1.2022.Boosted Radiation Bystander Effect of PSMA-Targeted Gold

nanomaterials
Article
Boosted Radiation Bystander Effect of PSMA-Targeted Gold
Nanoparticles in Prostate Cancer Radiosensitization
Daiki Hara 1,2 , Wensi Tao 1,3 , Ryder M. Schmidt 1,2 , Yu-Ping Yang 3,4 , Sylvia Daunert 3,4 , Nesrin Dogan 1,2 ,
John Chetley Ford 1,2, *, Alan Pollack 1,3 and Junwei Shi 1,3, *
1 Department of Radiation Oncology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA
2 Department of Biomedical Engineering, College of Engineering, University of Miami, Miami, FL 33146, USA
3 Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami,
Miami, FL 33136, USA
4 Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami,
Miami, FL 33136, USA
* Correspondence: [email protected] (J.C.F.); [email protected] (J.S.)
Abstract: Metal nanoparticles are effective radiosensitizers that locally enhance radiation doses
in targeted cancer cells. Compared with other metal nanoparticles, gold nanoparticles (GNPs)
exhibit high biocompatibility, low toxicity, and they increase secondary electron scatter. Herein, we
investigated the effects of active-targeting GNPs on the radiation-induced bystander effect (RIBE) in
prostate cancer cells. The impact of GNPs on the RIBE presents implications for secondary cancers
or spatially fractionated radiotherapy treatments. Anti-prostate-specific membrane antigen (PSMA)
antibodies were conjugated with PEGylated GNPs through EDC–NHS chemistry. The media transfer
technique was performed to induce the RIBE on the non-irradiated bystander cells. This study
focused on the LNCaP cell line, because it can model a wide range of stages relating to prostate
cancer progression, including the transition from androgen dependence to castration resistance and
Citation: Hara, D.; Tao, W.; Schmidt,
bone metastasis. First, LNCaP cells were pretreated with phosphate buffered saline (PBS) or PSMA-
R.M.; Yang, Y.-P.; Daunert, S.; Dogan,
N.; Ford, J.C.; Pollack, A.; Shi, J.
targeted GNPs (PGNPs) for 24 h and irradiated with 160 kVp X-rays (0–8 Gy). Following that, the
Boosted Radiation Bystander Effect collected culture media were filtered (sterile 0.45 µm polyethersulfone) in order to acquire PBS- and
of PSMA-Targeted Gold PGNP- conditioned media (CM). Then, PBS- and PGNP-CM were transferred to the bystander cells
Nanoparticles in Prostate Cancer that were loaded with/without PGNPs. MTT, γ-H2AX, clonogenic assays and reactive oxygen species
Radiosensitization. Nanomaterials assessments were performed to compare RIBE responses under different treatments. Compared
2022, 12, 4440. https://doi.org/ with 2 Gy-PBS-CM, 8 Gy-PBS-CM demonstrated a much higher RIBE response, thus validating the
10.3390/nano12244440 dose dependence of RIBE in LNCaP cells. Compared with PBS-CM, PGNP-CM exhibited lower
Academic Editor: Wen-Huei Chang cell viability, higher DNA damage, and a smaller survival fraction. In the presence of PBS-CM,
bystander cells loaded with PGNPs showed increased cell death compared with cells that did not
Received: 3 November 2022
have PGNPs. These results demonstrate the PGNP-boosted expression and sensitivity of RIBE in
Accepted: 9 December 2022
prostate cancer cells.
Published: 14 December 2022
Publisher’s Note: MDPI stays neutral Keywords: gold nanoparticle; radiosensitization; radiation-induced bystander effect; prostate LNCaP
with regard to jurisdictional claims in cancer cells; prostate-specific membrane antigen; active targeting
published maps and institutional affil-
iations.
1. Introduction
Copyright: © 2022 by the authors.
Prostate cancers contributed to about 34,500 deaths in the United States in 2022,
Licensee MDPI, Basel, Switzerland. according to American Cancer Society’s estimates [1]. As a mainstay of cancer treatment,
This article is an open access article radiotherapy (RT) is commonly used to offer both definitive and palliative strategies for
distributed under the terms and prostate cancer management [2]. The efficacy of RT in cancer therapy stems from the
conditions of the Creative Commons fact that ionizing radiation can directly and indirectly damage DNA and disrupt the
Attribution (CC BY) license (https:// atomic structure of biomolecules in the cellular environment [3]. In recent decades, we
creativecommons.org/licenses/by/ have witnessed a development boom in terms of high-precision RT techniques, such as
4.0/). intensity modulated radiotherapy (IMRT) and real-time adaptive MRI-guided radiation;
Nanomaterials 2022, 12, 4440. https://doi.org/10.3390/nano12244440 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2022, 12, 4440 2 of 18
these techniques allow improved dose conformity to the tumor target as well as a decreased
dose to adjacent healthy tissues [4]. Nevertheless, due to the similar mass energy absorption
properties of both cancer and healthy tissues, physical radiation dose escalation and beam
conformality has approached an upper limit with regard to prostate cancer external beam
RT. Radiosensitizers, including small molecules, macromolecules, and nanomaterials, are
promising agents that offer the means for further tumor dose escalation with improved
normal tissue sparing [5].
Due to their high mass energy absorption coefficient relative to soft tissue, nanoparti-
cles of high atomic number (Z) materials (such as: iodine, gadolinium, hafnium, tantalum,
tungsten, bismuth) have been implemented to improve the contrast between tumors and
healthy tissues, thus enabling tumor-specific radiosensitization with reduced side effects [6].
As a promising high-Z nano-radiosensitizer, gold nanoparticles (GNPs) have lately gar-
nered attention due to their special properties; these include high biocompatibility with
low toxicity, and the facile attachment of a variety of biological ligands [7]. The efficiency of
GNP radiosensitization has been extensively validated in both in vitro and in vivo scenarios
using numerous types of ionizing radiation, including kilovoltage (kV) and megavoltage
(MV) photons as well as charged particles [8]. Compared with other nanoparticles, GNPs
have been well studied for their efficient radiosensitizing effects, their multitude of mecha-
nisms which allows radiosensitization to be carried out, and their comparatively limited
toxicity [9]. Although dose enhancement factors vary based on radiation source, Jones et al.
reported Monte Carlo simulated microscopic dose enhancement factors of 80× in 50 kVp
photon beams and 9.8× in 6 MV photon beams, up to 100 µm, from the GNP surfaces [10].
Similarly, Lin et al. measured a dose enhancement factor of up to 14× in 150 MeV protons
at a distance of 10 µm from the nanoparticle surface [11]. Aside from the incident beam,
further optimization studies on GNP shape and size suggest that spherical nanoparticles
between 10 nm to 20 nm provide the most optimal increase in secondary electron scatter
and they minimize the level of toxicity in normal tissue [12].
Blood circulation pathway/time and the extent of tumor accumulation dictates the
level of biodistribution, toxicity, and radiosensitization from GNPs. Polyethylene glycol
(PEG) is ubiquitously used to coat GNPs, which significantly reduces nonspecific binding
with cells and serum proteins, improves GNPs’ stability and biocompatibility under physi-
ological conditions, and it greatly lengthens the circulation half-life of GNPs in vivo [13].
GNPs passively leak into the tumor interstitium from blood vessels feeding the tumor
via the enhanced permeation and retention (EPR) effect; this occurs due to the tumors’
leaky vasculature and poor lymphatic drainage [14]. The EPR effect is the rationale behind
the passive targeting approach; however, the efficiency of passive targeting is low, and
it is still controversial as to whether the EPR effect is relevant in humans [15]. Therefore,
the improved accumulation of GNPs with regard to tumors, beyond reliance on the EPR
effect, is critical for GNP-based therapies. GNPs are ideal candidates for conjugating tumor-
targeting agents because GNP surface chemistry enables a multitude of chemicals to bind at
high densities [16]. Among potential targeting agents for prostate cancer, prostate specific
membrane antigen (PSMA) ligands have been employed with great success in preclinical
and clinical studies for PET imaging and radionuclide therapies; this is because prostate
cancers consistently overexpress PSMA [17,18]. Our previous studies have shown the effec-
tiveness of PSMA-targeted GNPs and their ability to accumulate at higher concentrations
and to be retained for longer in prostate tumors [19].
Multiple studies have confirmed the radiosensitization of tumor-targeted GNPs in
a multitude of tumor tissues, including prostate cancers [8]. It is worth mentioning that
the radiosensitization observed in both in vitro and in vivo studies is often significantly
greater than the dose enhancement predicted by Monte Carlo computational models [20].
These suggest that complex chemical and biological components, including enhanced ROS
production, change during cell cycle distribution. Moreover, these components also affect
the overall toxicity levels in cancer cells, and they are involved in GNP-induced radiosen-
sitization [20]. Of these biochemical effects, the GNPs’ effects on the radiation-induced
bystander effect (RIBE) have recently received attention. RIBE is the phenomenon by which
non-irradiated cells exhibit similar ionizing radiation damage as a result of signals received
from nearby irradiated cells [21]. The bystander signals involved in this process may cause
altered gene expression, DNA and chromosomal damage, cell proliferation alteration, or
cell death in non-irradiated cells [22]. RIBEs have been demonstrated using a range of exper-
imental systems with multiple biological endpoints; this has been the case since they were
first identified by Nagasawa and Little in 1992 [23]. The underlying mechanisms mediating
RIBE responses have been extensively studied and it has been shown that reactive oxygen
and nitrogen species (ROS/NOS), DNA repair proteins, cytokines, ligands, extracellular
DNA (ecDNA), microRNA (miRNA), and membrane molecules are the main materials that
are released from targeted cells. Moreover, they are transferred to non-targeted cells via
gap junction intercellular communication (GJIC) and the media/circulatory system [24].
In standard radiotherapy, where uniform fields are delivered and all cells are directly
exposed to radiation, RIBE phenomena can be neglected; however, the role of RIBEs may
become more influential when heterogeneous or non-uniform fields are considered. Al-
though most clinical radiotherapy focuses on uniform exposures, there are some examples
of non-uniform plans being utilized in the clinic, including spatially fractionated radio-
therapy (or GRID), mini-beam radiotherapy (MBRT), microbeam radiotherapy (MRT), and
dose-painting radiotherapy [25]. Healthy tissue has been shown to be more tolerant of
spatially fractionated dose fields than tumor tissue, thus allowing for a high dose to be
delivered in a single fraction. Spatially fractionated GRID purposely irradiates the tumor
with highly non-uniform dose fields containing steep dose gradients [26,27]. Similarly to
GRID therapy, MBRT and MRT are also characterized by alternating distributions of high
and low doses, but on a much smaller scale (a hundred micrometers) [28]. Dose-painting
radiotherapy allows for the heterogeneous delivery of high radiation doses within the
tumor by targeting one or more regions of interest that are defined by functional imag-
ing [29]. Furthermore, Monte Carlo simulation studies have shown that GNP-induced dose
enhancement can be increased by a factor of 3000 compared with doses originating from a
hypothetical water nanoparticle, but only at microscopic distances of 10 µm [10,11]. The
heterogenous distribution of GNPs, combined with the highly localized energy depositions
made by GNPs, resemble the dose pattern of spatially fractionated radiotherapy [30]; thus,
RIBE plays an important role in GNP-enhanced radiation therapy.
RIBE responses have been observed in many cell types such as lymphocytes, fibrob-
lasts, endothelial, and cancer cells [31]. Rostami et al. first investigated the effects of
glucose-coated GNPs (Glu-GNPs) on the RIBE in MCF-7 (human breast cancer) and QUDB
(human lung cancer) cell lines [32]. Their results demonstrated Glu-GNPs’ enhanced RIBEs
in QUDB cells, but there was no RIBE enhancement in MCF-7 cells. This observation
suggests that the impact of GNPs on the RIBE is cell type specific (i.e., some cell types are
unable to produce bystander signals whereas others are unable to respond to bystander
signals). To the best of our knowledge, there are no studies focusing upon the impact of
GNP-induced radiosensitization on the RIBE in prostate cancer cells. The present study
was carried out to investigate how actively targeting GNPs impacts the RIBE response
in prostate cancer LNCaP cells. LNCaP cells were selected as the focus in this study be-
cause they can model a wide range of prostate cancer stages, including the transition from
androgen dependence to castration resistance and bone metastasis. Moreover, the high
expression of PSMA means that the LNCaP cancer model is a good choice with which to
develop active-targeted nanotherapeutic strategies for prostate cancer. Anti-PSMA anti-
bodies were conjugated with PEGylated GNPs to develop PSMA-targeted GNPs (PGNPs).
A conditioned medium transfer technique was employed to evaluate the RIBE responses.
The yield of the RIBE signal from irradiated cells, and the sensitivity of non-irradiated cells
with regard to the RIBE signal, was used to investigate the impact of PGNPs on the RIBE in
prostate cancer cells. As control groups, RIBE responses were also implemented for use on
other prostate cancer cell lines (including PC3, and 22Rv1) as well on normal prostate cell
lines (RWPE-1).
2. Materials and Methods

