL solution of 1:1
(Registration #:93-R-0438) and the PHS Policy on Humane Care and
Use of Laboratory Animals (PHS Assurance # A3865-01) and were
reviewed and approved by the University of California Santa
Barbara Institutional Animal Care and Use Committee as part of
protocol 6-16-916. After marrow isolation, cells were then
suspended in a 90% FBS / 10% dimethyl sulfoxide (DMSO) and
frozen for later use according to previous reports.61
acetonitrile/deionized (DI) water that was then added to a 2 mL
volume of acetonitrile (ACN) in which was dissolved NaBPh4 (66.7
mg, 0.195 mmol). The resulting mixture was sonicated for 3 min
after which most of the solvent was removed under reduced
pressure. The resulting hydrophobic black solid was suspended in
aqueous solution then collected by filtration, washed with DI water
and dried under vacuum. The solid was then dissolved in DCM and
recrystallized by vapor diffusion of ether to produce crystalline
black needles of I.
Unless otherwise specified, all culture ware used with
macrophages was non-TC treated plastic. Bone marrow
macrophages were produced via slight modification of previously
published methods.60 Cryo-preserved bone marrow was thawed
IgG-modified microcarriers.
The procedure for forming the polymer micro-emulsions was and added to 10 mL of DMEM/F-12 media supplemented with 10
modified from the literature.37-39 Acid-terminated PLGA (100 mg) mM GlutaMAXTM, 10% FBS, 1000 U/mL P/S, and 20 ng/mL MCSF-1
and ~80 μL of a solution of Nd-UCNPs in hexane (~10 mg UCNPs) (media denoted as DMEMb) cells were centrifuged at 400 g for 10
were added into 500 μL DCM, and the mixture was sonicated for 45 min, resuspended in DMEMb at 2.3 x 106 live cells/mL. DMEMb (24
min at room temperature. A 14 mg sample of I was dissolved in a mL) and 1 mL of bone marrow were added to a flask and put in the
mixture of ACN (150 μL) and DCM (400 μL) to form a dark purple incubator for 7-8 days to allow the bone marrow to mature into
solution that was then transferred into the PLGA solution, and the BMMs. On day 3, 25 mL of additional DMEMb was added to each
mixture was sonicated until homogenous. If the volume of as- flask to replenish the MCSF-1 in solution. After cells had matured
prepared solution was lower than 1.1 mL, more DCM was added. into BMMs, DMEMb was removed from the flask and cells were
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washed with DPBS. To dislodge the cells, the flask was treated with cells/cm2 in a 100-mm diameter non-TC treated petri dish in 17 mL
ice cold AccumaxTM (10-15 mL) for 20-30 min, and thumped once of DMEMb to make a 100 µg/mL solution. Dishes were put in a
with the palm of the hand. Cells were collected, washed with culture incubator for 2 h to allow for phagocytosis. Cells were
DMEMb and centrifuged at 400
g
for 10 min. Cells were washed once with warm DPBS after which, ice cold AccumaxTM (5
resuspended in BambankerTM cell cryopreservation media at 5 x 106 mL) was added and incubated at 37 ºC for 15-30 min. Cells were
cells/mL. BMMs were kept at -80 ºC overnight and were transferred then pipetted up and down gently to release them from the plate.
to liquid nitrogen afterwards. Confirmation of macrophage DMEMb (5-7 mL) was added to the cell suspension. Cells were
maturation was done using CD11b staining as described by Zhang et centrifuged once at 400g for 10 min and resuspended in 2 mL
al.60
DMEMb. Cells were then centrifuged 3 times at 42g for 5 min to
For experiments, BMMs were thawed and added to non-TC remove free particles in solution. In cases where macrophage
treated dishes for 24 h. Cells were then detached with AccumaxTM tracking was desired, 2 µM CellTrackerTM deep red in serum free
and counted. These cells were then replated at 62,500 BMM/cm2 DMEMb was added to cells for 45 min before AccumaxTM was
on plastic ware 18 h prior to the experiment.
applied.
NIH-3T3 murine fibroblast cells, 4T1 murine breast cancer cells,
and bEnd.3 murine brain endothelial cells between passage 3-15
were cultured on TC-treated plastic ware with Dulbecco’s modified
eagle medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 1000 U/mL of penicillin/streptomycin (P/S) and 1 mM sodium
pyruvate (media mixture known as DMEMpy). Cells were passaged
with 0.25% trypsin/EDTA once they reached a confluency of >80%.
Quantification of particle loading and macrophage uptake.
