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\DOI{10.5802/crchim.309}
\datereceived{2023-03-17}
\daterevised{2023-11-28}
\datererevised{2024-01-10}
\dateaccepted{2024-04-03}
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\dateposted{2024-07-11}
\begin{document}

\begin{noXML}

%\makeatletter
%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Breaking Barriers in Chemical Biology -- Toulouse 2022}
%\def\cdr@specialtitle@french{Briser les barri\`eres entre la chimique et la biologie -- Toulouse 2022}
%\makeatother

\begin{DefSom}
%\usepackage{upgreek}
\let\upgamma\gamma
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\CDRsetmeta{articletype}{review}

\title{Release of biologically relevant substrates from prodrugs and
nanocarriers using X- and \tralicstex{γ}{${\upgamma}$}-rays as trigger
signals}

\alttitle{Lib\'{e}ration de substrats biologiquement pertinents \`{a}
partir de prom\'{e}dicaments et de nanovecteurs en utilisant les rayons
X et \tralicstex{γ}{${\upgamma}$} comme signal de d\'{e}clenchement}

\author{\firstname{Amit} \lastname{Kumar}}
\address{Laboratoire de Chimie et Biochimie Pharmacologiques et
Toxicologiques, CNRS, Universit\'{e} Paris Cit\'{e}, Paris, France}

\author{\firstname{Peter Istv\'an} \lastname{Dalko}\CDRorcid{0000-0003-2294-3643}\IsCorresp}
\addressSameAs{1}{Laboratoire de Chimie et Biochimie Pharmacologiques
et Toxicologiques, CNRS, Universit\'{e} Paris Cit\'{e}, Paris, France}
\email[P. I. Dalko]{peter.dalko@parisdescartes.fr}

\keywords{\kwd{Redox probes}
\kwd{Smart nanomaterials}
\kwd{Radiolysis}
\kwd{Reactive oxygen species (ROS)}
\kwd{Combination therapy}}

\altkeywords{\kwd{Sondes redox}
\kwd{Nanomat\'{e}riaux intelligents}
\kwd{Radiolyse}
\kwd{Esp\`{e}ces r\'{e}actives de l'oxyg\`{e}ne (ROS)}
\kwd{Polyth\'{e}rapie}}

\begin{abstract} 
X- and \tralicstex{γ}{${\upgamma}$}-ray-excited theranostic techniques are increasingly
being developed for biomedical applications, as they overcome the
limitations of light penetration and tissue attenuation. Beyond the
historic opportunities that X-rays have opened up for medical imaging
and cancer therapy, this modality is increasingly used for drug
delivery from pro-drugs and nanomaterials. This brief review covers
several strategies that have been developed for biomedical applications
\end{abstract} 

\begin{altabstract} 
Les techniques th\'{e}ranostiques activ\'{e}es par les rayons X et
\tralicstex{γ}{${\upgamma}$} sont de plus en plus d\'{e}velopp\'{e}es pour des
applications biom\'{e}dicales, car elles surmontent les limites de la
p\'{e}n\'{e}tration de la lumi\`{e}re et de l'att\'{e}nuation dans
les tissus. Au-del\`{a} des possibilit\'{e}s historiques offertes par
les rayons X pour l'imagerie m\'{e}dicale et la th\'{e}rapie du
cancer, cette m\'{e}thode est de plus en plus utilis\'{e}e pour
l'administration de m\'{e}dicaments \`{a} partir de
prom\'{e}dicaments et de nanomat\'{e}riaux. Cette br\`{e}ve revue
couvre plusieurs strat\'{e}gies qui ont \'{e}t\'{e} d\'{e}velopp\'{e}es
pour des applications biom\'{e}dicales.
\end{altabstract} 

\maketitle

\vspace*{.2pc}

\twocolumngrid

\end{noXML}

\section{Introduction} \label{sec1}

\begin{figure*}
\vspace*{3pt}
\includegraphics{fig01}
\caption{Electromagnetic map and modeled soft-tissue penetration
capacity (calculated values). \label{fig1}}
\vspace*{2pt}
\end{figure*}

Biomedical applications of X-rays were and still are closely linked to
cancer imaging and therapy. Radiotherapy (RT) is an extremely important
first-line treatment for cancer therapy which, unlike other treatments
such as chemotherapy or immunotherapy which affect the whole body, is
generally a local treatment.~Despite the spectacular progress made in
the field of X-ray sensitization and improvement, radiotherapy as a
monotherapy generally fails to eliminate the entire tumor, as cancer
cells can undergo DNA repair and regrow~\cite{bib1,bib2}. For this
reason, current research is gradually shifting from monotherapy to
multimodal X-ray therapy, combining two or more therapeutic modalities,
producing synchronous/synergistic anticancer effects \cite{bib3,bib4}.
Increasing synergistic effect also optimizes several \mbox{shortcomings} of
drug-based therapies by improving systemic effect, nonspecific
biodistribution, poor bioavailability and limited circulation time,
which can lead to severe side effects and low therapeutic index.
Chemoradiotherapy has been widely used clinically, offering substantial
advantages in terms of treatment and prognosis. Research on the
simultaneous administration of drugs and the application of therapeutic
beams dates back several decades~\cite{bib5,bib6}  and is used to treat
certain stages of head and neck cancer, lung cancer, and gynecological
cancers. Now the field has reached a next-level maturity to experiment
with locally activable prodrugs and drug carriers, thus limiting the
general chemotoxicity and efficiency of the treatment.
Stimuli-responsive biodegradable nanoagents, which can not only deliver
and control chemotherapies, but also attenuate hypoxia, or play as
antimitotic agent to enhance chemoradiotherapy, have enormous
potential. Key to the use of these smart materials is the presence of
an interplay that is able to switch from one state to another,
triggering the release of the covalently or non-covalently attached
compound. Intelligent drug delivery systems can be designed around
chemical functions or fragments of molecules that modify their
structural or functional properties in engineered materials in response
to specific external or internal factors~\cite{bib7,bib8}. These
dynamic properties can be activated by chemical/biochemical,
electrical, magnetic, electromagnetic/optical, ultrasonic, dielectric,
redox or piezoelectric signals, some of internal (biological) origin,
others of external origin. These interplay units, which are small
chemical compounds, may undergo isomerization, rearrangement or
cleavage of chemical bonds, such as UV-sensitive \textit{azobenzene}
and $o$-nitrobenzyl derivatives, or coumarin esters, leading to direct
or indirect drug release. Some of these molecules were historically
developed for neuroscience where these ``caged'' compounds revolutionized
functional neurophysiology from the late 1970s. They are now commonly
used to design prodrugs and light-controlled drug delivery systems,
although their application in therapy is
limited~\cite{bib9,bib10,bib11}. Light activation has inherent
shortcomings. While the tissue penetration capacity of visible and
near-infrared (NIR) light is superior to the more often used UV
activation, their penetration in deep tissues does not extend beyond a
few hundred micrometers using intensities below cell toxicity. The main
obstacles are strong absorption and diffraction in turbid tissues.
Fortunately, longer and especially shorter wavelengths than UV/Vis/NIR
penetrate tissues better (Figure~\ref{fig1}). As longer waves access
vibrational and translational excitations, thus have not enough energy
to break a chemical bond, shorter waves can provide sufficient energy
to induce structural changes at the molecular level. As organic
compounds have no electronic transitions in this energy window, they
are transparent. Several ingenious strategies have recently been
developed to translate short-wave photons into a trigger signal
applicable to organic compounds that will be discussed in this 
review~\cite{bib12,bib13,bib14,bib15,bib16,bib17,bib18,bib19}. This
strategy also opens up new perspectives for personalized medical care,
bringing the dream of tailor-made medical treatments for individuals
closer to reality.

