Yields (in 10‐15 electron‐1 molecule‐1) for the formation of SSB and DSB induced by 1, 10, and 100 eV electron impact on 5 ML DNA films and 60 keV electron impact on 2900 nm DNA films deposited on a tantalum substrate.
This chapter focuses on the fundamental processes that govern interactions of low‐energy (1–30 eV) electrons with biological systems. These interactions have been investigated in the gas phase and within complex arrangements in the condensed phase. They often lead to the formation of transient molecular anions (TMAs), and their decay by autoionization or dissociation accompanied by bond dissociation. The damage caused to biomolecules via TMAs is emphasized in all sections. Such damage, which depends on a large number of factors, including electron energy, molecular environment, and type of biomolecule, and its physical and chemical interactions with radiosensitizing agents are extensively discussed. A majority of recent findings resulting from experimental and theoretical endeavors are presented. They encompass broad research areas to elucidate important roles of TMAs in irradiated biological systems, from the molecular level to nanoscale cellular dimensions. Fundamental aspects of TMA formation are stressed in this chapter, but many practical applications in a variety of radiation‐related fields such as radiobiology and radiotherapy are addressed.
- ionizing radiation
- low‐energy electrons
- dissociative electron attachment
- strand breaks
High‐energy ionizing radiation (e.g., γ‐ and X‐rays, electrons, and ions) affects biological materials, via a chain of physical, chemical, and biological processes. A complete understanding of these processes in living cells and tissues is a challenging task because of the multiple sequences of events, which lead to cell mutation or death. Nonetheless, such knowledge enhances our ability to cause death or inhibit growth of cancer cells in radiation therapy and to save healthy cells by radiation protection. As shown by many studies , cellular deoxyribose nucleic acid (DNA), containing genomic information, is the primary target for cell damage from ionizing radiation. The fundamental mechanisms involved in the induction of damage to DNA by radiation have therefore been subjects of intense investigations during the past decades [2, 3]. When exposed to ionizing radiation, large biomolecules such as DNA and proteins in the cell can be ionized and/or excited. This may effectively cause changes in their molecular structures by inducing bond ruptures and successive fragmentations, which then affect the function and metabolism of the cell. In DNA, the resulting damages may lead to incomplete repair, misrepair, or unrepair of the molecule. The displaced, mismatched, or damaged DNA bases may be misinterpreted during the replication cycle, deterring cell replications and causing accumulation of cancer predispositions for mutations [4–6].
Ionizing radiation damage to DNA can be induced directly by the interactions of primary quanta of radiation via ionization or excitation of individual components of the DNA itself and by secondary particles, including radicals, electrons, and ions generated along the track, after the interaction of primary radiation with molecules surrounding DNA, that is, water and other cellular components [7, 8]. It is by now well established that the consequences of radiation exposure of biological matter at the molecular level are largely due to secondary electrons (SEs), which are formed with a yield of about 5 × 104 per MeV of deposited radiation energy. SEs are the most abundant secondary species generated by the transfer of energy from ionizing radiation into the medium and essentially comprise slow electrons with kinetic energies below 30 eV. The energy distribution of SEs has a most probable energy around 9–10 eV , and those electrons of higher initial energy undergo successive energy losses via inelastic collisions, for example, electronic excitation and ionization. These later create further generations of electrons of significantly lower energies. As all electrons necessarily reach the low‐energy range (
The main purpose of this chapter is to describe the phenomena related to reactions of LEEs, which may produce biological effects in the cell, such as apoptosis and cell cycle arrest. Since utmost of the harmful mutagenic and lethal damages of ionizing radiation result from chemical modifications in the nucleus of living cells, sustained studies have been focused on the ultrafast mechanisms involved in the direct interaction of LEEs with DNA and its different subunits, as well as indirect processes which are associated to the interactions of electrons with the principal cellular components nearby DNA. An ultimate understanding of LEE damage mechanisms and their role in DNA damage due to radiation can be obtained from experiments with molecular targets of increasing complexity, that is, from simple gaseous and condensed phase biomolecules to plasmid and cellular DNA. This wide range of target structures is essential to systematically understand how the fundamental principles of the LEE interaction with simple biomolecules and DNA components intervene in more complex ones up to and including cellular DNA .
In the first two sections of this chapter, the formation of TMAs and their decay into DEA and autoionization processes are extensively reviewed for simple molecules in the gas and condensed phases. The next section exclusively concerns the interactions of LEEs with basic DNA subunits, that is, the bases, the sugar–phosphate unit, and its two basic constituents in the gas phase. Such studies are necessary to understand how SSBs and DSBs and base release in the much more complex DNA molecule can occur by LEE impact. Additionally, gas‐phase DEA to radiosenisitizers (halogenated nucleobase, Pt‐ and nitrogen‐based compounds) is discussed in Section 4.
While most of the simple DNA building blocks can be readily vaporized for experimental study to the gas phase, most of the larger units, that is, nucleosides (containing a DNA base + sugar) and entire nucleotides (sugar + base + phosphate group) undergo decomposition during evaporation . Electron attachment to the short oligonucleotides and single‐stranded oligomers containing different bases is reviewed in Section 5. Such molecules with a strong tendency to capture electrons and formation of electronically stable anions simplified the analysis of degradation products relative to longer single‐ and double‐stranded configurations . Since histones and the other chromosomal proteins present in the nucleus are in close contact with DNA, reactive species resulting from the interactions of LEEs with nearby amino acids may also interact with DNA, causing indirect damage. There is thus considerable interest in studying the fragmentation of chromosomal proteins induced by LEEs , and Section 6 is devoted to the investigations of the action of LEEs on building blocks of proteins, more particularly on amino acids and peptides.
Despite the significance of the gas‐phase and condensed‐phase experiments in revealing the major interactions of LEE with DNA, the results of these experiments do not essentially correspond to those obtained in the dynamic existent situation of the cell, where cellular DNA lies in a medium containing essentially water with proteins, ions, and vitamins dissolved in the aqueous environment. Section 7 thus reviews recent studies in more complex systems, where a DNA molecule is embedded into more realistic environments containing water, oxygen, histones, and DNA‐binding proteins that mimic cellular conditions.
The role of secondary LEEs in radiosensitization and radiation therapy is discussed in the final section of this chapter. LEEs have subcellular ranges (on the order of 10 nm) in biological materials and interact strongly and destructively with chemical bonds; so, they are ideal for promoting local (i.e., nanoscopic) increases of radiation damage in cells, particularly for targeted cancer therapies. We review a wealth of experimental data on LEE‐induced lesions in DNA bound to radiosensitizing gold nanoparticles and the platinum‐chemotherapeutic agents. This final section links the effects of radiation and chemotherapy, showing that by modulating the radiation chemistry, chemotherapeutic agents can become radiosensitizers. It also explains how our fundamental understanding of LEE‐induced DNA damage can be applied to optimize concomitant chemoradiation therapy (CRT) by modifying the action of LEEs or by increasing their numbers in cancer cells.