2.1. Synthesis of PSMA-Targeted GNPs (PGNPs)
Commercially available PEGylated GNPs (Creative Diagnostics, NY, USA) were used
as substrates for functionalization in this study. The GNP concentration was quanti-
fied by optical absorption spectra which were determined using UV-vis spectroscopy
(240 nm–780 nm, Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA). To realize ac-
tive and passive prostate cancer targeting, anti-PSMA and mouse IgG antibodies (Creative
Diagnostics, NY, USA) were coupled with PEGylated GNPs using EDC/NHS chemistry.
First, 0.2 M 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (#22980;
Thermo Fisher Scientific, Waltham, MA, USA) and 0.2 M N-hydroxysuccinimide (NHS)
(#24500; Thermo Fisher Scientific, Waltham, MA, USA) were simultaneously added to
a solution that included OD = 1 GNPs, 0.1 mg/mL antibody, and 0.1 M sodium borate
buffer. This mixture was incubated at room temperature for 24 h. Then, the conjuga-
tion solution was washed out by three centrifugations at 15,000× g for 30 min, and the
final GNP pellet was stored in Milli-Q water (Millipore, Bedford, MA, USA) to obtain the
desired concentration.
2.2. Characterization of PGNPs

Transmission electron microscopy was used to determine the shape and size of the
developed PGNPs. PGNPs were cast onto a carbon Formvar-coated copper grid for 30 min.
Excess liquid was absorbed using filter paper, and the grid was allowed to air dry overnight.
The grids were viewed at 80 kV in a JEOL JEM-1400 transmission electron microscope (JEOL
USA, Peabody, MA, USA) and images were captured with an AMT BioSprint digital camera
(Advanced Microscopy Techniques, Woburn, MA, USA). Additional characterizations
of nanoparticles have been detailed in our previously published works that focus on
PGNPs [19].
2.3. Cell Culture

Human prostate cancer cells (LNCaP, PC3, and 22Rv1) were obtained from ATCC.
Cells were cultured in RPMI1640 media (ThermoFisher Scientific, Waltham, MA, USA)
with 10% fetal bovine serum (FBS, GeminiBio, Sacramento, CA, USA) and 1% penicillin-
streptomycin (ThermoFisher Scientific, Waltham, MA, USA). The cells were grown in a CO2
incubator at 37 ◦ C and subcultured when they reached 80–90% confluency. Normal prostate
cells (RWPE-1) were cultured in the same conditions, although keratinocyte serum-free
media (ThermoFisher Scientific, Waltham, MA, USA) was used instead of RPMI1640.
2.4. PGNP Cytotoxicity

A MTT viability assay was used to assess the cytotoxicity of treatments on LNCaP
cells. Moreover, 24 h after the treatments, LNCaP cells in 96-well plates were stained with
1 M yellow tetrazolium substrate (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide) (MTT, Thermo Fisher Scientific, Waltham, MA, USA) in serum-free RPMI1640,
for 4 h, at 37 ◦ C. The supernatant was removed from the wells and formazan crystals were
dissolved with the addition of 100 µL DMSO. This was added to each well and left for
30 min at 37 ◦ C. An additional column of empty wells received 100µL of DMSO, which
were to be read as blanks. Microplates were read on a Clariostar microplate (BMG Labtech,
Ortenberg, Germany) reader for absorbance at 570 nm. MTT assay results were normalized
to ensure no treatment absorptions.
2.5. Cellular PGNP Uptake Assay

LNCaP cells were treated with 50, 100, 150, 250, 500, and 750 µg/mL PGNPs in
serum-free RPMI1640 media for 24 h. Serum-free media were used to avoid changes to
the nanoparticle surface as a result of FBS proteins. After 24 h, the cells were washed
three times with PBS, and they were replenished with fresh media supplemented with
FBS and pen-strep prior to running assays. PGNP concentrations were measured using
UV-Vis spectrophotometry on a Nanodrop Spectrophotometer before and after treatment.