Particles of a known concentration containing the NO-donor
and UCNPs were digested in 1:3 HNO3:HCl for 24 h to assure all
manganese was completely dissolved. Inductively coupled plasma
atomic emission spectroscopy (ICP-AES) was conducted with a
Thermo iCAP 6300 and was used to measure the manganese
content of each sample. Samples were compared to manganese
standard. For quantification of particle uptake, 3x106 cells with
Tumor spheroids
Tumor spheroids were grown with a procedure based on particles were analyzed in the same manner. These cells were
previous research.48 Confluent plates of 4T1 and NIH-3T3 cells were compared to cells without particles
split and resuspended in DMEMpy and then mixed at a ratio of 1:5
Macrophage-particle compatibility.
4T1:NIH-3T3 at a total cell concentration of 1.11x 104 cells/mL. To
Macrophages loaded with microparticles as described above
prevent evaporation of the hanging drops, the liquid reservoirs on
with various particle incubation concentrations were plated at
96 well hanging drop plates from 3d Biomatrix were filled with hot
62,500 cells/cm2 in 96 well plates in 100 µL DMEMb. Cells were put
0.5% w/v agarose and allowed to cool to room temperature. A 45
in a cell incubator and left for 24 or 48 h. A mixture containing 100
µL of DMEMb and 20 µL of alamarBlue® was added to the media in
µL aliquot of the cell mixture was added to the top of each well.
Plates were sealed with Parafilm® and put in the cell incubator for 7
each well. The plate was incubated for 3.5 h. A volume of 110 µL of
days. Media was replenished on days 4 and 6 by removing 15 µL of
media from each droplet and adding 15 µL of fresh DMEMpy. This the sensitivity of the fluorescence measurement. Solution
each well was transferred to black bottomed well plates to improve
fluorescence was measured on a TECAN M220 Infinite Pro plate
reader (λex = 550 nm, λem = 590 nm). Fluorescence from wells
without cells was subtracted as baseline.
procedure was done 2 times in a row during each media change
instead of removing 30 µL of media all at once which can
compromise the droplet. Spheroids were grown in droplets for 7
days.
Qualitative determination of NO release.
To incorporate macrophages, spheroids were transferred into
200 µL 96 well PCR plates used to allow for their easy manipulation
and inversion on the rotisserie without spilling. Spheroids were
washed 3 times with 100 µL of DMEMpy. DMEMpy (50 µL) was
added to each spheroid after the final wash. A 50 µL aliquot of a
solution of BMMs with blank or NO-donor loaded particles at 4x105
cells/mL in DMEMpy was added to each spheroid. Plates were put
on a rotisserie in a cell incubator for 3 days to assure optimal
interaction between macrophages and spheroids. On day 1.5, 100
µL of additional DMEMpy was added to each spheroid to replenish
solution nutrients. After 3 days, spheroids were washed 3 times
with DMEMpy to remove macrophages that had failed to infiltrate
the spheroid. Spheroids were then used for various downstream
applications.
Human fibronectin at 150 µg/mL in DPBS was added to glass
confocal dishes and incubated for 2 h at 37 °C to improve its cell
adhesion properties. Wells were washed 3 times with DPBS.
Macrophages with particles were plated in the glass confocal dishes
at 62,500 cells/cm2 and incubated for 8 h in DMEMpy. Cells were
incubated with L-NAME at 75 µg/mL in DMEMpy for 1 h to inhibit
biological NO production from nitric oxide synthases. Wells were
aspirated
and
9.3
µM
4-amino-5-methylamino-2’,7’-
difluoroflurescein (DAF-FM-2DA), a dye that detects intracellular
NO release, with L-NAME in DMEMpy was added to the cells and
incubated for 1 h. Wells were washed five times with phenol red
free, FBS free DMEMpy with L-NAME to remove uninternalized
DAF-FM. Wells were exposed to a 794 nm laser for 90 s at 13.1
W/cm2. NucBlue® was added to the wells to label cell nuclei. Wells
were imaged 20 min later. Images were taken with an Olympus
Fluoview 1000 Spectral Confocal. Care was taken to image cells as
quickly as possible to prevent the release of extra NO. Samples
were compared to macrophages exposed to the laser without
particles
Particle loading into BMMs.
Particles with or without NO-donor and UCNPs in DI water at a
concentration of 5 mg/mL in a glass vial were sonicated for 10 min
to break up particle aggregates. A volume of 340 µL of the
microparticle solution was added to macrophages plated at 62,500
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Quantification of macrophage chemotaxis.
HBSS solution in a modified cuvette and closely placed at the corner
in order to get full exposure with a 794 nm diode laser. Medical-
grade air was purged into the cuvette through plastic tubing but not
directly purged into the solution. Laser intensity 13.1 W/cm2 was
applied to all spheroid-involved measurement. During the period of
laser irradiation on these spheroids, the medical air purging was
only above the solution. But once the irradiation was stopped, the
purging tubing was manually immersed into the solution carefully in
order to avoid agitating these spheroids. The flowing gas conveyed
the NO released to the NOA.