2. Decay of Gaseous Transient Anions into Dissociative Electron Attachment (DEA) and Autoionization
In electron–molecule collisions, a TMA is formed, when an incoming electron occupies a previously unfilled orbital of a molecule for duration greater than the usual scattering time [15–17]. Since such an orbital exists at a precise energy , TMAs are formed at specific energies usually below 15 eV and rarely above 30 eV [15, 16]. Because of the uncertainty principle, the transient state has a width in energy, which characterizes and identifies the process in the dependence on incident electron energy of cross sections for particular energy‐loss processes or the formation of the products or damage yields (i.e., the yield functions). Thus, at the resonance energy, corresponding to the formation of the TMA, yield functions exhibit pronounced maxima that can be superimposed on monotonically increasing background, which results from nonresonant or direct scattering.
The formation of TMAs is well described and reviewed in the literature [15–21]. There are two major types of TMAs or “resonances” [15–17]. The first, known as a single‐particle resonance, occurs when the additional electron occupies a previously unfilled orbital of a molecule (or subunit of a large biomolecule) in its ground state. Here, the electron is temporarily trapped within an angular momentum barrier by the shape of the electron–molecule potential. Such TMAs are thus also termed shape resonances. Core‐excited resonances or “two‐particle, one‐hole” states form when electron capture is accompanied by electronic excitation, such that two electrons occupy previously unfilled orbitals. The incident electron is in effect captured by the positive electron affinity of an excited state of the molecule or basic subunit in the case of a large biomolecule, which for DNA might include a base, sugar, or phosphate group. If a momentum barrier in the electron–molecule or electron‐subunit potential contributes to the retention of the electron in the electronically excited molecule or subunit, the transitory anion is referred to as core‐excited shape resonance. If the TMA state is dissociative and the resonance lifetime is greater than about half of the vibration period of the anion, the latter dissociates. This process is called DEA.
The decay of a TMA into dissociative channels can be understood by considering the hypothetical internuclear potential‐energy curve of a diatomic molecule AB and one of its TMA state AB‾ shown in Figure 1. While the following description is rigorously applicable only to diatomic molecules, it is still qualitatively valid along a specific bond of a polyatomic molecule. Assuming that only Franck–Condon (F–C) transitions are possible and that the AB‾ state is dissociative, we see from the consideration of the ground‐state nuclear wave function that electrons with energies of between
When the TMA state lies above the electronically excited states of the molecule, this later can acquire electronic energy, after autoionization of the anion, in addition to vibrational and rotational motion. If the electronic excited state is dissociative, then fragments A and B (Figure 1) are produced. Thus, both decay by autoionization into dissociative electronically excited states and DEA cause the molecule to fragment.
Within a local complex potential–curve–crossing model, the DEA cross section may be expressed as
For further information on the mechanism of TMA formation and its effects on isolated electron–molecule systems, the reader is referred to previous works [15, 16, 22–26]. Information on resonance scattering from single layer and submonolayers of molecules physisorbed or chemisorbed on conductive surfaces can be found in the review by Palmer and Rous . The following section provides information essentially on TMA formation in the condensed phase (i.e., in molecules in solids, condensed onto a dielectric surface or forming a molecular or biomolecular thin film).
3. Modification of electron capture and decay of transient anions in the condensed phase
In principle, the formation and decay of TMAs of condensed molecules can be described using a modified gas‐phase picture. For molecular solids or sufficiently thick molecular films condensed onto a metallic substrate or a dielectric surface, the target molecules are unaffected by the substrate, and they exist in the physisorbed state . This weak form of adsorption is characterized by a lack of a chemical bond between molecules, so that the electronic structure and vibrational frequencies of the condensed molecule are essentially unchanged from those in the gas phase [17, 27]. Conversely, electron–molecule scattering is modified in the condensed phase as well as the properties of TMAs [17, 20].
Low‐energy (0 – 30 eV) electrons have wavelengths comparable to the distance between molecules in condensed media. Hence, they interact within molecular solids via delocalized processes, predominantly including static and correlation interactions with neighboring molecules, excitation transfer, and coherent scattering [28–31]. Such conditions make it difficult to transfer electron scattering and attachment data from the gas phase to the condensed phase. Even though theoretical models have tried to approximate these processes, the resulting calculations differ substantially from the available experimental data [31–34]. For example, in the gas phase, the incoming electron wave function is a plane wave, whereas scattering events in the condensed phase are those of a diffracted electron wave function that depends on the ordering of the solid. It can be readily seen from Eq. (2) that this change in the partial wave content of the scattered electron wave modifies the capture cross section. Furthermore,
The increase in DEA cross section resulting from the shift of the curve crossing point in Figure 1 from
Within this energy range there exists a single structure in the
In the condensed phase, TMAs differ from their gas‐phase counterparts, in the following ways: (1) the electron energies required for their formation are usually lower by 0.5–1.5 eV, dependent on the local polarization of the solid and/or changes to the anion's symmetry; (2) due to their lower energies, they usually have fewer intramolecular decay channels, although, new intermolecular channels via electron emission into the dielectric may appear; (3) the lifetimes will be longer or shorter due to the changes in the number of decay channels, energy, and symmetry; (4) the initial electron capture probability, and the cross sections for decay into particular intermolecular and intramolecular excitations or for DEA may vary by orders of magnitude, as these are dependent on energy, intramolecular and intermolecular electron transfer, and symmetry. In summary, when a TMA is formed on a molecule located inside or at the surface of a molecular or biomolecular solid, its gas‐phase characteristics are usually considerably affected by the local environment.
4. DEA to gaseous DNA subunits and radiosensitizers
4.1. DNA bases
A large number of DEA studies have been performed on gas‐phase DNA bases and their derivatives over the last two decades [2, 39]. Briefly, DEA is the resonant process that involves the LEE capture by a molecule (AB) to produce gaseous TMAs ((AB)‾), described in Section 2, which then dissociate into an anion (A‾) and a neutral radical or radicals (B•), according to the following reaction:
In general, the low‐energy resonances in nucleobases are present either at subexcitation (< 3 eV) energies or in the energy range 5–12 eV . The yield function for the DEA processes for thymine resulting in multiple fragment formation is shown in Figure 3. To analyze the formation of the negative ions, yield functions were usually recorded by scanning the incident electron energy, while potential voltages applied to the quadrupole mass spectrometer were set for a given ion mass. The ion yields were detected by a channeltorn and plotted as a function of the incident electron energy.