Spectrophotometry was conducted using a range between 280 nm to 700 nm, where the
peak intensity at 520 nm was used to quantify PGNP concentration; this was achieved
by benchmarking against the manufacturer’s recorded GNP optical density. The total
cellular uptake of PGNP was calculated as the PGNP’s concentration difference in media
before and after 24 h of treatment. Cells plated on coverslips were used to identify the
intracellular distribution of PGNPs with TEM. PGNP-treated cells were fixed overnight
in 2% glutaraldehyde, in 0.1 M phosphate buffer; then, they were post-fixed for 1 h in
2% osmium tetroxide in 0.1 M phosphate buffer, dehydrated through a series of graded
ethanols, and embedded in an EM-bed (Electron Microscopy Sciences, Fort Washington,
PA, USA). The glass coverslip was dissolved in hydrofluoric acid. In addition, 100 nm
sections were cut on a Leica Ultracut EM UC7 ultramicrotome and stained with uranyl
acetate and lead citrate. Images were captured on the same setup as the PGNPs on grids.
2.6. RIBE on LINCaP Cells

Conditioned media was collected from LNCaP cells to induce RIBE in LNCaP by-
stander cells. Prior to irradiation, cells were washed three times with PBS, and serum-free
RPMI1640 was added to the culture plates. Cells were irradiated between 0–8 Gy at
2.0 Gy/min on a 160 kV (0.0775 Å) Radsource RS-2000 Biological Irradiator (Rad Source
Technologies, Buford, GA, USA) with cells placed at 28 cm SSD (25 cm diameter circular
field size). Following irradiation, cells were incubated at 37 ◦ C for 1 h. Media from sham
and irradiated cells were harvested and filtered through a 0.45 µm polyethersulfone filter
(VWR, Radnor, PA, USA). All conditioned media were prepared immediately before use.
2.7. Effects of PGNPs on RIBE

To assess the effect of PGNPs on the RIBE, LNCaP cells were divided into three groups;
bystander cells treated with conditioned media from irradiated cells (PBS-CM), bystander
cells treated with conditioned media from PGNP treated irradiated cells (PGNP-CM), and
PGNP treated bystander cells treated with conditioned media (PBS-CM+PGNP).
2.8. Clonogenic Assay

LNCaP cells were grown in 6-well dishes, and they were treated as mentioned above.
Irradiated or PBS-CM treated cells were trypsinized, then replated on 6 cm dishes with
varying cell numbers. Following 3 weeks of growth, plates were washed three times
with cold PBS, fixed in 4% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA,
USA), and stained with 0.5% crystal violet (Thermo Fisher Scientific, Waltham, MA, USA)
for 30 min. Plates were washed with tap water, imaged on a Chemidoc touch (Bio-rad,
Hercules, CA, USA), and counted using particle counting on ImageJ.
2.9. γH2AX Assay

LNCaP cells were grown in chamber slides (Thermo Fisher Scientific, Waltham, MA,
USA), treated as mentioned above, and fixed in 4% neutral buffered formaldehyde, perme-
abilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin in PBS-tween
containing 5% goat serum. Slides were incubated with an antibody in order to hosphor-
H2AX (1:500, CST). This was followed by incubation with goat-anti-rabbit Alexa488 (1:2000,
Invitrogen, Waltham, MA, USA) and the slides were then mounted with a Prolong gold
antifade reagent with DAPI (Invitrogen, Waltham, MA, USA). Cells were analyzed on a
Leica confocal microscope (Leica Microsystems, Deerfield, IL, USA) with ×63 magnification.
All γH2AX foci were counted using ImageJ particle counting.
2.10. ROS Quantification

LNCaP cells in 96-well plates were preloaded with 20 µM of DCFH-DA (Sigma-
Aldrich, St. Louis, MO, USA) in serum-free RPMI1640 for 45 min. Afterwards, cells
were washed three times with PBS and fresh serum-free RPMI1640 media; alternatively,
conditioned media was added. For direct irradiation ROS studies, 0–8 Gy of radiation was
administered to the cells. DCFH-DA fluorescence at 480 nm excitation/535 nm emission
was scanned on a Clariostar microplate reader 1 h post irradiation or post conditioned
media treatment.
2.11. Statistical Analysis

GraphPad Prism version 9.0 software was used for all statistical analyses. Differences
between experimental groups were assessed using an unpaired t-test during a comparison
of the two groups.
3. Results
3.1. Characterization, Cell Uptake, and Cytotoxicity of PGNPs
The performance of PGNPs with respect to internalization and biocompatibility was
assessed for effective usage as radiosensitizers. A TEM image shows the spherical monodis-
Nanomaterials 2022, 12, x FOR PEER REVIEW 7 of 18
persed nanoparticles following conjugation with the PSMA-antibody, with a mean diameter
of 17.1 nm ± 0.28 nm (Figure 1A,B).
Figure
Figure1.1.(A)
(A)TEM
TEMimage
imageofofPSMA-antibody
PSMA-antibodyconjugated
conjugatedgoldgoldnanoparticles
nanoparticles(PGNPs).
(PGNPs).(B) (B)Histogram
Histogram
of PGNP diameters (mean = 17.1 nm ± 0.28 nm) from TEM images. (C) Representative TEM image
of PGNP diameters (mean = 17.1 nm ± 0.28 nm) from TEM images. (C) Representative TEM image of
of a LNCaP cell treated with 250 µ g/mL of PGNPs. (D) Magnified region with arrow heads
a LNCaP cell treated with 250 µg/mL of PGNPs. (D) Magnified region with arrow heads indicating
indicating internalized PGNPs. (E) PGNP uptake in LNCaP cells after 24 h of treatment at various
internalized
PGNP PGNPs.
doses (50 µ g/mL,(E)
100PGNP uptake
µ g/mL, 150 µ in LNCaP
g/mL, 250 µcells
g/mL,after
50024 h of treatment
µ g/mL, 750 µ g/mL). at (F)
various PGNP
Percentage
doses (50
viability of µg/mL,
the LNCaP100cells
µg/mL, 150 µg/mL,
incubated 250 µg/mL,
with different 500 µg/mL,
concentrations 750 µg/mL).
of PGNP (50 µ g/mL,(F)100
Percentage
µ g/mL,
viability
150 of the
µ g/mL, 250LNCaP
µ g/mL,cells
500incubated
µ g/mL, 750with different
µ g/mL). (G)concentrations
Western blotof forPGNP
PSMA (50expression
µg/mL, 100 inµg/mL,
whole
cell lysate of250
150 µg/mL, LNCaP,
µg/mL,PC3, 22Rv1 750
500 µg/mL, (prostate
µg/mL). cancer), and RWPE1
(G) Western (normal
blot for PSMA prostate)in cells.
expression whole(H)
cell
Quantification
lysate of LNCaP,ofPC3,
PSMA22Rv1expression in the western
(prostate cancer), and RWPE1 blot(normal
normalized
prostate)to cells.
a GAPDH signal. (I)
(H) Quantification
Fluorescence microscopy
of PSMA expression image
in the of LNCaP,
western PC3, 22Rv1,
blot normalized to a and
GAPDHRWPE1 cells(I)
signal. treated with 250microscopy
Fluorescence µ g/mL of
passive targeting (mouse IgG conjugated) GNPs and active targeting (PSMA-antibody
image of LNCaP, PC3, 22Rv1, and RWPE1 cells treated with 250 µg/mL of passive targeting (mouse conjugated)
PGNPs. (J) Quantification of fluorescence microscopy normalized to the highest signal in each cell
IgG conjugated) GNPs and active targeting (PSMA-antibody conjugated) PGNPs. (J) Quantification
line. * Denotes p<0.05 from Welch’s t-test.
of fluorescence microscopy normalized to the highest signal in each cell line. * Denotes p < 0.05 from
Welch’s t-test.
3.2. Effect of Radiation Dose or PGNP Concentration on Radiosensitization
PGNP-induced radiosensitization in LNCaP cells was assessed through a clonogenic
cell survival assay and γ-H2AX assays. To examine the effects of radiation doses and
PGNP concentrations on in vitro radiosensitization, the LNCaP cells were incubated with
PGNPs at different concentrations (0, 50, 100, 150, 250 µ g/mL) and irradiated with
different doses (0, 2, 4, 6, 8 Gy). Clonogenic cell survival assay results (Figure 2A)
Incubating PSMA-expressing LNCaP cells with the developed PGNPs revealed the
internalization of PGNPs in small clusters throughout the cell (Figure 1C,D). Cellular uptake
analysis (Figure 1E) demonstrated the enhanced PGNP levels at increasing concentrations,
with the maximum saturation point starting from a concentration of 250 µg/mL. On
average, a single LNCaP cell accumulated 12 picograms PGNP under 250 µg/mL of
PGNP treatment (Figure 1E). The percentage cell viability (Figure 1F) demonstrated that
there exists no statistically significant cytotoxicity for PGNPs with concentrations up to
250 µg/mL; therefore, the biocompatible concentration of the 250 µg/mL PGNPs was
applied to the following RIBE experiments, which falls under the calculated IC50 value of
420 µg/mL.
To validate the specificity of active-targeting PGNPs, GNPs conjugated with mouse
IgG (passive-targeting) and anti-PSMA IgG (active-targeting) were used for treatment
on prostate cancer cell lines (including LNCaP, PC-3, and 22Rv1) and normal prostate
endothelial cell lines (RWPE-1). First, we compared the expression of PSMA with different
prostate cancer cell lines, as well as normal prostate cell lines. Western blot analysis
demonstrated that LNCaP and 22Rv1 express significant levels of PSMA, whereas PC-3 and
RWPE-1 are PSMA negative (Figure 1G,H). Then, we compared the targeting efficiency of
PSMA-passive and -active GNPs on different cell lines by labelling GNPs with fluorophores
and treating each cell line with 250 µg/mL of GNPs for 24 hrs. Compared with PSMA-
passive GNPs, PSMA-active GNPs exhibited significantly increased levels of fluorescence
signals in LNCaP and 22Rv1 cell lines, but not in PC3 and RWPE-1 cells; therefore, moving
forward, LNCaP cells were identified as the optimal cell line with which to study the RIBE
because of their high PSMA expression (Figure 1I,J).
3.2. Effect of Radiation Dose or PGNP Concentration on Radiosensitization