\subsection{On the nature of X- and $\gamma$-rays}

Although X- and ${\upgamma}$-rays are both electromagnetic
radiation (photons), there is no consensus on how to precisely
differentiate between them. They differ mainly in their origin:
${\upgamma}$-rays are produced during the nuclear decay of atomic
nuclei from the atomic core and particle annihilation, while X-rays are
produced when the velocity of electrons (or other charged particles)
slows down, and the energy difference between these two kinetic states
is dispersed as high-energy photons by Bremsstrahlung and
characteristic radiation~\cite{bib20,bib21}. X-rays are generally
generated in a vacuum tube, where electrons emitted by the cathode,
accelerated under high voltage, collide with a tungsten metal anode.
X- and ${\upgamma}$-rays are ionizing radiations but less so than
alpha and beta particles. The energy ranges of X- and
${\upgamma}$-rays overlap considerably, with X-rays essentially
covering the ``lower'' part of the spectrum with energies above a few
tens of eV, typically between 120~eV and 120~keV (thus the shortest
ultraviolet spectrum) and ${\upgamma}$-ray energies extending up to a
few tens of MeV. In other words, X-rays (generally) have longer
wavelengths than ${\upgamma}$-rays, mainly ranging from 0.01 to 10~nm.
Both ${\upgamma}$- and X-rays have a high penetration power in soft
tissue, whose extent depends on their respective wavelengths.
Penetration is greater at higher energies but also depends on the
tissue type. The impact of tissue thickness on X-ray penetration is
dramatic. Small increases in tissue thickness led to large attenuations
in the intensity of the incident beam. Because of the critical
importance of penetration into (biological) media, highly accurate
transport equations were developed from the early 1950s and are
currently used to estimate the exposure of biological tissues to
radiation~\cite{bib22}. Indeed, an utmost important value in biomedical
applications is the dose absorbed by the living body. The gray
(symbol:~Gy) is the unit of ionizing radiation dose that measures the
energy deposited/absorbed (in joule per kilogram of matter).

Radiotherapy is used in cancer therapy as ionizing radiation can break
down the DNA of cancer cells more effectively than that of healthy
cells. This is made possible by the natural repair mechanism, which is
more effective for normal cells than for cancer cells, which function
in an intrinsically dysfunctional way. The upper limit of a ``safe
dose'' has been defined as a total dose of 55--60~Gy administered to a
focal field with fractions of 1.8--2~Gy per day. This means that the
total irradiation dose is delivered in small pieces to be gentler on
normal tissues and to allow normal cells to repair DNA damage after
each treatment. The radiation threshold depends sensitively on the
tissue/organ/type of cancer/general condition of the patient and
is assessed individually for each patient. An example of a
dose-response relationship is illustrated in the treatment of stage III
non-small cell lung cancer, where the administration of a total dose of
40~Gy is applied in fractionated treatment (4~Gy/day for five days,
followed by a two-week break and a further dose of 4~Gy/day for five
days), or the administration of a total dose of 40~Gy, 50~Gy or 60~Gy
is applied in continuous treatment (2~Gy/day)~\cite{bib23}. For this
reason, drug delivery probes for simultaneous activation must meet the
threshold of 2 to 4~Gy, meaning that they must release the maximum
payload within this dose for \mbox{biomedical} use. 

How can this high-energy radiation be used for prodrug activation and
for drug delivery? Three conceptually different approaches have been
developed, based either (i) on transformations mediated by {energy} or by
an electron transfer-promoted change in the electronic structure (thus
in the stability) of the carrier; (ii) on the incorporation of
ROS-sensitive functions into the drug delivery matrix that react
chemically and undergo fragmentation; (iii) on the upregulation of
enzymes that have recently been explored for selective prodrug
activation, but the scope of such activation is beyond the scope of
this study~\cite{bib24}.

\subsection{Drug release triggered by energy- or electron
transfer-mediated structural changes} 