The high‐energy resonances lead to transient anion fragmentation via opening of the ring structure, while the low‐energy resonances are primarily due to loss of one or two neutral hydrogen, which maintains the ring structure.
The DEA yield functions for nucleobases and their related compounds show a remarkable feature that can be recognized as a common phenomenon, that is, site selectivity [40–42]. By tuning the energy of the incoming electron, it is possible to control the location of the bond cleavage. That is, a specific chemical bond in a molecule can be targeted by electrons followed by fragmentation. As an illustration of this site selectivity in nucleobases, DEA to thymine with deuterated and methylated substitutions is described. This phenomenon was observed for other nucleobases and their derivatives, for example, adenine  and hypoxanthine .
where is the TMA of thymine (T) and is the closed‐shell anion formed by the ejection of a neutral hydrogen radical . This dehydrogenation process depends on the site from which the H atom is removed. Experimental studies with partially deuterated thymine, in which the deuterium is at either nitrogen or carbon sites, showed that hydrogen loss occurs exclusively from the N sites. H loss from the C sites is thermodynamically accessible within this energy range, but has not been observed experimentally. Moreover, in employing methylated thymine and uracil, it has been shown that by adjusting the electron energy, the loss of H can be made even site‐selective with respect to the N1 and N3 positions. Although 1 eV electrons induce H loss at the N1 position (N1‐H), the process can be switched at 1.8 eV to N3‐H (Figure 3b). These results have significant consequences for the molecular mechanism of DNA strand breaks induced by LEEs. Within DNA, the N1 position of thymine is coupled with the sugar moiety and thus forms thymidine, which is one of the nucleosides. Because the shapes of the signals from thymine and the more complex thymidine resemble each other, it can be concluded that H abstraction in thymidine predominantly occurs at the thymine moiety and, more precisely, at the N3 position (Figure 3b) .
In addition to the detection of anions and the energies at which they are formed, much effort has been expended to matching particular types of DEA process to specific resonant peaks observed in DEA ion yields. In the case of the most abundant anion formed for all nucleobases, it has been proposed that these resonant peaks can be assigned to vibrational Feshbach resonances (VFRs) [46, 47]. VFRs usually occur at low energies, when vibrational levels of the transient anion lie below the corresponding vibrational states of the neutral, and are more expected in greatly polarized molecules with very large dipole moment, which leads to a long‐range attractive interaction. They may serve as a gateway for dissociation at low energies if they are coupled with a dissociative valence state. This can be the case for the formation of dehydrogenated ions from nucleobases, where resonances arise from coupling between the dipole bound state and the transient anion state associated with the occupation of the lowest
Further to the observations of site selectivity in DEA processes leading to single‐bond cleavage within a nucleobase, site selectivity also occurred in multiple‐bond cleavage. As in the cases of both dehydrogenation of nucleobases and its complementary channels, which resulted in the Hˉ formation, site selectivity was demonstrated when multiple‐bond cleavage was involved, for example, for the formation of NCO‾ from thymine and its derivatives . This anionic fragment was formed in a sequential decay reaction, in which the dehydrogenated anionic nucleobase acts as an intermediate product. In this case, the remarkable resonances, which were observed for dehydrogenation and for H reaction channels in nucleobases, were preserved for the subsequent decay reaction, leading to the formation of NCO‾ as the final product.
In general, the total cross sections for DEA to nucleobases exhibited comparable magnitudes in the energy range for TMA formation . However, these cross sections were up to 10 times smaller than those for the formation of single‐strand breaks, while the cross sections for sugar and phosphate group analogs (see Section 4.2) were even smaller in magnitude.
4.2. Sugar and phosphate group
The high fragility of the DNA backbone with respect to the impact of LEEs with low kinetic energy was observed for 2‐deoxy‐D‐ribose and its RNA equivalent (i.e., ribose), along with their analogs . In principle, the dissociation of any of P–O–C bonds in the sugar–phosphate backbone or C–C bond within the sugar could result in a DNA strand break. If such breakages were to occur via the DEA process in DNA, then DEA would represent an important pathway through which the direct interaction of LEEs could affect biologically significant damage.
The DEA to 2‐deoxy‐D‐ribose results in a strong decomposition of the sugar at electron energies near 0 eV, indicating the loss of one or more molecules of water . Similar findings were observed for D‐ribose and other sugars , indicating that DEA at 0 eV is a common property of all monosaccharides. However, the mechanisms for DEA reactions leading to loss of neutral water are more complex in comparison to the dehydrogenation of the nucleobases, because they involve the dissociation of multiple bonds and/or atom rearrangement with simultaneous formation of new bonds. Therefore, the mechanism of DEA to sugars near 0 eV is not fully understood. It is however proposed to occur via the formation of a “shape” resonance. In a sugar molecule, the extra orbital can be a σ* orbital of the O–H bond. As was observed for alcohols , the σ* orbital of the hydroxyl group for the dehydrogenation channels appears at higher energy for simple alcohols than for cyclic alcohols. Moreover, it was found that larger numbers of hydroxyl groups present in a molecule could enhance the dissociation of an O–H bond, by decreasing the energy of the thermodynamic threshold. This mechanism has been suggested for 2‐deoxy‐D‐ribose and D‐ribose, which contain three and four hydroxyl groups, respectively . In addition, experiments with the ribose analogs tetrahydrofuran and 3‐hydroxytetrahydrofuran showed that DEA cross sections were greatly enhanced by the presence of OH groups . However, for alcohols, their molecular dissociation involved simple bond cleavage, while in sugars, fragmentation of several different bonds occurs. One of the proposed models for sugar dissociation was provided from ab initio calculations of VFRs formed initially by a dipole‐bound state of sugar due to a large dipole moment . Other quantum chemical calculations confirmed this model, showing that the sugar ring can convert into an open chain by intramolecular charge transfer. This mechanism can lead to dissociation by loss of a water molecule, assuming that the barrier for such a transfer is sufficiently low . It was also calculated through quantum dynamics scattering theory that the formation of shape resonances for D‐ribose is excluded at low energies, but they can be formed at higher energies .
In the case of thymidine, in which sugar is covalently bound to thymine, the DEA study showed two resonant structures (Figure 3b) . The one at lower energy was associated with a reaction in which the excess electron is initially localized in the sugar moiety, leading to the glycosidic bond cleavage. The second resonance was attributed to a reaction in which the excess electron was localized on the thymine moiety, resulting in the loss of a neutral H atom from the N3 site, as was mentioned for thymine. Since nucleosides can be easily decomposed due to the elevated temperatures necessary for evaporating samples, no experimental data for other gas‐phase nucleosides or nucleotides are reported, besides those for thymidine , cytidine , and 2‐deoxycytidine 5‐monophosphate .