PGNP-induced radiosensitization in LNCaP cells was assessed through a clonogenic
cell survival assay and γ-H2AX assays. To examine the effects of radiation doses and PGNP
concentrations on in vitro radiosensitization, the LNCaP cells were incubated with PGNPs
at different concentrations (0, 50, 100, 150, 250 µg/mL) and irradiated with different doses
(0, 2, 4, 6, 8 Gy). Clonogenic cell survival assay results (Figure 2A) demonstrated enhanced
radiosensitization levels in LNCaP cells when subjected to increased PGNP concentrations.
Moreover, for a specific PGNP concentration, a higher radiosensitization level was observed
at a higher radiation dose. Figure 2B compares the radiosensitization of PGNPs, using
various concentrations, under 2-Gy irradiation. When treated with 150 µg/mL PGNPs
or more, there was a statistically significant difference regarding the radiosensitization
levels between the groups that were treated without and with PGNPs, thus suggesting that
150 µg/mL was the minimum concentration required for PGNP-induced radiosensitization
under 2 Gy radiation.
γ-H2AX foci were assessed as biomarkers for double-strand DNA damages. As
shown by the fluorescence images in Figure 2C, γ-H2AX foci could be clearly distinguished
after the irradiation (2 Gy) of LNCaP cells incubated with/without PGNPs (250 µg/mL).
Additionally, the non-irradiated LNCaP cells that were incubated with 250 µg/mL PGNPs
showed no signs of foci formation, thus showing that these nanoparticles have no DNA
damage effects without radiation. The average number of γ-H2AX foci per cell was counted
during 2-Gy irradiation at different PGNP concentrations (0, 50, 100, 150, 250 µg/mL), and
the results are presented in Figure 2D. PGNP treatment showed a dose dependent increase
in foci count with an increase in GNP concentration (Figure 2D). A statistically significant
increase in foci count was seen at 250 µg/mL.
have no DNA damage effects without radiation. The average number of γ-H2AX foci per
cell was counted during 2-Gy irradiation at different PGNP concentrations (0, 50, 100, 150,
250 µ g/mL), and the results are presented in Figure 2D. PGNP treatment showed a dose
Nanomaterials 2022, 12, 4440 dependent increase in foci count with an increase in GNP concentration (Figure 2D). 8 of A
18
statistically significant increase in foci count was seen at 250 µ g/mL.
Figure
Figure 2.
2.(A)
(A)Representative
Representativesurvival
survivalcurve
curvefor
forLNCaP
LNCaP cells irradiated
cells with
irradiated various
with doses
various (0, (0,
doses 2, 4,2,6,4,
86,Gy) following a 24 h pretreatment of PGNPs at different concentrations. (B) Survival fraction
8 Gy) following a 24 h pretreatment of PGNPs at different concentrations. (B) Survival fraction of 2
Gy-irradiated LNCaP cells, 24 h after the treatment with PGNPs at different concentrations. (C) γ-
of 2 Gy-irradiated LNCaP cells, 24 h after the treatment with PGNPs at different concentrations.
H2AX staining of LNCaP cells pretreated with 0 or 250 µ g/mL PGNPs and irradiated with 0 Gy or
(C) γ-H2AX staining of LNCaP cells pretreated with 0 or 250 µg/mL PGNPs and irradiated with
2 Gy. (D) Comparison of γ-H2AX foci counts for groups with different PGNP concentrations after
0 Gyirradiation.
2Gy or 2 Gy. (D)* Comparison of γ-H2AX
Denotes p < 0.05 foci counts
from Welch’s t-test.for groups with different PGNP concentrations
after 2Gy irradiation. * Denotes p < 0.05 from Welch’s t-test.
3.3.
3.3. Effect
Effect of
of PGNP
PGNP on on RIBE
RIBE Signaling
Signaling Intensity
Intensity
The effect of PGNPs on RIBE
The effect of PGNPs on RIBE signaling signaling was assessed
was assessed through through the conditioned
the conditioned medium
medium (CM) transfer procedure (Figure 3A), where the bystander
(CM) transfer procedure (Figure 3A), where the bystander LNCaP cells were treated LNCaP cells were
with
treated
PBS- or with
PGNP-CM PBS- that
or PGNP-CM
was extracted thatfrom
wasirradiated
extractedcellsfromthatirradiated cells that
were pretreated withwere
PBS
pretreated
or 250 µg/mL withPGNPs.
PBS or 250 µ g/mL
Figure PGNPs.the
3B shows Figure 3B shows
radiation the radiation
response of PBS-CM response of PBS-
treatments on
CM treatments on LNCaP/PC3/22Rv1/RWPE-1
LNCaP/PC3/22Rv1/RWPE-1 bystander cells. bystander cells. The of
The RIBE response RIBE
the response
bystanderofcellsthe
bystander
treated with cells
thetreated
PBS-CMwith fromthethePBS-CM from
irradiation the irradiation
groups (2, 4, 6, 8 Gy)groups (2, 4, 6, 8 Gy)
were statistically were
different
statistically different from the non-irradiation group (p < 0.05); this
from the non-irradiation group (p < 0.05); this indicated the radiation dose dependenceindicated the radiation
dose
of thedependence
RIBE in LNCaP of thecells.
RIBE This
in LNCaP cells. This dose-dependence
dose-dependence effect was also observed effect was also
in PC-3
observed in PC-3 and 22Rv1 cells, but not in RWPE-1 cells. Moreover,
and 22Rv1 cells, but not in RWPE-1 cells. Moreover, 22Rv1 cells showed a weaker RIBE 22Rv1 cells showed
aresponse
weaker RIBE response thanprostate
than LNCaP/PC3 LNCaP/PC3 cancerprostate cancer
cell lines. MTT cellassays
lines. MTT
(Figure assays (Figure
3C) showed
3C)
the showed the statistically
statistically different celldifferent
viabilitycell viability
between between
LNCaP LNCaP cells
bystander bystander
treated cells
withtreated
2 Gy-
with
PGNP-CM 2 Gy-PGNP-CM
and 2 Gy-PBS-CM. and 2This Gy-PBS-CM. ThisPGNP-enhanced
aligns with the aligns with the PGNP-enhanced
radiosensitization (as
radiosensitization
shown in Figure 2)(as andshown in Figure
the radiation 2) and
dose the radiation
dependence of thedose
RIBEdependence
(as shown inofFigurethe RIBE3B).
(as shown
γ-H2AX in Figure
assays (Figure3B).
3D)γ-H2AX assaysthe
demonstrated (Figure 3D) demonstrated
significantly different number the of significantly
foci, or the
different number of foci, or the DNA damage between bystander
DNA damage between bystander cells, treated with 2 Gy-PGNP-CM and 2 Gy-PBS-CM. cells, treated with 2 Gy-
PGNP-CM
Furthermore, andthe
2 Gy-PBS-CM.
clonogenic cell Furthermore,
survival assaythe showed
clonogenic cell survival
significantly assay showed
decreased colony
significantly
formations ofdecreased
bystander colony formations
cells treated withof2 bystander
Gy-PGNP-CM cells compared
treated with with2 Gy-PGNP-CM
the 2 Gy-PBS-
compared
CM group. with (Figurethe3E);2 therefore,
Gy-PBS-CM group. (Figure
PGNP-induced 3E); therefore,
radiosensitization further PGNP-induced
enhanced the
RIBE signaling in LNCaP cells. In Figure 3C–E, no significant differences were observed
between the LNCaP bystander cells treated with 0 Gy-PGNP-CM or 0 Gy-PBS-CM, which
further illustrates the non-cytotoxicity of the 250 µg/mL PGNPs.
Nanomaterials 2022, 12, x FOR PEER REVIEW
radiosensitization further enhanced the RIBE signaling in LNCaP cells. In Figure 3C

significant differences were observed between the LNCaP bystander cells treated
Gy-PGNP-CM or 0 Gy-PBS-CM, which further illustrates the non-cytotoxicity of t
µ g/mL PGNPs.
Figure 3. (A) Workflow