Organic probes are transparent to X-rays, as organic materials do not
exhibit absorption transitions in this high-energy spectrum.
Consequently, irradiation can be used for sterilization to eliminate
microbial growth and prepare pyrogen-free samples without altering the
structure of the biomedical sample or equipment~\cite{bib25,bib26}.
To be able to explore X/${\upgamma}$-rays as a trigger, an intermediate
transduction mechanism is needed, which bridges the gap between
high-energy electromagnetic radiation and what organic compounds can
``understand'' to react. Fortunately, there are a few such
interactions. Noteworthily not necessarily the ``simplest'' solutions
are the most effective. For example, the light produced by Cerenkov
radiation, which is emitted when a charged particle passes through a
dielectric medium at a speed greater than the phase velocity of light
in that medium, would be useful as a local light trigger signal if the
intensity of the UV light generated by this phenomenon could be
increased~\cite{bib27}. Similarly, parametric down-conversion (an
instantaneous nonlinear optical process that converts a higher-energy
photon into a lower-energy photon pair, in accordance with the law of
conservation of energy and the law of conservation of momentum) would
be an interesting solution, although it cannot yet be used due to the
lack of efficient generation of idler photons at optical
wavelengths~\cite{bib28}. The conversion of X-rays to UV can be
achieved more easily using scintillators (see
above)~\cite{bib29,bib30}. These materials play an important role in
X-ray-induced photodynamic therapy (PDT), enabling low doses of
radiation to be applied, as do a variety of well-designed irradiation
modes and \mbox{intelligent} strategies for modifying the tumor
microenvironment~\cite{bib31}. This activation modality generates
$^{1}\mathrm{O}_{2}$ and ROS similarly to
conventional photosensitizers in PDT under ultraviolet (UV) or visible
light (scintillator-derived strategies will be discussed in the
paragraph on redox/electron transfer reactions).\looseness=-1

The most frequently used electron/energy transfer-related activation
strategies are based on inelastic scattering, which involves a loss of
energy from the incident primary photon: the shorter wavelength primary
photon is transformed to a longer wavelength secondary photon.
Inelastic scattering occurs via a number of mechanisms. The energy is
transferred to the sample, generating a series of useful signals that
can be exploited in a number of analytical methods and chemical
transformations. The interaction of X- and ${\upgamma}$-rays with
matter is described by the photoelectric effect, Compton scattering,
and pair production. The photoelectric effect is a phenomenon whereby
electrically charged particles are released from or within a material
when it absorbs electromagnetic radiation (Figure~\ref{fig2}). The
effect is often defined as the ejection of electrons from an atom when
light falls on it, and called the Auger--Meitner effect at higher
frequencies, such as X- and ${\upgamma}$-rays. The effect consists
in the occupation of a cavity in the inner shell of an atom,
accompanied by the emission of an electron from the same atom. When an
electron is removed from the atom, leaving a vacancy, an electron of a
higher energy level can fall into the vacancy, resulting in a release
of energy (Figure~\ref{fig2}). Although this energy is most often
released in the form of an emitted photon, it can also be transferred
to another electron, which is ejected from the atom; this second
ejected electron is called an Auger electron. Since an electron vacancy
initially produced by the incident photon leads to two new electron
vacancies, which in turn can produce the same number, a complete
cascade of electrons can be produced according to tabulated
probabilities. The atom is finally left in a state of multiple
ionizations. The Auger effect (non-radiative relaxation) is the
predominant mode of relaxation in light target atoms such as C, N, O 
($Z<20$) or for L-layer ionizations.

\begin{figure}
\vspace*{2pt}
\includegraphics{fig02}
\vspace*{2pt}
\caption{The photoelectric effect is the consequence of inelastic
scattering between the photon and the electron, which generates a
non-radiative relaxation (Auger effect) and a radiative relaxation in
the target atom. \label{fig2}}
\end{figure}

The radiative mode corresponds to the emission of fluorescence
radiation, whose spectrum of discontinuous lines is characteristic of
the material. In heavy materials ($Z>50$) and for K-layer vacancies,
fluorescence is the main relaxation mode.

Compton scattering results from the collision of a photon with an
electron at rest: this is the combination of an incident X-ray photon
and a weakly bound electron in the atom, whose binding energy is
negligible compared to the incident energy. During this interaction,
the incident X-ray photon disappears: part of its energy is given to
the peripheral electron, which is then ejected: this is known as a
Compton electron; if the other part of its energy is transmitted in the
form of a new X-ray photon, this is known as a scattered or recoil
photon. At the energies used in radiology, the Compton effect is
favored in low-density, water-equivalent (low $Z$) media (fat, soft
tissue, etc.). The Compton effect also dominates the interaction
processes of photons with energies between 0.2 and a few MeV, whatever
the atomic number of the medium through which they pass. The
probability of occurrence of these scatterings decreases as the energy
of the incident photon increases. The photoelectric effect and Compton
scattering can be enhanced by the presence of a captive sensor and can
also be exploited by F\"{o}rster-type energy transfer, which can take
place between the sensor (often derived from a metal) and the organic
effector, resulting in a change in the electronic configuration of the
organic probe.

Pair production only occurs at high energies and is statistically rare
under normal biomedical conditions. Through this effect, the
${\upgamma}$-ray is transformed into matter in the form of a pair of
negatively and positively charged particles (electron and positron).
Since an electron has a rest mass equivalent to 0.511~MeV of energy, a
minimum ${\upgamma}$ energy of 1.02~MeV is required to produce this
pair. Any excess energy of the ${\upgamma}$-ray producing the pair is
given to the electron--positron pair in the form of kinetic energy. In
most cases, the positron will undergo annihilation by reaction with an
electron in the detector material, creating two ${\upgamma}$ photons of
0.511~MeV each.


Photoelectric absorption dominates at low energies, followed by Compton
scattering, then pair production as energy increases. Absorption of
very high-energy photons leads to nuclear decay. The above processes
(with the exception of photodisintegration) all result in the
production of electrons (or their antimatter equivalent, positrons) and
lower-energy X-rays, which undergo further absorption and scattering.
In addition, these heavy nanoparticles can selectively scatter and/or
absorb high-energy radiation~\cite{bib32,bib33,bib34}, leading to
energy transfer to photosensitizers, often porphyrin derivatives, metal
complexes, scintillators or quantum dots that are sufficiently stable
under radiative conditions~\cite{bib35}.