Similarly, due to experimental difficulties, the phosphate group in the gas phase could not easily be investigated as an isolated compound. Its simplest analog, H3PO4 (phosphoric acid), is not easily vaporized for gas‐phase experiments or molecular deposition for thin film experiments . Therefore, to understand the DEA process within the phosphate group, several compounds involving phosphoric acid derivatives, for example, dibutylphosphate and triethylphosphate , were examined. DEA to these compounds lead to P–O and C–O bond cleavages, which correspond to a direct single‐strand break. As for sugars, many fragmentation channels occurred close to 0 eV; however, these low‐energy channels are most likely driven by the large electron affinity of PO3 (4.95 eV). The cross sections for DEA to the sugar and phosphate group analogs were relatively small, that is, about one magnitude lower than those for nucleobases . These gas‐phase results on sugars and phosphate units revealed that LEE attachment can induce single‐strand breaks by electron localization either on the sugar moiety followed by the electron transfer to the backbone or directly on the phosphate group.
An important characteristic of many current and potential radiosensitizers used in radiotherapy (or potential ones) is a high cross section for DEA. Since halogenated pyrimidines, mainly substituted uracil derivatives, exhibit high sensitivity to electron attachment and a rich fragmentation pattern from DEA, they have attracted considerable interest as radiosensitizers. From a medical point of view, the substitution of pyrimidines in the genetic sequence of cellular DNA does not affect the gene expression, and additionally enhances the sensitivity of living cells to radiation. A large number of gas‐phase experimental and theoretical studies of several halogenated pyrimidines (e.g., 5‐bromouracil [58–63], 5‐chlorouracil [58, 59, 61, 64, 65], 5‐fluorouracil [58, 59, 61, 65], 5‐iodouracil [59, 62], 6‐chlorouracil [58,66]) were performed in recent years and report orders of magnitude of higher cross sections for DEA relative to their nonsubstituted precursors. Further to the DEA studies, other electron spectroscopic techniques and theoretical calculations at the ab initio and density functional theory levels were utilized to characterize electronic structure and reveal the fragmentation mechanisms of halogenated pyrimidines . These studies elucidated the energies of vertical transitions to π* and σ* orbitals, showing that the ground TMA state of pyrimidine with the additional π* electron is a few tens of eV more unstable than the neutral ground state, whereas the vertical electron affnities of the halogenated derivatives were found to lie close to 0 eV. Moreover, DEA studies revealed that the lowest π* anion states of the halogenated pyrimidines follow similar fragmentation channels, resulting in the formation of the halide fragment anion. These studies also revealed that the total anion yields for bromopyrimidine were much larger than those measured for the chloro‐derivatives. These results indicate that bromopyrimidines carry the greatest potential as radiosensitizers for damage by SEs, which, via DEA to bromo‐substituted DNA, will enhance radiation‐induced damage to the cell. Recently, gas‐phase DEA studies on halogenated purines (e.g., chloroadenine ) and fluorinated nucleosides (2‐deoxy‐5‐fluorocytidine and 2,2‐difluorocytidine (gemcitabine) ) have been initiated to determine in what ways their radiosensitizing properties are derived from LEE‐driven chemistry.
In addition to the halogenated nucleobases, several aromatic compounds containing nitro groups have been recently investigated in the gas phase. For instance, DEA studies performed for 5‐nitrouracil showed the formation of a long‐lived parent anion, as well as a rich fragmentation pattern via formation of either “shape” or “core‐excited” resonances at low electron energies [70, 71]. The properties of 5‐nitrouracil showed a radiosensitizing nature similar to that of the halogenated pyrimidines. Interestingly, while in the case of halogenated pyrimidines, the most dominant fragment formed was a halide anion, that for 5‐nitrouracil is an anion of the pyrimidine without a nitro group. Therefore, the counterpart fragment of this dissociation channel is the formation of the NO2 radical, which is formed in close vicinity to DNA and can lead to the activation of lethal cluster damage in living cells.
There is also a great potential for other nitro‐containing compounds such as nitroimidazolic compounds to be used in radiotherapy, since LEEs effectively induce their dissociation [72, 73]. Similarly, their decomposition via DEA involves a range of unimolecular fragmentation channels from simgle‐bond cleavages to complex reactions, possibly leading to a complete degradation of the target molecule. However, these studies revealed that the entire rich chemistry induced by DEA was completely suppressed by methylation in the electron energy range below 2 eV.
In recent years, platinum‐based drugs were also investigated regarding their decomposition by LEEs. It was suggested that in concomitant treatment in which chemotherapeutic drugs and radiotherapy are combined, one possible mechanism responsible for the observed synergy between treatments is the enhancement in the number of secondary species induced by primary radiation in the vicinity of the binding site of the platinum compounds in DNA (see Section 8). The gas‐phase DEA studies of PtBr2 in the electron energy range between 0 and 10 eV showed the formation of the Br anion via two possible channels. The most dominant channels were assigned to the Br‾ + PtBr dissociation limit reached at ∼1 eV and the higher energy channel to Br‾ + Pt + Br .
The observation that all these radiosenisitizers exhibit DEA with high effciency, even close to 0 eV, may have significant implications for the development and use of these drugs in tumor radiation therapy. Considering their use as radiosensitizers, their fragmentation and the resulting generation of radicals at very low electron energies may be a key in understanding their action and the molecular mechanisms necessary to improve radiotherapy.
5. Electron attachment to short single‐stranded and plasmid DNA
Cellular DNA consists of a double‐stranded helical structure, composed of two long polynucleotide chains . Thus, as already mentioned in the Introduction section, in order to systematically understand LEE damage mechanisms and their role in radiation DNA damage, molecular targets of increasing complexity were studied, from simple molecules containing just two of the basic subunits (e.g., a phosphate group coupled with a sugar or a nucleoside having a DNA base + sugar), via synthetic, single‐ and double‐stranded oligonucleotides, containing multiple nucleotides to plasmid and other cellular DNA with many thousands of base pairs.
Even though most simple DNA components may be easily vaporized for experimental investigation in the gas phase, the larger units such as nucleosides and nucleotides usually decompose during evaporation . In any case, the condensed phase is certainly the more appropriate environment to study problems relevant to radiation damage in biomolecular systems. The experimental methods and techniques, used in the condensed phase, differ from those in the gas phase. Most condensed phase experiments are achieved by bombarding thin films (2–10 nm) of oligonucleotides or plasmid DNA with an energy‐selected beam of LEEs from an electron gun or an electron monochromator. To prevent excessive charging, these thin‐film biological samples are deposited onto a metal substrate by spin‐coating, lyophilisation (freeze‐drying), or molecular self‐assembly, as in the case of thiolated DNA on gold substrates  and 1,3‐diaminopropane layer plasmid on graphite . The LEE‐induced damage to plasmid and linear DNA films has then been investigated by (1) measuring electron‐stimulated desorption (ESD) of anions, (2) imaging the breaks by atomic force and scanning tunneling microscopies, and (3) analyzing, after bombardment, the change of DNA topology by gel electrophoresis or the molecular content by high‐ performance liquid chromatography (HPLC) and mass spectroscopy [35, 77].