Figure of 3. the
(A)conditioned
Workflow of medium transfer procedure
the conditioned mediumfor a RIBE procedure
transfer response investi-
for a RIBE re
gation. (B) MTTinvestigation.
cell viability assays of LNCaP/PC3/22Rv1 (prostate cancer)
(B) MTT cell viability assays of LNCaP/PC3/22Rv1 and RWPE1
(prostate(normal
cancer) and R
(normal
prostate) bystander prostate)
cells treated bystander
with PBS-CM cells treated
extracted with
from PBS-CM
their extracted
respective from
irradiated celltheir
line respective
cells irra
cell line cells under different radiation doses. (C–E) MTT cell viability,
under different radiation doses. (C–E) MTT cell viability, γH2AX, and clonogenic assays of bystander γH2AX, and clon
assays
cells treated with of bystander
0 Gy-PBS-CM, cells treated0 with
2 Gy-PBS-CM, 0 Gy-PBS-CM,
Gy-PGNP-CM, and 22 Gy-PGNP-CM.
Gy-PBS-CM, 0 Gy-PGNP-CM,
* Denotes and
PGNP-CM.
p < 0.05 from Welch’s t-test. * Denotes p < 0.05 from Welch’s t-test.
3.4. Effect
3.4. Effect of PGNP on the of PGNP onofthe
Sensitivity Sensitivity
Bystander of Bystander
Cells to the RIBECells to the RIBE
The influence of PGNPs
The on the
influence of sensitivity
PGNPs on of thebystander
sensitivitycells to the RIBE
of bystander wastoassessed
cells the RIBE was as
on the PBS-CM ontreated LNCaPtreated
the PBS-CM cells that werecells
LNCaP pretreated withpretreated
that were or without 250µg/mL
with or without 250µ
PGNPs. The detailed
PGNPs. Theworkflow
detailed is workflow
outlined in Figure 4A.
is outlined Moreover,
in Figure 2 Gy irradiation
4A. Moreover, 2 Gy irradiatio
was adopted here because
adopted here2 because
Gy was 2the Gyminimum RT dose that
was the minimum RT enabled
dose thatthe generation
enabled the generatio
of a significantsignificant
RIBE response in LNCaPinbystander
RIBE response cells, as shown
LNCaP bystander cells, asinshown
Figurein3B. The 3B. The
Figure
RIBE responseresponse
was evaluated in the following
was evaluated four groups:
in the following 0 Gy-PBS-CM
four groups: on no-PGNPs-
0 Gy-PBS-CM on no-PGNPs-l
loaded cells (0 cells
Gy-PBS-CM), 0 Gy-PBS-CM
(0 Gy-PBS-CM), treatment
0 Gy-PBS-CM on PGNPs-loaded
treatment on PGNPs-loadedbystander cells cells
bystander
(0 Gy-PBS-CM+PGNPs), 2 Gy-PBS-CM on no-PGNPs-loaded cells (2 Gy-PBS-CM+PGNPs),
PBS-CM+PGNPs), 2 Gy-PBS-CM on no-PGNPs-loaded cells (2 Gy-PBS-CM+PGNPs
and 2 Gy-PBS-CM on PGNPs-loaded
2 Gy-PBS-CM cells (2 Gy-PBS-CM+PGNPs).
on PGNPs-loaded MTT assays inMTT
cells (2 Gy-PBS-CM+PGNPs). Figure 4B in Figu
assays
demonstrated that there was significantly lower cell viability in the 2 Gy-PBS-CM+PGNPs
demonstrated that there was significantly lower cell viability in the 2 Gy-PBS-CM+P
group (85%) compared withcompared
group (85%) the 2 Gy-PBS-CM
with the group (92%). Additionally,
2 Gy-PBS-CM group (92%).compared withcompare
Additionally,
the 2 Gy-PBS-CM the 2treatment
Gy-PBS-CM group, the 2 Gy-PBS-CM+PGNPs
treatment group showedgroup
group, the 2 Gy-PBS-CM+PGNPs both anshowed bo
increased foci count in the γH2AX assay and decreased colony formation in
increased foci count in the γH2AX assay and decreased colony formation i the clonogenic
assay (Figure 4C,D), thus indicating
clonogenic assay (Figurethe PGNP-boosted
4C,D), thus susceptibility
indicating theof PGNP-boosted
LNCaP bystander susceptibil
cells to RIBE damage.
LNCaP bystander cells to RIBE damage.
Nanomaterials 2022, Nanomaterials
12, 4440 2022, 12, x FOR PEER REVIEW 10 of 18
Figure 4. (A) Workflow

Figure 4.of(A)
the Workflow
conditioned
of medium transfermedium
the conditioned procedure in order
transfer to investigate
procedure in orderthe
to investig
effect of PGNPs effect
on theofsusceptibility
PGNPs on the susceptibility of LNCaP bystander cells to RIBE MTT
of LNCaP bystander cells to RIBE damage. (B–D) cell (B–D) MT
damage.
viability, γH2AX,viability,
and clonogenic
γH2AX, assays of bystander
and clonogenic cells under
assays different
of bystander treatments:
cells 0 Gy-PBS-CM:
under different treatments: 0 G
CM: 0 Gy-PBS-CM on bystander cells pretreated without PGNPs;
0 Gy-PBS-CM on bystander cells pretreated without PGNPs; 0 Gy-PBS-CM+PGNPs: 0 Gy-PBS-CM 0 Gy-PBS-CM+PGNPs: 0G
CM treatment on bystander cells pretreated with 250 µ g/mL PGNPs;
treatment on bystander cells pretreated with 250 µg/mL PGNPs; 2 Gy-PBS-CM+PGNPs: 2 Gy-PBS- 2 Gy-PBS-CM+PGNPs
CM on bystanderPBS-CM on bystander
cells pretreated withoutcells pretreated
PGNPs; without PGNPs;2 2Gy-PBS-CM
2 Gy-PBS-CM+PGNPs: Gy-PBS-CM+PGNPs:
on bystander 2 Gy-PBS-C
bystander cells pretreated with 250 µ g/mL PGNPs. * Denotes p < 0.05 from Welch’s t-test.
cells pretreated with 250 µg/mL PGNPs. * Denotes p < 0.05 from Welch’s t-test.
3.5. on
3.5. Effect of PGNP Effect
ROS of Production
PGNP on ROS Production
As a common and As apotent
common typeand potent
of RIBE type of ROS
mediator, RIBEwas mediator,
measured ROSwith
wasDCFDA
measured in with DC
in order
order to study the impact to study
of PGNPsthe impact
on ROS of PGNPs
productionon ROS production
in relation to thein RIBE
relation to the RIBE resp
response.
Figure 5A shows Figure 5A shows the
the percentage percentage
of ROS change of ROS change
between LNCaP between LNCaP
cells treated withcells treated with P
PGNPs
at various nontoxic concentrations (10, 50, 100, 150, 200, 250 µg/mL). There were noThere we
at various nontoxic concentrations (10, 50, 100, 150, 200, 250 µ g/mL).
significant
significant differences differences
between betweentreatment
the different the different treatment
groups, groups, thus
thus indicating thatindicating
no th
clear
clear relationship relationship
between PGNPbetween PGNP and
concentration concentration
ROS productionand ROS production
exists. Figure 5B exists. Figu
compares the
compares the intracellular ROSintracellular ROS levels
levels of irradiated of irradiated
LNCaP cells thatLNCaP
were cells that were
pretreated withpretreated
and without PGNPsand without PGNPs
(250 µg/mL). As(250
shown,µ g/mL). As shown, irradiation-induced
irradiation-induced ROS production that ROS was productio
approximately was1.2× approximately
higher at every 1.2× radiation
higher doseat (Figure 5B) in LNCaP
every radiation dose cells pretreated
(Figure 5B) in LNCaP
with PGNPs, compared
pretreatedwith with LNCaP
PGNPs, cells pretreated
compared without
with LNCaP PGNPs; this indicates
cells pretreated that PGNPs
without
PGNPs boostedindicates
ROS production
that PGNPsduringboosted
radiation ROStreatment. Figure
production 5C exhibits
during the effect
radiation of
treatment. Figu
PGNPs on ROSexhibits
production in LNCaP
the effect bystander
of PGNPs on ROS cells in the following
production in LNCaP groups: treatedcells
bystander with in the follo
PBS-CM, treatedgroups:
with PGNP-CM,
treated with incubated
PBS-CM, with PGNPs
treated for PGNP-CM,
with 24 h, and treated with PBS-CM
incubated with PGNPs for
(PBS-CM+PGNPs). and For eachwith
treated treatment
PBS-CM group, the bystander cells
(PBS-CM+PGNPs). Fortreated with conditioned
each treatment group, the byst
media harvested from
cells irradiated
treated cells that were
with conditioned subjected
media to higher
harvested fromdoses produced
irradiated cells more
that were sub
ROS, which highlights
to higher doses produced more ROS, which highlights the radiation dosetodepende
the radiation dose dependence of ROS production with regard
the RIBE. Due ROSto the GNP-induced
production radiosensitization,
with regard to the RIBE. Due the bystander cells treated
to the GNP-induced with
radiosensitizatio
PGNP-CM showed a larger
bystander ROS
cells fold with
treated change, compared
PGNP-CM with athe
showed cellsROS
larger treated
foldwith PBS-
change, compared
CM. Comparedthe with
cellsthe PBS-with
treated andPBS-CM.
PGNP-CM treatment
Compared withgroups,
the PBS- the
and PBS-CM+PGNP
PGNP-CM treatment gr
Nanomaterials 2022, 12, x FOR PEER REVIEW
the PBS-CM+PGNP group demonstrated a much higher level of ROS productio