\subsection{Chemical transformation of ROS-sensitive functions}


Materials sensitive to reactive oxygen species (ROS) can form covalent
bonds with the transiently generated intermediates formed by the
ionizing radiation. This can be facilitated by the presence of electron
donor shuttles, or simply the presence of large quantities of water,
ubiquitous in living organisms, which produce a large variety of
reactive intermediates by irradiation~\cite{bib36}. When exposed to
light or ionizing radiation, water undergoes a series of
transformations involving electronic excitations, ionization of
solvated species and the formation of free radicals and solvated
electrons~\cite{bib37,bib38,bib39,bib40,bib41,bib42}. The radiolysis
of water generates thus various reactive particles, in which the
$^{\bullet}$OH and the e$^-_{(\mathrm{aq})}$ are the dominant
products. These processes occur, for example, in (photo)electrochemical
cells~\cite{bib43,bib44}, biological molecules~\cite{bib45}, as well
as in atmospheric water~\cite{bib46}. Many of these are governed, at
least in part, by the nature of electron transfer and binding energies
in aqueous solutions. Among the many reactive species formed during
this reaction are hydrated electrons and ROS~\cite{bib47} such as
peroxides, singlet oxygen and oxyradicals, which are considered the
chemically active component of radiation therapy. The
HO$^{\bullet}$ radical (hydroxyl radical) is the
most abundant radical, which can then react with surrounding tissues to
create organic radicals (usually alkyl radicals). Historically, iodine
and gold-based nanoparticles (AuNPs) were the first radiosensitizers
due to their high X-ray absorption, synthetic versatility and unique
chemical, electronic and optical \unskip\break {properties} \cite{bib48}. The
radiosensitization dates back to the pioneering work of Hainfeld, who
demonstrated that gold nanoparticles behave as effective radioactive
agents, since tumor-bearing animals survive longer when treated with
radiotherapy after intravenous injection of ultra-small gold
nanoparticles~\cite{bib49}. The energy of the initial photon is thus
transferred to electrons, which create ionization that produces
\textit{in fine} ROS in the presence of water and oxygen, leading to
significant chemical and biological effects such as DNA degradation.
The range of radiosensitizers now extends to other heavy elements with
high atomic numbers, such as hafnium, gadolinium or bismuth
atoms/nanoparticles~\cite{bib34,bib50,bib51,bib52,bib53} which are
well-known radiosensitizers, capable of amplifying radiation doses in
tumor tissue~\cite{bib54,bib55,bib56}.

As mentioned in the previous paragraph, ${\upgamma}$/X-ray activation
enables ``second-generation'' photodynamic therapy (PDT), which relies
essentially on ROS generation and is driven by the activation of a
photosensitizer (PS), followed by the transfer of excitation energy to
molecular oxygen to result in the generation of cytotoxic ROS.
Noteworthy, the metal--photosensitizer combination is superior to the
use of metal particles alone for ROS generation in terms of biological
efficacy, and has a wider reach than the more conventional UV- and
IR-triggered ROS generation, as it overcomes the penetration limits of
UV or IR into tissue~\cite{bib16,bib57,bib58}.

Fragmentation of ROS-sensitive bonds, such as sulfide/disulfide or
selenide/diselenide bonds, or other chemical functions, as well as
changes in the mechanical properties (rigidity) of the nanomaterial
under the influence of radiation, can lead to the release of the
sequestered ligand.

\section{Activation of prodrugs or nanocarriers by electron or energy
transfer}


The prodrug strategy that was developed steadily in the second half of
the last century to improve how the drug is absorbed, distributed,
metabolized, and excreted in the living body involves masking one of
the key parts (usually polar functions) of a biologically active
compound, necessary for the activity, and restoring it when the masking
group is cleaved~\cite{bib59,bib60,bib61}. This strategy was also
developed in neurosciences, physiology, and molecular biology by using
light as trigger signal, making it possible to \mbox{control} drug activation
in space and time. By analogy with light, tissue-penetrating X-ray can
be used under particular conditions. 

\subsection{Electroreduction of indolequinones} 

Historically, the first report on X- and ${\upgamma}$-ray molecular
fragmentation by electron transfer was reported by the Tanabe group
using tethered 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FdUrd)
with 2-oxoalkyl groups covalently linked to radiation-sensitive quinone
derivatives enabling the release of the antitumor agents 5-FU and
5-FdUrd, respectively (Scheme~\ref{sch1})~\cite{bib62,bib63}.


\begin{scheme}
\vspace*{4pt}
\includegraphics{sc01}
\vspace*{16pt}
\caption{\label{sch1}Activation of indolequinone-{\ubreak}tethered
5-fluorodeoxyuridine by electron transfer and subsequent drug release.}
\vspace*{-7pt}
\end{scheme}

Mechanistic studies have shown that these prodrugs undergo reduction by
capturing hydrated \unskip\break electrons (e$^-_{(\mathrm{aq})}$) generated by the
radiolysis of water. Optimization of the anchoring group structure has
yielded indolequinones that can release the covalently attached ligand
by bioreduction or under radiolytic
conditions~\cite{bib64,bib65,bib66,bib67}. This transformation was
presented as the first prototype transformation for the release of
complex structures from encapsulated compounds sensitive to
irradiation~\cite{bib68}.

\subsection{Intramolecular electron or energy transfer from heavy
elements}

The Gd-sensitized aminoquinolone probe was found to undergo radiolytic
fragmentation with a covalently attached model dihydrocinnamate as
substrate~\cite{bib4,bib69}. Although the precise mechanism of the
fragmentation has not been established, electron transfer or electronic
coupling between the irradiated Gd complex and the quinolinium probe
has been postulated. Radiolysis of the covalently attached probe by
17.5~keV and 1.17~MeV X-rays resulted in the liberation of the
dihydrocinnamate model (Scheme~\ref{sch2}).

\begin{scheme*}
\includegraphics{sc02}
\vspace*{4pt}
\caption{\label{sch2}Radiolysis of gadolinium-bound quinoline cage with
release of dihydrocinnamate ligand.}
\vspace*{-5pt}
\end{scheme*}

In a somewhat analog entry, a Gd chelate and azobenzene were combined
in a radio-switch\unskip\break (Scheme~\ref{sch3})~\cite{bib70}. Azobenzene was
isomerized by low-dose X-rays, offering the possibility of implementing
trigger systems based on radiation penetrating the isomerization. 

\begin{scheme*}
\includegraphics{sc03}
\vspace*{4pt}
\caption{\label{sch3}Activation of \textit{cis}-GdAzo to trans-GdAzo
using a 1~keV source.}
\vspace*{-6pt}
\end{scheme*}

The activation efficiency (i.e., part of the isomerized
(\textit{trans}) compound compared to the starting (\textit{cis})
material) was 33\% and 69\% at irradiation doses of 2 and 20~Gy,
respectively, by using 1~keV source (${\sim}$1.24~nm). No compounds other
than \textit{trans}-GdAzo were observed spectrophotometrically. This
molecular activation based on a radio-switch seems promising and opens
the way to new types of applications such as actionable tools in
radiotherapy.