Oligomers of single‐stranded DNA containing the four bases (e.g., G, C, A, and T), which are among the simplest forms of DNA, have made the analysis of degradation products much simpler than would be the case for longer single‐ and double‐stranded configurations. Short oligomers deposited onto metal surfaces (e.g., tantalum, platinum, and gold) as films of different thicknesses (1–5 ML) were bombarded with LEEs and produced fragments analyzed by HPLC . The results for the GCAT oligonucleotide indicated that strand breaks occur preferentially by cleavage of the C–O bond rather than the P–O bond, with two maxima at electron energies of 6 and 10 eV [78, 79].
Recently, Bald and co‐workers demonstrated the visualization of LEE‐induced bond cleavage in DNA origami‐based DNA nanoarrays on the single‐molecule level using atomic force microscopy (AFM) [80–82]. This novel method has a number of advantages: (1) only miniscule amounts of material are required to create submonolayer surface coverage, because of the facility to detect the DNA strand breaks at a single‐molecule level; (2) within a single experiment, more than one oligonucleotide sequence with various arrangements can be irradiated to efficiently compare a number of different DNA structures; (3) the method represents a simple way to obtain absolute strand break cross sections, thus providing benchmark values for further experimental and theoretical studies, and finally (4) this technique is not limited to single strands, but can be extended to quantify DSBs and to investigate higher order DNA structures.
Applying this technique, Bald and coworkers compared the absolute strand break cross sections of different 13‐mer oligonucleotide sequences (i.e., 5'‐TT(
In order to increase the complexity of targeted biomolecules, several studies have investigated the damage induced by LEEs in double‐stranded plasmid DNA. Due to the supercoiled arrangement of plasmid DNA, a single‐bond rupture in a DNA with a few thousand base pairs can produce a conformational change in the topology of the entire molecule. These changes include base alterations, abasic sites, intra‐ and inter‐strand base cross‐links, DNA adducts, and SSBs or DSBs; hence, these can be detected efficiently by techniques such as gel electrophoresis. This technique can identify supercoiled (SC), nicked circular (C), full‐length linear (L), cross‐linked (CL), and short linear forms of DNA, which can be assigned to undamaged DNA, SSBs, DSBs, several types of cross‐linked DNA, and multiple double‐strand breaks (MDSBs), respectively .
Though it has been established that most of the strand breaks induced by ionizing radiation have been repaired by a DNA ligation step, a DSB represents a particularly detrimental lesion that poses a serious threat to the cell, since it usually cannot be easily repaired . Indeed, even a single DSB can lead to cell death if left unrepaired or, more worryingly, it can cause mutagenesis and cancer if repaired improperly .
The results obtained for LEE‐irradiated supercoiled plasmid DNA in several investigations are well described in the literature and summarized in authoritative review articles [10, 11, 35]. These studies have shown that SSBs can occur as a result of DEA at electron energies well below electronic excitation and ionization thresholds (0.8–10 eV) [83, 90]. The results of Martin et al.  reveal two resonant peaks at 0.8 and 2.2 eV in the SSB yield function (i.e., the number of strand breaks versus the incident electron energy) via the formation of TMAs. These findings are consistent with theoretical calculations indicating that SSBs induced by near‐zero energy electrons are thermodynamically feasible [91–93]. Theoretical simulations of electron scattering and electron capture via “shape” resonances support the role of LEEs in DNA strand breaks . Theoretical calculations on scattering and attachment of LEEs to DNA components up to supercoiled plasmid DNA have been intensively reviewed in recent years [95, 96].
Another spatially resolved technique that exploits the use of graphene‐coated Au thin films and surface‐enhanced Raman spectroscopy (SERS) has recently emerged. Utilizing this technique, the sequence dependence of DNA damage at excitation energies < 5 eV can be studied . Currently, Ptasińska and coworkers are performing a quantitative and qualitative investigation of the various types of damages to dry and hydrated DNA induced by exposure to helium and nitrogen atmospheric pressure plasma jets (APPJs). Since an APPJ contains multiple reactive species, including LEEs, also found in radiation chemistry, exposure to these plasma jets provides information on both the direct and indirect pathways to damaging DNA. Ptasińska and coworkers have employed nitrogen APPJ to induce DNA damage in SCC‐25 oral cancer cells, and have thus provided new insight into radiation damage to a cellular system .
6. LEEs interaction with protein building blocks
It is well known that within the cells, DNA is in close contact with, and packed by, chromosomal proteins (histones). The attachment of proteins protects DNA from damage by compaction (e.g., which restricts easy access by free radicals to DNA) and repairs some of the damage of electron/hydrogen donation . LEE damage to proteins within cells should not, by itself, cause significant long‐term biological damages, because proteins can be replaced. However, due to the presence of histones and other chromosomal proteins in the vicinity of DNA, reactive species produced from LEE interactions with protein constituents (e.g., nearby amino acids) may in turn interact with DNA, causing indirect damage. Thus, from a radiobiological point of view, there is considerable interest in studying the action of LEEs on this important class of biomolecules . Recent work has focused on the building blocks of proteins, that is, amino acids and small peptides, since the size and complexity of chromosomal proteins prevent direct detailed analysis of the fragmentation processes induced by LEEs [11, 39]. Indeed, measuring the fragmentation of amino acids and their analogs is no more complex than it is for DNA constituents (see Sections 4 and 5) [101–103], and can help elucidate the effects of electron irradiation in larger more complex proteins .
In the recent years, several investigations have employed soft ionization techniques, such as matrix‐assisted laser desorption ionization (MALDI) [104–107], electrospray ionization (ESI) [108, 109], and collision‐induced dissociation (CID) [110–114], to study the ionization and fragmentation of different amino acids and small peptides in the gas phase. Gas‐phase investigations of LEE‐induced damage to protein subunits have been reported for the amino acids alanine , tyrosine , glycine [117, 118], proline [119, 120], cysteine , and serine [122,123], as well as small peptides, such as dialanine  and amino acid esters . For all cases, the anion yield functions (i.e., ion yields measured as a function of electron energy) exhibited localized maxima at energies below 15 eV, indicating the formation of TMAs. It has been established that no intact parent anion is observable on mass spectrometric timescales after capture of a free electron, and that the most probable reaction corresponds to the loss of a hydrogen atom from a carboxyl group to form for a molecule “M,” the dehydrogenated anion (M–H)‾ at energies of around 1.5 eV [120, 123, 126, 127]. Early DEA studies ascribed this process to initial electron attachment into a
In the condensed phase, analyzing LEE‐stimulated desorption of anions from physisorbed thin films of glycine, alanine, cysteine, tryptophan, histidine, and proline [128, 129] indicated that H‾ was the major desorption fragment, as CH3‾, O‾, and OH‾ were the fragments produced with lower signals in all named amino acids. Similar results were observed in ESD experiments from LEE‐bombarded chemisorbed films, prepared by self‐assembled monolayers (SAMs) of two different chains of Lys amide molecules . For this model of a segment of a peptide backbone, the desorbed signals were dependent on the length of the amino acid sequence.