group demonstrated a much
indicating thehigher level
effect of of ROS production,
internalized PGNPs on thethussensitivity
indicatingofthe effect ofcells to th
bystander
internalized PGNPs on the sensitivity of bystander cells to the RIBE response.
response.
Figure 5. DCFDAFigure
total ROS
5. production
DCFDA total measurements
ROS productionnormalized in accordance
measurements with the measure-
normalized in accordance w
ments of the untreated group. (A)
measurements ofPercentage
the untreatedof ROS
group.change in LNCaP of
(A) Percentage cells
ROSincubated
change in with PGNPs
LNCaP cells incubat
under various concentrations.
PGNPs under (B)various
Total ROS change induced
concentrations. by irradiation
(B) Total ROS changein LNCaP
induced cells
bypretreated
irradiation in LNC
with and withoutpretreated
250 µg/mLwith and (p
PGNPs without
< 0.05 250
at 4,µ6,
g/mL
and PGNPs (p <Total
8 Gy). (C) 0.05 ROS
at 4, change
6, and 8in Gy). (C) Total ROS ch
bystander
bystander
LNCaP cells in the followingLNCaP
groups:cells in the
treated following
with PBS-CM, groups: treated
treated with PBS-CM,
with PGNP-CM, andtreated with PGNP-C
bystander
bystander
cells incubated with PGNPs cells
for 24incubated withwith
h and treated PGNPs for 24(PBS-CM+PGNPs)
PBS-CM h and treated with (p PBS-CM (PBS-CM+PGN
< 0.05 at 2, 4, 6,
0.05 at 2, 4, 6, and 8 Gy). * Denotes p < 0.05 from Welch’s t-test.
and 8 Gy). * Denotes p < 0.05 from Welch’s t-test.
4. Discussion 4. Discussion
Actively targeting tumors
Actively is crucialtumors
targeting for GNP-aided
is crucialradiosensitization; indeed, it en-
for GNP-aided radiosensitization; ind
hances tumor specific accumulation and cell killing while sparing surrounding
enhances tumor specific accumulation and cell killing while sparing surrounding h healthy
tissue. In this study, internalization
tissue. In this study, analysis on TEM images
internalization demonstrated
analysis on TEM the images
intracellular
demonstrat
biodistribution intracellular
of the developed PGNPs inofcell
biodistribution thecytoplasm.
developed PGNPsAlthough nuclear
in cell localiza-
cytoplasm. Although n
tion would theoretically
localization induce
woulda greater number
theoretically of DNA
induce doublenumber
a greater strand breaks
of DNA through
double strand
increased secondary electron
through scattersecondary
increased around GNPs, electronrecent studies
scatter haveGNPs,
around suggested
recentmitochon-
studies have sug
drial damage poses an additional threat to long-term cancer cell proliferation
mitochondrial damage poses an additional threat to long-term cancer cell prolif [33]; therefore,
the cytoplasmic[33];
effects of radiosensitization
therefore, the cytoplasmic areeffects
equally of valuable in terms of
radiosensitization aredirect
equallyDNA valuable in
damage. Furthermore, uptake experiments indicated that cellular GNP
of direct DNA damage. Furthermore, uptake experiments indicated that cellulasaturation occurred
at approximately 250 µg/mL,
saturation withatminimal
occurred toxicity250
approximately occurring
µ g/mL,within the LNCaP
with minimal cells,
toxicity occurring
and accumulations reachingcells,
the LNCaP 12 pg/cell. This value indicates
and accumulations reachinglower toxicityThis
12 pg/cell. levels thanindicates
value
other studies performed with GNPs
toxicity levels and LNCaP
than other studiescells, which with
performed reported
GNPs IC50
and values
LNCaP between
cells, which re
100–200 µg/mLIC50 aftervalues
24 h [34–36]. This difference is mainly because
between 100–200 µ g/mL after 24 h [34–36]. This difference of the media used
is mainly b
during incubation with GNPs. In other studies, complete growth media were
of the media used during incubation with GNPs. In other studies, complete growth used, which
allows the control group
were to continue
used, which allowsgrowing; however,
the control in ourtostudy,
group continue serum free media
growing; was in our
however,
used, which significantly reduces cell proliferation in the control cells. GNPs are known to
serum free media was used, which significantly reduces cell proliferation in the
induce cell cycle arrest; therefore, viability studies using complete media generate an IC50
cells. GNPs are known to induce cell cycle arrest; therefore, viability studies
based on a control cell line with cell proliferation, whereas GNP treated cells can become
complete media generate an IC50 based on a control cell line with cell prolife
senescent [37]. However, this study employs serum-free media, which ensures that the MTT
whereas GNP treated cells can become senescent [37]. However, this study em
assay specifically measures cell death. This toxicity and uptake data agree with previous
serum-free media, which ensures that the MTT assay specifically measures cell deat
targeted nanoparticle studies [38]. Furthermore, this study confirmed that the anti-PSMA
toxicity and uptake data agree with previous targeted nanoparticle studie
antibody functionalized PSMA-targeting GNPs are effective radiosensitizers for prostate
Furthermore, this study confirmed that the anti-PSMA antibody functionalized
cancer cells through a clonogenic assay and γ-H2AX assay. Both the clonogenic assay and
targeting GNPs are effective radiosensitizers for prostate cancer cells through a clon
γ-H2AX results showed a GNP dose dependent response to LNCaP cell radiosensitivity.
assay and γ-H2AX assay. Both the clonogenic assay and γ-H2AX results showed
The focus of the LNCaP prostate cancer cell line in the present study was motivated by
dose dependent response to LNCaP cell radiosensitivity. The focus of the LNCaP p
the high expression of PSMA in LNCaP cells, and studies showing the high sensitivity of
LNCaP cells to cancer cell lineWestern
radiotherapy. in the present study was
blot confirmed the motivated by the high
highest expression expression
of PSMA in of PS
LNCaP cells, and studies showing the high sensitivity
LNCaP cells compared with other prostate cancer cell lines (including PC3 and 22Rv1), of LNCaP cells to radioth
Western
as well as a normal blot confirmed
prostate the RWPE-1).
cell line (i.e., highest expression
Both LNCaP of PSMA in LNCaP
and 22Rv1 cell cells
lines compare
other prostate cancer cell lines (including PC3 and
demonstrated an elevated uptake of PGNPs. Interestingly, the normal prostate cells (i.e., 22Rv1), as well as a normal p
RWPE-1) demonstrated a high uptake of the GNPs used in this study, under the sameelevated
cell line (i.e., RWPE-1). Both LNCaP and 22Rv1 cell lines demonstrated an
of PGNPs.
in vitro incubation conditions Interestingly, the normal
as the prostate cancer prostate cells During
cell groups. (i.e., RWPE-1) demonstrated
GNP-assisted
radiotherapy, tumor specificity occurs because 5–150 nm-sized GNPs accumulate in the condit
uptake of the GNPs used in this study, under the same in vitro incubation
tumor tissues as a result of the enhanced permeation and retention (EPR) effect; however,
in contrast to the in vitro scenario, normal prostate tissues accumulate significantly fewer
GNPs than prostate tumors in vivo due to their low levels of EPR. Therefore, in the in vivo
scenario, normal prostate cells only accumulate a small number of GNPs, even though they
do show a high uptake of GNPs in vitro.
Moreover, GNPs affected the RIBE in LNCaP cells. LNCaP bystander cell viability de-
creased with increased radiation doses up to 8 Gy when using the media transfer technique.
The dose dependence of the RIBE response was also observed in other bystander prostate
cancer cells (PC3, 22Rv1), but not in normal prostate cells (RWPE-1). As expected, radiore-
sistant 22Rv1 cells showed a minimal response to the RIBE compared with LNCaP and PC3
cells. Additionally, PGNP-CM treatment induced increased cell death in bystander cells
compared with bystander cells treated with PBS-CM. The increased cell death in bystander
cells after PGNP-CM treatment suggests that the GNPs increased the radiation damage in
irradiated cells, and thereby increased RIBE signaling because the RIBE in LNCaP cells was
dose dependent for a wide range of cells. Previous studies on breast cancer cells showed
that the dose dependence of cell lines in relation to the RIBE is critical to a GNP-enhanced
RIBE [32]. γ-H2AX and clonogenic cell survival assays further confirmed the radiosensi-
tization of LNCaP cells through the RIBE. This study only investigated the effects of the
cytotoxicity of active-targeting PGNPs on LNCaP cells. Future relevant studies are required
to investigate the effects of the cytotoxicity of PGNPs on PC3, 22Rv1, and RWPE-1 cell lines,
which would enable an exploration of the impact of PGNPs on different prostate cancer
cells as well as normal prostate cells.
PGNPs also increased the LNCaP cells’ sensitivity to RIBE signals in a cell viability
assay, γ-H2AX, and a clonogenic assay. The increased total cellular ROS following condi-
tioned media exposure in PGNP-treated bystander cells demonstrated that imbalanced
cellular redox potential plays a role. Previous studies indicate that metal nanoparticle
treatment augments antioxidant and ROS-generating enzyme levels and leads to increased
cellular ROS production [39]. Culcasi et al. and Wilhelmi et al. identified an upregulation in
ROS-generating NADPH oxidase (NOX) enzymes that played a key role in metal nanoparti-
cle cytotoxicity through increased cellular ROS, which thus led to DNA fragmentation [40].
Enzymes such as NOX enzymes, which can induce oxidative stress, play a key role in
radiation therapy and the RIBE because studies show that the inhibition of NOX and other
ROS-generating enzymes can significantly reduce DNA damage [41,42]. Furthermore, ROS
clearance through enzymes such as superoxide dismutase (SOD), glutathione peroxidase
(GPx), and catalases maintain cellular redox potential. A variety of metal nanoparticle stud-
ies in various tissues demonstrate a decrease in the expression of these enzymes, including
a decrease in GPx in TiO2 , a decrease in SOD and catalases in CuO, and a decrease in SOD in
iron oxide [39]. Although there is a dearth of experiments with regard to GNPs and antioxi-
dant enzyme changes, it is a potential parameter requiring further investigation; therefore,
the dose dependent increase in total cellular ROS upon the irradiation of PGNP-treated
cells likely occurs as a result of a two-fold effect. First, the physical dose enhancement of
PGNPs increased local dose deposition, leading to physical radiation dose dependent ROS
production, and PGNP-impaired cellular redox maintenance with biologically boosted ROS