\begin{scheme*}
\includegraphics{sc04}
\vspace*{1pt}
\caption{\label{sch4}Activation of pyrenebutyric acid immobilized by
USPIO by X- or ${\upgamma}$-ray irradiation.}
\vspace*{-2pt}
\end{scheme*}

Solvated electrons and transiently formed radical species generated by
X/${\upgamma}$-rays have been exploited in the fragmentation of
sensitive redox probes derived from picolinium and immobilized on the
surface of ultra-small superparamagnetic iron oxide nanoparticles
(USPIO)~\cite{bib71}. The iron-derived nanoparticle enabled
Auger/Compton electron generation upon X-ray or ${\upgamma}$
irradiation, as well as monitoring of probe biodistribution by
T2-weighted MRI and by the appearance of fluorescence from the released
probe (Scheme~\ref{sch4}). The functionalized particles showed high
colloidal stability at physiological pH. The {picolinium} group
effectively quenched the fluorescence of the covalently attached pyrene
reporter in the starting material, probably via F\"{o}rster-type
quenching and dark complex formation. Probe activation was validated
under pulsed radiation and also by using a conventional Cs-137 source.
Although fragmentation was observed under both conditions, pulsed
sources proved considerably more effective: 82\% of the fluorescence of
the pyrene reporter was recovered upon 30~Gy irradiation, compared with
27\% of the fluorescence under conventional conditions.

\begin{scheme*}
\includegraphics{sc05}
\vspace*{5pt}
\caption{\label{sch5}The NAPC-ADC conjugate undergo fragmentation by
X-ray induced electron-transfer reaction {in vivo} to release a highly
toxic MMAE.}
\vspace*{-8pt}
\end{scheme*}

Several redox-sensitive compounds derived from picoline, quinolinine
and picolinium were examined for X-ray activation~\cite{bib72}.
4-Hydroxymethylene pyridinium was selected as the most efficient probes
for releasing client molecules in a dose gradient ranging from 0 to
60~Gy (Scheme~\ref{sch5}). The cytotoxic payload (monomethyl auristatin
E (MMAE)) was attached via a carbamate linker to the probe. \mbox{A~humanized}
antibody (sibrotuzumab) was attached to the release complex for better
targeting (NAPC-ADC) (Scheme~\ref{sch5}). The therapeutic efficacy of
the radiation-induced activation was then assessed {in vivo}. Mice were
irradiated with $3 \times 4$~Gy of X-rays on days 2, 4 and 6
post-injection. It should be noted that the overall 12~Gy of X-ray dose
was supposed to trigger the complete release of MMAE. Tumor growth
inhibition in the NAPC-ADC ${+}$ X-ray group was better: mean tumor size
was around $85 \pm 55~\mathrm{mm}^{3}$ at 21~days, whereas tumor size
in the control (PBS only) and {radiation-only} groups was up to $1387
\pm 463$ and $433 \pm 120~\mathrm{mm}^{3}$, respectively ($n = 5$).
Administration of NAPC-ADC (5~mg${\cdot}$kg$^{-1}$) alone had no significant
impact on tumor growth, suggesting that radiation-induced payload
release contributed to tumor treatment.

\subsection{Fragmentation of the quaternary\newline \mbox{ammonium} group}

Similar to the C--N bond cleavage of quaternary organoammonium salts
under photochemical \mbox{conditions,} quaternary ammoniums can be converted
to tertiary amines under X-ray radiative conditions~\cite{bib73a}.
Fragmentation alters the polarization of the central benzyl group
(i.e., through the transformation of an electron-withdrawing group
(EWG) into an electron-donating substituent), triggering the
1,6-fragmentation of the benzyl-bound carbamate, thereby releasing the
ligand (Scheme~\ref{sch6})~\cite{bib73b}.

\begin{scheme}
\includegraphics{sc06}
\vspace*{9pt}
\caption{\label{sch6}Radiolytic fragmentation of the quaternary
ammonium group.}
\vspace*{-4pt}
\end{scheme}

Carfilzomib, an anticancer drug with a morpholine motif, was released
as a model substrate to demonstrate the feasibility of the {in vitro}
strategy of radiation-induced prodrug release. Radiation-induced
cleavage was demonstrated in living cells and in tumor-bearing mice,
but high doses (60~Gy) were required to achieve satisfactory
conversions.

\begin{scheme*}
\includegraphics{sc07}
\vspace*{5pt}
\caption{\label{sch7}Radiolytic activation of doxorubicin.}
\vspace*{-2pt}
\end{scheme*}

\begin{scheme*}[t!]
\includegraphics{sc08}
\vspace*{5pt}
\caption{\label{sch8}Activation of VHL E3 ligase by removing the
tetrafluoroazidobenzyl carbonate protecting group by X-ray.}
\vspace*{-5pt}
\end{scheme*}

\section{Reduction of azides to amines} 

Prodrugs of pazopanib and doxorubicin encapsulated in sulfonyl azide
and phenyl azide were released at irradiation doses of up to 60~Gy
(Scheme~\ref{sch7})~\cite{bib74}. The driving force behind the
transformation is the high reducing potential of e$^-$ generated by
the radiolysis of water, with a standard electrode potential of ${-}$2.77~V. 
Although the exact reduction mechanism is unclear, it appears to
follow a similar pathway to the photolysis of sulfonyl azides in
alcohols and water to generate sulfonamides via the formation of
sulfonylamido radicals or nitrenes~\cite{bib75}.

In a similar strategy, a tetrafluorobenzyl azide cage was used to mask
the polar functions of the VHL E3 ligase via a carbonate linker.
Continuous irradiation up to 60~Gy resulted in conversion of the azide
group to an amine and triggered ligand release
(Scheme~\ref{sch8})~\cite{bib76}.