Human fibronectin (50 µL,150 µg/mL) in DPBS was added to
the top of 24-well sized FluoroBlokTM transwell inserts with 8 µm
pores and incubated in a cell incubator for 1.5-2 h to improve their
adhesion properties for endothelial cells. The top of the inserts was
washed 3 times with 200 µL of DMEMpy. A 200 µL aliquot of bEnd.3
cells at a concentration of 1x105 cells/mL were added to the top of
each well. Cells were incubated for 4.5 days with media changes
every other day. No media was added to the bottom of the insert
until the final day to prevent the growth of a monolayer in the
bottom of the insert which has been reported previously when Confocal imaging of UCNPs in live tumor spheroids.
media is added to both sides.62 On day 4.5, media in the top insert
Spheroids were grown and incubated with macrophages with
was replaced and 600 µL of DMEMpy was added to the bottom of
the insert. Transepithelial electrical resistance (TEER) values were
taken and all inserts with a value ≥ 0.33 Ω·cm2, which traditionally
represents a fully confluent monolayer, were accepted for the
experiment. Inserts were washed 3 times with DMEMpy. 200 µL of
either DMEMpy alone, or BMMs with or without particles at 5x105
cells/mL in DMEMpy were added to the top of the inserts and 600
µL of DMEMpy with 125 ng/mL MCP-1 was added to the bottom of
each well to serve as a chemoattractant for the macrophages. Cells
were incubated for 24 h in a cell incubator and washed once with
DPBS. NucBlue® in HBSS was added to the top and bottom of each
insert to visualize cells. After 20 min, images from 3 random
locations from the bottom of each insert were taken with an
Olympus CKX-41 inverted microscope. Cells were counted with the
particle analysis tool on ImageJ. Wells that contained only a bEnd.3
and without particles as described in a previous section. Calcein AM
(10 µM) was incubated with the spheroids for 2 h in DMEMpy and
to mark spheroid peripheries. Live spheroids were added to a petri
dish with HBSS. UCNPs were detected using a 3W 100 fs pulsed
laser (37.5 nJ/pulse) operating at 810 nm. Images were acquired
with an Olympus Flowview 1000MPE confocal microscope with a
25x objective (numerical aperture = 1.05). Images were recorded
over a 508.431µm x 508.431µm area (1024 x 1024 pixels) and were
acquired with a scan rate of 100 µs/pixel. Bandpass filters (420-460
nm and 495-540) nm were utilized to isolate fluorescence from
UCNPs and calcein AM, respectively. Images acquired every 10 µm
were combined to form 3D reconstructions with ImageJ.
Tumoricidal potential of high dose NO therapy.
Five spheroids containing microparticle loaded macrophages
monolayer were used as a baseline and were subtracted out of with and without NO-donor were transferred to the corner of a
macrophage wells.
cuvette containing DMEMpy. Spheroids were exposed to a 794 nm
laser at 13.1 W/cm2 for 6 min to release NO from particles inside
macrophages in the spheroids. This procedure was done to each
spheroid 1, 2, or 3 times with 2 min between each exposure. The
spheroids were transferred to a 96 TC-treated well plate and
incubated for 24 h. After incubation, media was removed and 100
µL of DPBS was added to each well. To measure spheroid viability, a
PNPP assay was used as previously described with slight
modifications.51 Briefly, one 10 mg PNPP substrate tablet was
added to 5 mL of a solution containing 0.1 M sodium acetate and
0.1% TritonTMX-100. A 100 µL aliquot of this solution was added to
the DPBS already in each well. The plate was incubated at 37 ºC for
3-3.5 h. NaOH (10 µL, 1M) was added to each well after which
absorbance was measured at 405 nm on a TECAN M220 Infinite Pro
plate reader within 10 min of NaOH addition to prevent loss of
signal.
Determination of macrophage/spheroid penetration.
Macrophages labeled with CellTrackerTM deep red were
allowed to penetrate spheroids as described above to allow for
visualization. Spheroids were fixed in ice-cold methanol for 30 min.
Spheroids were then embedded in Tissue-Tek® O.C.T compound and
sectioned with a 25 µm thickness. Spheroids were mounted with
Vectashield® hard set mounting media with 4,6-diamidino-2-
phenylindole (DAPI) to preserve samples and stain cell nuclei. For
hypoxia detection, some spheroids were incubated with 100 µM
PIMO in DMEMpy for 2 h prior to fixation with methanol (MeOH).