levels. The latter was observed in bystander cells with conditioned media transfer studies.
Ultimately, the elevated ROS levels occurred as a result of significantly increased enzymatic
ROS production, which produced a surplus, and the conditioned media treatment in the
bystander cells, which is likely to be the reason for the increased DNA damage seen in the
γ-H2AX assay; this is also likely to be the case for the increased radiosensitization seen in
the clonogenic survival assays.
A key element of this study was the use of conditioned media as a tool for inducing
RIBE. Conditioned media utilizes soluble cytokines such as IL-1, IL-2, IL-6, IL-8, TNF-alpha,
and TGF-beta [43]. Many of these cytokines are highly expressed in immune cells, such as
macrophages, but in this study, we only studied the soluble factors in conditioned media
produced by LNCaP cells, and we treated them to LNCaP cells; situations such as these
can only be observed locally within the tumor. Therefore, the impact of the RIBE in distal
regions or RIBE signals from immune cells could augment the observed effect. Furthermore,
using the conditioned media technique caused the loss of two major RIBE signals: free
radicals and gap junction signals [24]. Most free radicals are eliminated quickly before
the conditioned media is transferred. Additionally, the strongest known signaling of the
RIBE occurs through gap junctions. Previous RIBE studies have utilized partial culture
irradiation, whereby a few cells in a single culture dish are radiated [44]; however, this
RIBE is very localized, with an unclear, limited range [45]. Therefore, RIBE enhancement
with PGNPs may have a different effect if studied with the partial irradiation method.
Furthermore, recent studies focus on the impact of exosomes on the radiation-induced
bystander effect [46,47]. A major impact of filtering the conditioned media with 0.45 µm
sterile filter is the preservation of exosomes in the conditioned media. Lastly, the use of
conditioned media provided in vivo translation opportunities for this study. The injection
of conditioned media into mice could potentially show tumor treatment effects, and vice
versa, the serum from irradiated mice can be used in vitro. These studies reveal the degree
and range of RIBE signaling changes that GNPs can affect. The results presented here
suggest that the bystander prostate cancer cells would experience greater cell death rates
when these cells are enriched with GNPs and treated with RIBE signals from irradiated
cancer cells with internalized GNPs.
A potential caveat of this study is the fact that the RIBE enhancement of cancer cells
treated with kV photons is limited to LNCaP prostate cancer cells. Previous studies have
demonstrated that various cells have different responses to the RIBE [46]. For instance,
some cells (such as the breast cancer cell line, MCF7) are known to show a radiation-induced
bystander effect but not in a dose dependent manner. On the other hand, QUDB cells
have a dose dependent response [32]. Another study showed that there is a radiation-
induced cell growth inhibitory bystander response, but only at high radiation doses, not
low radiation does [48]. This is particularly interesting as LNCaP demonstrated a consistent
RIBE, including low and high radiation doses. To consider the full effect of the radiation-
induced signaling of the RIBE in prostate cancer cells, studies into other prostate cancer
cells, as well as normal tissue prostate fibroblast cells, can provide an understanding of how
the RIBE could be used in a clinical setting. Additionally, they could also demonstrate the
effect of GNPs on these bystander effects. Moreover, RIBEs have been known to differ based
on their radiation quality and LET. The RIBE has been studied using various radiation
sources, including photons, particles, and heavy ions at varying degrees of intensity [49–51].
Although many of these have not been compared against each other, several studies indicate
that the magnitude of bystander effect is dependent on LET [52]. Changes between radiation
sources have been shown to change dose dependence as well as relieve the RIBE burden
entirely [53]. This is particularly important when studying GNP-induced radiosensitization
because the mechanism of GNP-mediated radiosensitization is different depending on the
radiation source. For instance, interactions between kV energy beams and GNPs leads
to the emission of short-range secondary electrons and radiosensitization. The use of kV
energies in this study maximizes the effect of secondary electron scatter compared with
other modalities [8]. Moreover, MV irradiated GNPs induce radiosensitization through
increased mitochondrial toxicity, and Monte Carlo and in vitro studies suggest protons or
heavy ions generate a massive increase in ROS production [54]. All these modalities are
currently used clinically for prostate cancer treatments in the form of brachytherapy or
external beam radiotherapy, and the 2 Gy increments match the conventional fractionation
schemes of standard treatment schedules. As our study suggests, the RIBE contributes to
GNP-mediated radiosensitivity through ROS production, and the increase in sensitivity
may be further enhanced when protons and heavy ion-irradiated cells are the donors
for the conditioned media. Lastly, this study focuses on GNPs, but a multitude of metal
nanoparticles have demonstrated radiosensitization capabilities. A diverse group of these
metal nanoparticles induce cellular redox changes, which suggest that the effects observed
in this study are not limited to GNPs. In addition, several studies indicate that metal
nanoparticles introduce cellular toxicity through ion shedding heavy metal poisoning with
TiO2, carcinogenesis from ZnO, and inflammation from impurities in Ag [55–57]. GNPs
are free of these problems, but these toxicities relate largely to ROS generation from the
nanoparticles, which could further impact RIBE sensitivity in a similar manner to GNPs.
The clinical benefit of the bystander effect can be observed through a series of in vivo
experiments, including the use of spatially fractionated radiotherapy [25]. Spatially frac-
tionated radiotherapy is the delivery of a radiation dose to smaller fields without delivering
radiation to the entire tumor to reduce toxicity. Preclinical studies in mouse models demon-
strated that a 10 Gy dose to a small region of the tumor would cause radiation damage
to the surrounding tumor tissue [58]. Furthermore, this bystander effect showed changes
in gene expression for DNA repair, cell cycle arrest, and apoptosis, as is the case with
the changes seen in irradiated tumor cells. Although studies on spatially fractionated
radiotherapy have been conducted since the 1950s, inventions of multi-leaf collimators
have recently shown promising results regarding prostate tumor responses [59]. The im-
plication of enhanced bystander effects from GNPs suggests that they can be valuable in
combination with spatially fractionated radiotherapy to enhance the bystander effect. The
results presented here suggest that spatially fractioned radiotherapy would benefit from
GNPs, even if GNPs are not homogenously distributed throughout the tumor. Factors such
as hypoxia, nanoparticle targeting agents, and tumor microenvironments can affect the
distribution of GNPs, but a spatially fractionated study would enhance tumor treatment
efficacy, regardless of the GNP content in each beamlet. Furthermore, it is possible to use
imaging studies to target regions with high GNP concentrations in order to selectively
utilize the effects that GNPs have on the RIBE for dose painting. In vivo experiments
concerning GNPs, combined with spatially fractionated radiotherapy, could also reveal the
extent of the GNP-enhanced RIBE to the surrounding tissue. An additional weakness in this
study, for clinical reference, is the use of a single radiation dose to induce a bystander effect.
For clinical use, the bystander effect would require fractionated radiation. It is important to
note that fractionated radiation doses have had mixed results in RIBE studies [60]; however,
previous studies on the pharmacodynamics of gold nanoparticles indicate that fractionated
radiation therapy is completely feasible for GNP-enhanced radiation therapies [19].
Finally, this study suggested that the radiosensitization effects of off-targeted GNPs
could be a potential double-edged sword. The increased effect of RIBE sensitivity as a
result of nanoparticles could induce a greater number of secondary tumorigeneses, as
nanoparticles are known to accumulate in various organs such as the liver, spleen, and
kidneys [33,61]. These effects can be particularly worrisome since secondary cancers
in the lung are prevalent with prostate cancer treatments [62]. Previous studies have
demonstrated low accumulations of GNPs in the lung, which may therefore mitigate this
risk, but the high accumulations in the liver, kidney, and spleen may increase the risk of
secondary cancers in these organs when treated with GNPs. This form of distant organ
damage may be described more specifically as an ‘abscopal effect’, and it is not particular
to the bystander effect; however, the proper control of nanoparticle biodistribution with
targeted nanoparticles could be critical for GNP radiotherapy safety.
Studies on the RIBE present further biological explanations on the variances observed
between in silico GNP studies and in vivo/in vitro experiments. Several Monte Carlo
studies have demonstrated that GNPs can increase local dose deposition by up to 200× per
nanoparticle with 250 kvP photon beams [63]; however, the increased secondary electron
scatter fails to account for the total radiosensitization observed in vitro, especially for
megavoltage energies [20]. Studies show that GNP radiosensitization can occur due to
a variety of reasons, including physical, chemical, and biological [20]. Physically, GNPs
increase the secondary electron scatter, and chemically, the GNPs form free radicals to
make DNA more susceptible to radiation damage. A myriad of biological effects occur,
including cell cycle changes, mitochondrial damage, and DNA repair inhibition. Studies in
glucose-capped GNPs increased the cell population in the G2/M phase, which is the most
radiosensitive phase. Secondly, several studies on the GNPs’ impact on mitochondria show
radiosensitization through the loss of mitochondrial potential, thus leading to necrosis,
caspase-related mitochondrial dysfunction, and mitochondrial membrane polarization; this