\section{Activation of \textit{N}-oxide prodrugs}

The high electrode potential of electrons generated by local radiolysis
of water can also effectively reduce nitrogen oxides~\cite{bib77}. The
caging strategy involves oxidation of the target drug to $N$-oxide
\mbox{prodrugs,} generating drugs whose biological activity is silenced.
Radiolytic activation has been demonstrated from NO-imiquimod,
NO-ampiroxicam, NO-pranoprofen, NO-loratadine, and NO-camptothecin by
X-ray-induced electron transfer using radiation up to 60~Gy
(Figure~\ref{fig3}).

\begin{figure*}
\vspace*{2pt}
\includegraphics{fig03}
\caption{$N$-Oxide prodrugs for radiolytic activation.\label{fig3}}
\vspace*{12pt}
\end{figure*}

\begin{figure*}
\includegraphics{fig04}
\caption{Nanoparticles, made by the co-assembly of the hydrophobic
Au--LAHP NPs, amphiphilic oxidation-sensitive PPS--PEG polymers and
hydrophilic DOX were activated by {in situ}-formed hydroxyl radicals
($^{\bullet}$OH). The oxidation of the thioether chain to sulfoxide and
sulphone transformed the hydrophobic chain to hydrophilic, leading to
vesicle degradation and the release of the drug. \label{fig4}}
\end{figure*}

\section{Chemical transformation of ROS-sensitive functions}

\subsection{Oxidation of thioethers}

Nanovesicles were assembled from ROS-sensitive poly(propylene
sulfide)-poly(ethylene glycol) (PPS-PEG) amphiphilic polymers and
hydrophobic Au nanoparticles (Au NPs) linked to X-ray-labile linoleic
acid hydroperoxide (LAHP) (Figure~\ref{fig4})~\cite{bib78}. The
nanomaterial exhibited burst release of encapsulated doxorubicin (DOX)
by irradiation at 8~Gy, up to 46.7\%. Remarkably, drug release from
Au--LAHP--vDOX incubated in H$_{2}$O$_{2}$ progressively increased to
76.7\% 24~h after irradiation, while late drug release in PBS was
minimal. In contrast, drug release from Au--LA--vDOX showed little or
no response to X-ray irradiation. Irradiation (8~Gy) in a mouse
subcutaneous tumor model significantly retarded tumor growth in the
presence of Au--LA--vDOX and inhibited tumor growth even after
irradiation, which could be attributed to the slow release of DOX from
vesicles.

In an analogous study, radiation-generated ROS were the trigger signal
for the activation of doxorubicin (DOX)-loaded nanomicelles derived
from poly(propylene sulfide) (PPS) and hyaluronic acid
(HA)~\cite{bib57}. HA--PPS@DOX nanoparticles exhibited prolonged
circulation times of up to 24 h and showed decent accumulation in
tumors. When oxidation by X-ray irradiation (8~Gy) converted the
lipophilic thioether chain to polar sulfoxides, the ROS-sensitive
HA--PPS@DOX NMs disintegrated and released the cytotoxic cargo.


\subsection{Fragmentation of disulfide and diselenide bonds} 

Disulfide and diselenide are not particularly sensitive to direct
X-rays without the synergistic effect of a high concentration of ROS.
The Tanabe group devised a DNA amphiphile (DAM) composed of hydrophilic
oligodeoxynucleotides linked to hydrophobic alkyl chains by disulfide
bonds~\cite{bib79}. When DAM molecules are exposed to X-ray
irradiation, selective fragmentation of the disulfide bonds leads to
dissociation of the aggregates and release of\break the drug. 

\begin{figure*}
\includegraphics{fig05}
\caption{Cross-linked polysulfide shell of cysteine-decorated poly-G4.5
PAMAM dendrimer. \label{fig5}}
\end{figure*}

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig06}
{\vspace*{-2pt}}
\caption{Diselenide-linked micellar aggregates were cleaved in the
presence of ROS, generated by ${\upgamma}$-rays (5~Gy). \label{fig6}}
{\vspace*{-2pt}}
\end{figure*}

Radiolytic oxidation (5~Gy) resulted in the cleavage of the
cross-linked polysulfide shell of the cysteine-decorated poly-G4.5
PAMAM dendrimer \mbox{(a ``4th generation''} branched polyamidoamine \mbox{polymer}
containing 128 surface carboxylate groups), allowing release of the
anticancer drug (Figure~\ref{fig5})~\cite{bib80}.

Ma \etal~used PEG--PUSeSe--PEG, a radiation-sensitive diblock
copolymer, to construct a nanoscale drug delivery system
(Figure~\ref{fig6})~\cite{bib81}. On exposure to ${\upgamma}$-rays
(5~Gy), the generated ROS oxidized the \mbox{diselenide} groups by forming
selenic acid and triggering the fragmentation of the PEG--PUSeSe--PEG
micellar aggregates/release of the encapsulated drug (PUSeSe stands for
polyurethane diselenide hydrophobic block). Unfortunately the
PEG--PUSeSe--PEG polymer showed cytotoxicity to HepG2 cells.

Immunotherapy can be combined with radiotherapy and chemotherapy,
providing a new strategy for cancer treatment. A combination of
diselenide and pemetrexed has been developed to combine NK cell-based
cancer immunotherapy with radiotherapy and chemotherapy in a single
system~\cite{bib82}. The assemblies were prepared by co-assembly
between pemetrexed and cytosine-containing diselenide via hydrogen
bonds (Scheme~\ref{sch9}). ROS generated by ${\upgamma}$-radiation
(5~Gy) not only cleaved the diselenide bonds (converted to seleninic
acid), thereby suppressing human leukocyte antigen E (HLA-E) expression
in cancer cells and activating the immune response of NK cells, but
also released pemetrexed.

\begin{scheme*}
\vspace*{6pt}
\includegraphics{sc09}
\vspace*{8pt}
\caption{\label{sch9}Assembly of the diselenide--pemetrexed block.}
\end{scheme*}

\begin{figure}
\vspace*{2pt}
\includegraphics{fig07}
\vspace*{2pt}
\caption{ROS-sensitized mesoporous\newline \mbox{organosilica} nanoparticles
(MONs).\label{fig7}}
\vspace*{2pt}
\end{figure}

The Chen group has developed a strategy for the targeted delivery of
doxorubicin (DOX) to tumor sites using mesoporous organosilica (MON)
nanoparticles coated with a cancer cell membrane, which can be
activated by X-rays (Figure~\ref{fig7})~\cite{bib83}. This approach
involved loading DOX and a PD-L1 inhibitor into the pores of MONs
bridged by diselenic linkers. MONs were then coated with cancer
cell-derived membrane fragments to produce anti-PD-L1 CM@MON@DOX +.
Low-dose X-ray irradiation (less than 1~Gy) resulted in the
fragmentation of the diselenic bond, disrupting the structure of the
MONs, and releasing DOX and the PD-L1 inhibitor, resulting in
DOX-mediated immunogenic cell death at the tumor site~\cite{bib84}.