Amino acids are also suitable model molecules for investigating the interactions of biomolecules with metallic surfaces, particularly silver and gold. Of the 20 naturally occurring amino acids, only cysteine contains a thiol (-SH) group, which allows it to bind to the metal by forming a S-Metal bond [130, 131]. This characteristic makes cysteine an ideal model to investigate protein interactions with gold surfaces including those of gold nanoparticles [132, 133]. A detailed study on electron attachment to L‐cysteine/Au(111) was recently reported by Alizadeh et al. [134, 135] who measured anion yields desorbed from chemisorbed (SAMs) and physisorbed thin films bombarded with sub‐20 eV energy electrons. These ESD measurements showed that LEEs are able to effciently decompose this amino acid via DEA and dipolar dissociation (DD), when the molecule is chemisorbed via the SH group to a gold surface.
Regarding the protective effect of amino acids on DNA against LEEs, Solomun et al.  reported that the single‐strand DNA‐binding
Furthermore, by studying via first‐principles molecular dynamics simulations a model system composed of thymine and glycine, Kohanoff et al.  recently investigated the protection of DNA by amino acids against the effects of LEEs. They considered thymine–glycine dimers and a condensed‐phase model consisting of one thymine molecule solvated in amorphous glycine. These results indicated that at room temperature, the amino acid chemically and physically performs the role of a protective agent for the nucleobase. In a chemical mechanism, the excess electron is first captured by the thymine; then, a proton is transferred in a barrierless way from a neighboring hydrogen‐bonded glycine. Reducing the net partial charge on the thymine molecule stabilizes the excess electron. In the physical mechanism, glycine molecule acts as an electron scavenger to capture the excess electron directly, which prevents the electron to be localized in DNA. Protecting the nucleobase via the latter mechanism requires a predisposition for proton transfer to the oxygen in the carboxylic acid group of one of the involved amino acids. Consequently, raising the free‐energy barrier associated with strand breaks, prompted by these mechanisms, can halt further reactions of the excess electron within the strand of DNA, for instance, transferring the electron to the backbone which leads to induce a strand break in DNA. Increasing the ratio of amino acid to nucleic acid will enhance the protecting role of amino acids, and accordingly will decrease the induction of DNA strand breaks by LEEs, as shown experimentally [137, 139].
7. LEEs interaction and induced damage under cellular conditions
The gas‐ and condensed‐phase experiments with DNA and its constituents discussed previously were performed under ultrahigh vacuum (UHV) conditions to permit use of electron beams and mass spectrometry, and to better control the molecular environment. While such experiments provide information on the direct effects of LEEs, they do not reveal how LEEs can indirectly damage DNA. Comparatively, due to the experimental difficulties related to the production and observation of LEEs in aqueous media, studies on the indirect damage of LEEs to DNA have not been greatly developed.
Ideally, to understand how the fundamental mechanisms in LEE–DNA interactions are adapted in living cells, the experimental studies should be extended to the more complex dynamic molecular environment of the cell, or more realistic ones, for the DNA molecule that contains essentially water, oxygen, histones, and DNA‐binding proteins . For instance, in the work of Ptasińska and Sanche , the ESD yields of different anions desorbed by 3–20 eV electron impact on GCAT films were measured under an aqueous condition, corresponding to 5.25 molecules of water per nucleotide. Their experiments demonstrated that adding water to dry DNA results in the binding of the molecule to the phosphate group at the negatively charged oxygen , and then formation of a complex of tetramer and a water molecule (DNA•H2O). This complex permits the formation of a new type of dissociative core‐excited TMA located on the phosphate group, which decays by O‾ desorption under electron impact via a resonance at 11–12 eV and by OH‾ desorption from breaking the P–O bond. H‾ also desorbs by dissociation of a TMA of the complex which causes bond cleavage on the H2O portion. Moreover, LEE‐induced damage to DNA via DEA enhances by a factor of about 1.6 when an amount of water corresponding to 60% of the first hydration layer is added to vacuum‐dried DNA. Although the magnitude of this enhancement is considerable, it is still much smaller than the modification in yields of products produced by the first hydration layer surrounding the DNA during the radiochemical events that follow the deposition of the energy of LEE in irradiated cells. Theoretical and experimental studies were concurrently carried out on the diffraction of 5–30 eV electrons in hydrated B‐DNA 5'‐CCGGCGCCGG‐3’ and A‐DNA 5'‐CGCGAATTCGCG‐3’ sequences by Orlando et al. . They postulated that compound H2O•DNA states may contribute to the modification of strand breaks yield functions [142, 143]. Furthermore, Orlando et al. noted that lowering of the threshold energy for DSBs below 5 eV may be correlated with the presence of these compound states. In this case, an initial “core‐excited” resonance would autoionize, yielding electronically excited water‐derived states and a low‐energy electron. The electronically excited state dissociates forming reactive O, OH, and H, which can lead to sugar–phosphate bond breakage. The slow electron could moreover scatter inelastically within a limited mean free path and excite a “shape” resonance of a base on the opposite strand. The combination of these two energy‐loss channels could lead to a DSB. This type of DSB requires the presence of water and is difficult to be repaired due to the close proximity of damage sites.
Recent work using graphene‐coated gold thin films also signaled the significance of the existence of water molecules in DNA damage mediated by “shape” resonances . This is likely due to the influence of water on lowering the barrier for charge transfer from the base to the sugar–phosphate bond. In addition, the binding interaction of DNA with graphene allows direct coupling to the phosphates as well as more direct scattering with the guanine and adenine bases. Electrons that have not been captured by DNA bases can be captured by graphene and immediately transferred over 200 nm within < 0.36 ps. The environmental or graphene substrate interactions are critical, and at least two mechanisms occur simultaneously during DNA damage on monolayer graphene: direct base capture and ballistic transfer from the graphene.