leads to apoptosis when combined with radiation. Thirdly, H2AX studies have shown com-
plications with DNA repair when GNPs are present, based on increased double stranded
DNA damage to the foci after 24 h. RIBE changes from GNPs add to the list of biological
changes which lead to increased radiosensitization, and the biological factors affecting
radiosensitization from direct radiation further enhance the RIBE. Nanoparticles have
recently garnered attention in radiotherapy as valuable agents for imaging and radiosensi-
tization. As such, the direct effects of metallic nanoparticle radiosensitization and toxicity
have been heavily studied [38,64]; however, the nanoparticles’ impact on the RIBE has not
been well documented. The radiation-induced bystander effect can have a critical role in
improving the treatment efficacy of treatment modalities, such as spatially fractionated
radiotherapy, and it can also have detrimental effects in the form of off-target secondary
cancers. This study introduced the potential for combining GNPs to augment the effects of
the RIBE in prostate cancer treatment.
5. Conclusions
Prostate cancer-targeting GNPs were used to radiosensitize LNCaP prostate cancer
cells. This study elucidated the impact of the bystander effect on the biological radiosensiti-
zation of prostate cancer cells with targeted GNPs. Additionally, this study highlights the
increased signaling intensity of the bystander effect from GNPs in the irradiated cells, as
well as the greater sensitivity to the bystander effect in non-irradiated cells as a result of
GNP treatment. A plethora of studies have demonstrated that radiosensitization from metal
nanoparticles occurs as a result of a multitude of biological and chemical factors; these
factors impact a greater area than the originally proposed site for local dose deposition. The
findings from this study contribute to knowledge concerning another biological component
of GNP radiosensitization. Ultimately, these findings suggest further research should be un-
dertaken to investigate the GNP-enhanced bystander effects, abscopal effects, distant tissue
toxicity risks, and combinations of GNPs and heterogenous dose distribution treatments.
Author Contributions: Conceptualization, J.S. and W.T.; methodology, J.S., W.T., D.H. and Y.-P.Y.;
software, D.H. and R.M.S.; validation, D.H., W.T. and J.S.; formal analysis, D.H.; investigation, D.H.,
J.S., W.T., A.P. and J.C.F.; resources, J.S., A.P., J.C.F., S.D. and N.D.; data curation, D.H. and J.S.;
writing—original draft preparation, D.H. and J.S.; writing—review and editing, J.S., W.T. and J.C.F.;
supervision, J.C.F. and J.S.; funding acquisition, A.P., J.C.F., S.D. and J.S. All authors have read and
agreed to the published version of the manuscript.
Funding: Financial support was received from Department of Radiation Oncology at University of
Miami. This work was supported by Sylvester Comprehensive Cancer Center and the American
Cancer Society. This work was also partially supported by Sylvester CCSG transdisciplinary pilot
grant, and cancer center core grant SP30 240139-02.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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Nanomaterials 2022, 12, 4440. https://doi.org/10.3390/nano12244440 https://www.mdpi.com/journal/nanomaterials

2. Materials and Methods

2.2. Characterization of PGNPs

2.3. Cell Culture

2.4. PGNP Cytotoxicity

2.5. Cellular PGNP Uptake Assay

UV-Vis spectrophotometry on a Nanodrop Spectrophotometer before and after treatment.

2.6. RIBE on LINCaP Cells

2.7. Effects of PGNPs on RIBE

2.8. Clonogenic Assay

2.9. γH2AX Assay

2.10. ROS Quantification

2.11. Statistical Analysis

3.2. Effect of Radiation Dose or PGNP Concentration on Radiosensitization

radiosensitization further enhanced the RIBE signaling in LNCaP cells. In Figure 3C

Figure 3. (A) Workflow

Figure 4. (A) Workflow

the PBS-CM+PGNP group demonstrated a much higher level of ROS productio

caspase-related mitochondrial dysfunction, and mitochondrial membrane polarization; this

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