Diselenide-derived block copolymers have been used to load doxorubicin
for precision chemotherapy~\cite{bib85}. ROS generated by X-ray
irradiation (2~Gy) promoted release of the encapsulated drug under 
{in vivo} conditions in tumor-bearing mice, which produced a
more pronounced overall antitumor effect than the other test groups. 

\begin{scheme*}
\includegraphics{sc10}
\vspace*{10pt}
\caption{\label{sch10}Fragmentation of rhodamine DHBO--Me to
Me-Rhodamine (green) by ${\upgamma}$-ray irradiation.}
\vspace*{-5pt}
\end{scheme*}

{\vspace*{-2pt}}

\subsection{Dihydroxybenzyl functions sensitive to\newline
\mbox{oxidation}}

{\vspace*{-2pt}}

The Co-60 source has been tested in the radiative fragmentation of
several carbamate-bound photosensitive compounds~\cite{bib86}. Best
fragmentation kinetics were observed with resorcinol derivatives. The
hypothesis of radiation-induced \textit{ortho}- or
\textit{para}-hydroxylation of the aryl ring was put forward as the
operative fragmentation mechanism, followed by a 1,4 or 1,6 elimination
reaction releasing the covalently attached ligands (Scheme~\ref{sch10}).
Radiation activation has been studied in cell experiments as well as in
tumor-bearing mice, using the ligand MMAE as an anticancer agent with a
single-dose activation of 4~Gy. Details of oncological relevance were
not provided, however.

\vspace*{-2pt}

\subsection{Release of small molecules}

\subsubsection{NO synthesis triggered by ROS}

The mixing of hafnium tetrachloride (HfCl$_{4}$) and 2-nitroimidazol in
a microemulsion containing hexamethylene, hexyl alcohol, and a
surfactant (triton) resulted in the formation of a unique type of
nanoscale coordination polymer, HNP NPs~\cite{bib87}. NPs were coated
with DOPA (sodium salt of 1,2-dioleoyl-sn-glycero-3-phosphate) and then
mixed with 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC),
cholesterol and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene 
glycol))-5000] (DSPE--PEG5000). The nanomaterial was shown to act
both as a radiosensitizer and as an immune adjuvant for NO donors
(2-nIm), which released NO in a controlled manner under X-ray
irradiation. 

A template-free method was devised for the synthesis of Bi--SH
nanoparticles (Bi--SH NPs), which were then coated with SiO$_{2}$ to
improve aqueous solubility, and finally functionalized with the NO
precursor S-nitrosothiol (Bi--SNO NPs)~\cite{bib88}. The particular
advantage of this system is the local release of NO: the short
half-life of NO and its high sensitivity to biological substances
considerably limit the development of NO-based therapeutic platforms
for clinical application. The release of NO by X-rays (5~Gy) has been
validated in rodents.

\subsubsection{Carbon monoxide release triggered by X-rays}

A multifunctional X-ray-activatable theranostic nanoplatform has been
developed based on Ce-doped LiLuF$_{4}$ scintillant and UV-sensitive
Mn$_{2}${(CO)}$_{10}$ for the controlled generation of CO and manganese
dioxide (MnO$_{2}$) in tumors~\cite{bib89}. Under X-ray irradiation,
radioluminescent UV light from SCNPs triggered the photolysis of
Mn$_{2}$(CO)$_{10}$ by luminescent resonance energy transfer (LRET). In
this process, the Mn--Mn and Mn--CO bonds of Mn$_{2}$(CO)$_{10}$ were
cleaved photochemically to release CO forming MnO$_{2}$ as a
by-product. {In vitro} and {in vivo} experiments demonstrated the potent
antitumor activity and low systemic toxicity of this nanomedicine.
\looseness=-1

\subsubsection{Peroxynitrite generation}

Peroxynitrite (ONOO$^{\bullet -}$), a reaction product derived from
nitric oxide (NO) and superoxide ($\mathrm{O}_{2}^{\bullet -}$), is a powerful
oxidizing and nitrating agent that modulates complex biological
processes and promotes cell death. A multifunctional X-ray-controlled
ONOO$^{\bullet -}$ generation platform based on scintillating
nanoparticles (SCNPs) and UV-sensitive NO donors, Roussin black salt,
has been reported~\cite{bib90}. In this system, Ce-doped LiLuF$_{4}$
acts as a radiosensitizer by promoting ROS, including $\mathrm{O}_{2}^{\bullet-}$, 
under X-ray irradiation, and it also converts X-rays to UV light
to activate Roussin's black salt to release NO. The simultaneous
release of NO and $\mathrm{O}_{2}^{\bullet -}$ ensures efficient
X-ray-controlled production of ONOO$^{\bullet -}$ in tumors.

\section{Miscellaneous}

Doxorubicin (DOX), gold nanoparticles (AuNPs), DNA-coated AuNPs
(DNA--AuNPs), and DOX conjugated to DNA--AuNPs (DOX--DNA--AuNPs) were
synthesized and incubated with MCF-7 breast cancer cells~\cite{bib91}.
Strong internalization was observed with only residual leaching of
immobilized doxorubicin. The extinction efficiency of DNA
strand-attached DOX on AuNPs was estimated at 99.7\%. Under the
influence of X-rays, the DOX--DNA--AuNP DNA strands were cleaved,
probably by the presence of $^{\bullet}$OH radicals, and the DOX
molecules were removed from the AuNP surface. The drug-carrying
nanoparticles became more toxic as the X-ray dose increased, from 
${-}1\% \pm 15\%$ at 2~Gy to ${+}19\% \pm 13\%$ at 5~Gy and  ${+}33\% \pm
21\% $ at 10~Gy. Noteworthily the observed toxicity was greater than
the sum of the radiation toxicity and the toxicity of the drug-carrying
nanoparticles. Given the amount of AuNPs in the cell
(${\approx}$30~wt\%), the small increase in toxicity observed seems to
suggest that these nanoparticles remained in the cytoplasm, as this
amount of AuNPs in the cell would cause significant damage only if the
increase in X-ray absorption was taken into account. It should be noted
that this report is the first demonstration of increased cytotoxicity
due to X-ray-triggered release of chemotherapeutic drugs from
nanoparticles in cells.