An alternative approach to simulate cellular conditions has been recently developed by Alizadeh et al.  to investigate LEE‐induced DNA damage under atmospheric conditions and at various levels of humidity and oxygen. Thin films of plasmid DNA deposited on tantalum and glass substrates were exposed to Al Kα X‐rays of 1.5 keV. The general features of the photo‐ejected SE from the metallic surfaces exposed by primary X‐ray photons are well understood; in particular, more than 96% of SEs emitted from tantalum lie below 30 eV and the energy distribution peaks around 1.4 eV, with an average energy of 5.85 eV . Whereas the damages induced in DNA deposited on glass are due to soft X‐rays, those arising from DNA deposited on tantalum result from the interaction of X‐rays + LEEs. The difference in the damage yields measured in the samples deposited on two different substrates is ascribed to the interaction of LEEs with the DNA and its nearby atmosphere.
Alizadeh and Sanche  employed this technique to examine how the presence of several cellular components (such as, O2, H2O and O2/H2O) modulates the LEE‐induced damage to DNA molecules. They observed that for hydrated DNA films in an oxygenated environment, the additional LEE‐induced damage that results from the combination of water and oxygen exhibits a super‐additive effect, which produces a yield of DSB almost seven times higher than that obtained by X‐ray photons. More recently, they reported the formation of four radiation‐induced products from thymidine by soft X‐rays and LEEs, specifically base release, and base modification including 5‐HMdUrd, 5‐FordUrd, and 5,6‐DHT . Of the products analyzed, thymine release was the dominant channel arising from N‐glycosidic bond cleavage involving
Recently, novel decay mechanisms for electronic excitations and correlated electron interactions have become subjects of intense study. Just over a decade ago, Cederbaum et al. [148–150] proposed an ultrafast relaxation process in inner valence levels, which occurs in molecular systems with weakly bound forces, such as van der Waals forces or hydrogen bonding. This mechanism referred to as intermolecular Coulomb decay (ICD) is possible mainly due to the couplings and interactions induced by the local environment. Unlike most ionization processes, ICD results in the ejection of an electron from the neighbor of an initially ionized atom, molecule, or cluster . The energy of the ICD electron is low, typically less than 10 eV. ICD is expected to be a universal phenomenon in weakly bound aggregates that contain light atoms and may represent a hitherto unappreciated source of LEEs. Though most ICD measurements have concentrated on rare gas clusters, new sophisticated experimental approaches have detected ICD in large water clusters  or at condensed‐phase interfaces containing water dimers and clusters .
Random damage to cellular biomolecules such as DNA is associated with the onset of cancer, whereas the controlled targeted local release and interactions of LEEs can be used as effective therapeutic cancer treatment agents. Since ICD is a source for the ejection of slow electrons, it has been proposed that ICD could play a role in the induction of SSB and DSB in DNA . Estimation by Grieves and Orlando  indicated that ICD may represent up to 50% of the SSB probability for energy depositions >20 eV and ionization events directly at the DNA–water interface. Since the formation of DSBs requires excitation energies >5 eV, the impact on DSBs is expected to be much lower. If ICD contributes significantly to DNA damage, this could be exploited during X‐ray treatment of cancer. Figure 4 schematically shows that how utilizing of X‐ray interactions with gold nanoclusters within living cells, which subsequently results in releasing both Auger and ICD electrons, has been suggested as a potential strategy for targeted cancer treatment .
After such extensive studies on LEE‐induced damage under “near”‐cellular conditions, it was only very recently that the lethal effects of LEEs in cells have been demonstrated by Sahbani et al. , who investigated the biological functionality of DNA, via a simple model system comprising
8. Role of LEEs in radiosensitization and radiation therapy
A major impetus for achieving a better understanding of the action of ionizing radiation in biological systems relates to applications in radioprotection and radiotherapy. Since LEEs play a major role in transferring the energy of the initial high‐energy particle or photon to initiate all subsequent chemistry in irradiated media, understanding their interaction with biomolecules is now being recognized as a crucial and essential step toward such applications. As seen from Section 4, in the last decades, our expanding knowledge of LEE–DNA interactions has been applied to experiments involving known radiosensitizers. Both at the theoretical and experimental levels, this work served to suggest new compounds having radiation‐damage enhancing properties and to explain the details of their response to high‐energy radiation either alone or when bound to DNA (i.e., the main target for cell killing in radiotherapy).
8.1. Transient anions in halogen compounds
Bromouracil, which can replace thymine in DNA during cell replication, and bromouridine were the first radiosensitizing candidates to be investigated theoretically and experimentally with LEEs. The studies [157–167] confirmed the prediction of Zimbrick and coworkers  that the radiosensitizing properties of these compounds arose from DEA of solvated electrons, and further showed that DEA of higher energy (0–7eV) electrons was also involved in radiosensitization. Platinum bromide, aromatic compounds containing nitro group and other halogenated thymidine derivatives were found to play similar roles [58, 70, 71, 163–165, 169, 170]. Following early investigations with solvated electrons , a relatively large number of experiments have been performed both in the gas (see Section 4.4) and condensed phases [160, 161, 165] to study electron scattering from––and attachment to––halogenated pyrimidines. Several experiments were performed using SAMs of BrdU‐containing oligonucleotides [157, 158, 171]. These included the detection of the electron‐stimulated desorption of ion and neutral species and HPLC analysis of damaged films, as well as electronic and vibrational electron‐energy loss spectra for gaseous bromouracil . These studies revealed that the radiosensitization properties of halogen compounds are more complicated than previously anticipated . Within the 0–7 eV energy range, resonant electron scattering mechanisms with halouracils lead to more complex molecular fragmentation than that occurs with thymine, which produces a different range of anionic and neutral radical fragments. When formed within DNA, such fragments could react with local subunits, and thus lead to lethal clustered damage, further to that already occurring in unsensitized DNA. The most striking evidence of a huge enhancement of LEE damage obtained upon Br substitution in thymine is seen in the early results of Klyachko et al. , who found that, in the presence of water, DEA to bromouracil could be enhanced by orders of magnitude compared to the dry compound. Differences between wet and dry TMA states of halogenated pyrimidines have recently been investigated by Cheng et al. . They applied Koopman's theorem in the framework of long‐range corrected density functional theory for calculation of the TMA states and self‐consistent reaction field methods in a polarized continuum to account for the solvent. Their results indicate that the TMAs of these molecules are more stable in water, but to differing degrees.