This allowed PIMO to fully infiltrate spheroids and covalently bind
to cells in hypoxic regions. After sectioning, these samples were
blocked with PBS containing 4% FBS, 1% goat serum, and 0.05%
Tween® 20 (blocking solution) for 1 h. Hypoxyprobe Red549 (#HP7-
100Kit; Hypoxyprobe) in a 1:200 dilution in blocking solution was
added to each spheroid for 18 h at 4 °C to allow the antibody to
bind to the PIMO present in the sectioned spheroid. Sections were
Tumor microenvironment modulation with low dose NO therapy.
Spheroids with particle loaded macrophages were transferred
then washed three times with PBS with 0.05% Tween® 20. In other to a 96 well TC-treated plate with a pipette and were incubated for
experiments to demonstrate retention of particles after spheroid 12 h to allow them to adhere. Half of the spheroids were then
penetration, particles were labeled with Cy3.5®-IgG. Because of the exposed to a 735 nm LED at 0.58 mW/cm2 for 8.5 h. Spheroids were
overlap between Hypoxyprobe Red549 and Cy3.5®, they were not collected in groups of 6 and washed once with DPBS. AccumaxTM
used together. Images were acquired with an Olympus Fluoview (300 µL) was added and spheroids were incubated for 30-45 min.
1000 spectral confocal microscope and processed with ImageJ.
Spheroids were broken up with rapid pipetting to form a single cell
suspension. Cells were fixed with 4% paraformaldehyde (PFA) for 10
min followed by permeablization with ice cold methanol (MeOH)
for 10 min to allow for antibody labeling. Cells were washed three
times with DPBS with 1% BSA and 0.1% Tween 20. Rabbit anti-HIF-
NO release measurement.
The NO measurement followed the modified procedure from
the literature.16 All spheroids were carefully transferred to 1 mL
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0.1% Tween 20 was added to the cells for 1 h at RT. Cells were
washed once with DPBS with 1% BSA and 0.1% Tween 20. FITC
labeled Goat-anti-rabbit IgG(H+L) (1:250, #A27034; ThermoFisher
Scientific) was added to the cells for 30 min in DPBS with 1% BSA
and 0.1% Tween 20. Cells were washed with DPBS 3 times and
analyzed via flow cytometry on a FACSAria (Becton Dickinson) using
488-nm (HIF-1α) or 633-nm (CellTracker Deep Red) excitation. Cells
were analyzed for % of the cells with fluorescence signal greater
than background by setting the gate to exclude the signal from
unstained cells (negative control). Results were analyzed with FCM
Express 6 Plus.
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7
8
Acknowledgements
This research was supported by grants to PCF from the
Chemistry Division of the National Science Foundation (CHE-
156570) and to SM from the Department of the Defense, Defense
Threat Reduction Agency (HDTRA1-15-1-0045). We thank Aaron
Anselmo, Renwei Chen, Megan Chui John V. Garcia, Vinu Krishnan,
Elizabeth S. Levy, Agustin Pierri, Ivan Pina and Anusha Pulusari of
the University of California, Santa Barbara, who carried out
preliminary studies or provided technical help that advanced our
understanding of the systems reported here. UCNPs synthesis at
the Molecular Foundry was supported by the Office of Science,
Office of Basic Energy Sciences, of the U.S. Department of Energy
under Contract No. DE-AC02-05CH11231. Plate reader, dynamic
light scattering, and tissue culture work were conducted in the
Biological Nanostructures Laboratory within the California
NanoSystems Institute, supported by the University of California,
Santa Barbara and the University of California, Office of the
President. We acknowledge the use of the NRI-MCDB Microscopy
Facility and its single and multiphoton spectral laser scanning
confocal and supported by the Office of The Director, National
Institutes of Health of the NIH under Award # S10OD010610 and
S10RR02259501A1. Inductively coupled plasma atomic emission
spectroscopic measurements were conducted in the UCSB MRL
Shared Experimental Facilities which are supported by the MRSEC
Program of the NSF under Award No. DMR-1720256; a member of
the NSF-funded Materials Research Facilities Network.
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§
A reviewer raised the legitimate concern that Gd3+ has been shown to
be toxic to humans. Inclusion of the Gd3+ dopant in the UCNPs in the
present case was purely for synthetic convenience, since the only
function Gd3+ serves is to facilitate synthesis of the desired hexagonal
phase of NaYF4 (ref 34-36). There are other methods for synthesizing
hexagonal NaYF4 without Gd (ref 33), and these will be used in future
studies.
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a
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Table of Contents Illustration and Synopsis
Macrophage-mediated targeting and photochemical release
provides spatial-temporal control of nitric oxide delivery to tumor
spheroids
.
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