ROS can oxidize unsaturated lipids and destabilize liposomal membranes,
releasing the encapsulated cargo. The Deng laboratory devised a drug
delivery system using sensitized liposomes containing 3--5~nm gold
nanoparticles (GNPs) and the photosensitizer verteporfin (VP), and
X-rays to trigger liposome rupture~\cite{bib92}. This system takes
advantage of the intensity of the radiation and the ability of
liposomes to adapt to the environment. This system takes advantage of
the radiation intensification effect of GNPs to amplify the production
of singlet oxygen ($^{1}\mathrm{O}_{2}$) and other ROS from verteporfin
under X-ray irradiation, triggering thus the rupture of liposomes. The
results indicate that liposomes loaded with GNPs and VP exhibit higher
rates of $^{1}\mathrm{O}_{2}$ production than those loaded with GNPs or
VP individually. The antitumor efficacy of this drug delivery system
was validated {in vitro} and {in vivo}, demonstrating that LipoDOX, a drug
delivery system based on DOX-loaded liposomes, displayed superior tumor
inhibition efficacy following X-ray irradiation.

The liposome was prepared from dioleoyl-glycerophosphocholine (DOPC)
and dioctadecenoyl-trimethylammonium-propane (DOTAP)~\cite{bib92}.
Cellular uptake of the nanomaterial was facilitated by its positive
charge at neutral pH. {In vitro} gene inhibition by X-ray activation of
the liposome was demonstrated using an antisense oligonucleotide for a
pituitary receptor specific for adenylate cyclase-activating
polypeptide (PACAP) in rats. The system was also tested in
X-ray-triggered chemotherapy, using doxorubicin in liposomes on human
HCT 116 colorectal cancer cells.

It has been shown that low-dose X-rays (1--4~Gy at 6~MeV) can trigger
the release of doxorubicin (DOX) from liposomes co-loaded with gold
nanoparticles and photosensitizers~\cite{bib93}. ROS produced by gold
nanoparticles and photosensitizers under X-ray irradiation destabilized
the lipid membrane and led to the release of encapsulated DOX. 

In a similar system, the anticancer drug paclitaxel (PTX) and
radioluminescent CaWO$_{4}$ (CWO) nanoparticles were coencapsulated
with poly(ethylene glycol)-poly(lactic acid) (PEG--PLA) block
copolymers (``PEG--PLA/CWO/PTX NPs'')~\cite{bib93}. Upon exposure to
X-rays, the PEG--PLA coating layer degraded and PTX molecules were
released. It should be noted that radioluminescent CWO nanoparticles
emit UV-A/blue light (350--525~nm) under X-ray excitation, which also
produced radio-enhancement/radio-sensitization effects in tumor
tissues. \looseness=-1

GFP reporters were attached to silica-coated gold nanoparticles (AuNP@
SiO$_{2}$) by DNA strands as linkers~\cite{bib94}. The DNA strand is
broken by reaction with ROS, releasing the GFP reporters. Although
proteins also react with ROS at high rates, the low concentrations of
ROS rendered this damage (less than one reaction per protein)
undetectable in this work. The authors hypothesized that the efficiency
of AuNP@SiO$_{2}$ release could potentially be improved if the
thickness of the SiO$_{2}$ layer was reduced and DNA strand breaks
could be chemically enhanced. This work has shown that radiation
activation can be compatible with the delivery/activation of
therapeutic proteins in biological systems. 

Alkyne-modified dextran hydrogel (500 kDa) was irradiated with
${\upgamma}$-rays (14.4~kGy), resulting in densification of
cross-linking by the formation of reactive terminal alkyne radicals,
generated directly by irradiation or indirectly by reaction products
from radiolysis of water~\cite{bib95}. Polymerization controlled
hydrogel rigidity, contraction, release, and fusion. Hydrogel stiffness
increased twofold for hydrogels swollen with H$_{2}$O and threefold for
hydrogels swollen with phosphate buffer after ${\upgamma}$ irradiation,
due to the increase in the cross-linking density.

\section{Conclusion and outlooks}

Tissue-penetrating X- and ${\upgamma}$-rays have the potential to
selectively reach specific tissues. Simultaneous release of organic
compounds with the therapeutic beam presents a conceptually different
approach compared to standard protocols that combine radiotherapy and
chemotherapy. This approach may allow further reducing radiation doses
and systemic cytotoxicity compared to current protocols. Since organic
compounds are transparent at this wavelength, and direct irradiation is
inefficient, the search for different transduction mechanisms has led
to many ingenious drug delivery strategies. To overcome the
difficulties associated with the biocompatibility, degradability and
cellular toxicity of several nanocarriers, strategic solutions are
still needed. One of the main challenges is to minimize the X-ray doses
required to trigger cargo release close to unity. Even though the
release efficiency is one of the most important parameters, in most
reports the quantitative analysis of the drug release is neglected
claiming technical difficulties. This should be certainly improved in
the near future. \looseness=-1

The X-ray activation method is likely to be applied clinically in the
near future, provided that issues of toxicity (of nanomaterials) and
efficacy (enabling the necessary activation doses to be reduced) are
optimized. Also, the nanoparticle formulation can serve as a
simultaneous multifunctional platform for chemo-, thermo-and
immuno-radiotherapy; it can not only enhance tumor irradiation
responses during RT but also leverage combination therapy to achieve a
synergistic effect. We believe that this mode of activation is still in
its infancy, while opening up the potential of nuclear medicine to the
control of smart materials. Future developments can be extended to
proton and carbon ion beams, although they have a very narrow Bragg
peak, meaning that they do not scatter along the radiation path and
reach the biological region without energy loss~\cite{97}. This young
field is developing rapidly and, with access to increasingly powerful
and safe experimental methods and \mbox{equipment,} promises a
breakthrough in research and clinical applications. 

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

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