The radiosensitization properties of halouracils depend not only on hydrated electrons, but also on LEEs and on DEA. However, the high propensity of LEEs of very low energies (i.e., <1 eV) to fragment bromouracil and deoxybromouridine (BrUdR) may, according to the theory, exist only in single‐stranded DNA . This important prediction was confirmed by Cecchini et al.  for the case of solvated electrons and was commented upon by Sevilla . Solutions of single‐ and double‐stranded oligonucleotides, and of double‐stranded oligos containing mismatched bubble regions, were irradiated with
8.2. Transient anions in DNA bound to platinum chemotherapeutic agents
Considering that it can often take years, if not decades, before potential new radiosensitizers arrive in the clinic, Zheng et al.  hypothesized that present clinical protocols involving high‐energy radiation and platinum (Pt) chemotherapeutic agents could be improved by considering the fundamental principles of energy disposition, including the results of LEE experiments. Their initial goal was to explain the superadditive effect occurring in tumor treatments, when cisplatin and radiation were administered in concomitance [176, 177]. Zheng et al.  found that, with cisplatin bound to DNA as in the cancer cells, damage to the molecule increases by factors varying from 1.3 for high‐energy electrons to 4.4 at 10 eV. Considering the much higher enhancement factor (EFs) at 10 eV, the increase in bond dissociation was interpreted as being triggered by an increase in DNA damage induced by LEEs.
In their experiments, Zheng et al.  deposited lyophilized films of pure plasmid and plasmid–cisplatin complexes on a clean tantalum foil. The films were bombarded under UHV with electrons of 1–60 keV. Under these conditions, 90% of the plasmid–cisplatin complexes consisted of a cisplatin molecule chemically bound to DNA, preferentially at the N7 atom of two guanines producing an interstrand adduct. The films had the necessary thickness to absorb most of the energy of the electrons. The different forms of DNA corresponding to SSBs and DSBs were separated by gel electrophoresis, and the percentage of each form quantified by fluorescence. Exposure response curves were obtained for several incident electron energies for cisplatin bound or not to plasmid DNA. Table 1 gives the results for exposure to 1, 10, 100, and 60,000 eV electrons of films of pure DNA and cisplatin/plasmid complexes with a ratio (
This type of radiosensitization was investigated in more detail by irradiating with a
|Form of damage||SSB||DSB|
|5 ML||2900 nm||5 ML||2900 nm|
|27± 3||33 ± 3||57 ± 5.5||1.2 ±0.1||ND||10 ± 1||13 ± 2||0.4 ± 0.2|
|38 ± 3||120 ± 11||150 ± 15||2.4 ±0.3||5 ±1||17 ± 1||36 ± 4||0.5 ± 0.2|
|52 ± 5||143 ± 14||199 ± 18||3.0 ±0.4||5 ±2||29 ± 2||44 ± 4||0.7 ± 0.1|
These results suggest two major pathways by which hydrated electrons interact destructively with TTTTTGTGTTT–cisplatin [181, 182]. First, the hydrated electron is captured initially on a thymine base and is transferred to the guanine site by base to base electron hopping, where DEA detaches the cisplatin moiety from the oligonucleotide. Alternatively, the hydrated electron interacts directly with the platinum–guanine adduct, and cisplatin is detached via DEA. These hypotheses were consistent with those proposed by Rezaee et al.  for LEE‐induced damage to plasmid DNA. Additionally, Rezaee et al. suggested that in the double‐stranded configuration, the cisplatin molecule weakens many of the DNA chemical bonds and changes the topology of the molecule; these modifications render DNA much more sensitive to damage over large distances . Of course, under high‐energy irradiation conditions, the increase in ionization cross section, due to the presence of the Pt atom, also increases the quantity of LEEs near cisplatin and therefore may indirectly contribute to the increase in damage.
More recently, the energy dependence of conformational damage induced to pure plasmid DNA  and cisplatin–plasmid DNA complexes  was investigated in the range 2–20 eV. In addition to the strong resonances (i.e., TMAs) in pure DNA around 5 and 10 eV, further TMA specific to cisplatin‐modified DNA were observed in the yield function of SSBs at 13.6 and 17.6 eV. Moreover, the presence of cisplatin lowered the threshold energy for the formation of DSBs to 1.6 eV, considerably below that observed with electrons in pure DNA films. In all cases, the measured yields were larger than those measured with nonmodified DNA. To reconcile all existing results starting from those obtained with hydrated electrons to those generated up to 20 eV, Bao et al.  suggested a single mechanism that could apply to shape and core‐excited resonances, depending or not if electronic excitation of the Pt or guanines was involved in TMA formation. This mechanism, previously proposed for shape resonances by Rezaee et al. , can be explained with reference to Figure 6. When the TMA is formed on the Pt adduct, the extra electron is delocalized and occupies simultaneously, with identical wave functions, the two bonds linking the Pt atom to guanine bases on opposite strands. Occupancy of the dissociative
The LEE enhancement mechanism of damage in DNA–Pt drug complexes acts on a femtosecond timescale, which quite unlike other biological mechanisms of radiosensitization, act over macroscopic times that can range from hours to days. These considerations imply that the mechanism (e.g., physicochemical vs biological) of radiosensitization by Pt agents in concomitant chemoradiation therapy may be sensitive to the timing between the injection of the drug to the patient and the irradiation. Thus, if TMA formation in DNA plays a major role in radiosentization by Pt drugs, maximal cancer cell killing should be achieved, if these cells are irradiated when the maximum amount of Pt is bound to their nuclear DNA.
Led by this hypothesis, Tippayamontri et al. [185, 186] determined the optimal conditions for concomitant chemoradiation treatment of colorectal cancer with cisplatin, oxaliplatin, and their liposomal formulations Lipoplatin and Lipoxal [187, 188]. Using an animal model of human colorectal cancer, they determined the time window for maximum radiosensitization and synergy with irradiation, by studying the pharmacokinetics and time‐dependent intracellular distribution of the Pt drugs. This, in turn, is determined by the reaction kinetics of the drug with DNA and the DNA repair kinetics.
In nude mice bearing HCT116 colorectal carcinoma, treated with the Pt drugs, they measured by inductively coupled plasma mass spectrometry, the platinum accumulation in blood, serum, different normal tissues, tumor, and different tumor cell compartments, including the amount of Pt bound to nuclear DNA [185, 186] Figure 7a indicates the positions of binding of cispelatin to DNA. Examples of the amount of cisplatin and Lipoplatin binding to the DNA of HCT116 colorectal cancer cells in mice are shown in Figure 7b as a function of time after injection of the drug. Radiation treatment (15 Gy) was given 4, 24, and 48 h after drug administration. The resulting tumor growth delay was reported and correlated with apoptosis analyses. Optimal survival of the mice and highest apoptosis were observed when radiation was given at 4 or 48 h after drug injection. These times corresponded to the times of maximal platinum binding to tumor DNA, as shown in Figure 7b for cisplatin and Lipoplatin. When tumor irradiation was performed at 48 h, the ratio of tumor growth delay for the group having the combined treatment compared to delay for the group treated with chemotherapy alone varied from 4.09 to 13.00, depending on the drug. The most efficient combination treatment was observed when the amount of Pt drug binding to DNA was highest, as predicted from fundamental considerations [178–182]. Such results testify our fundamental understanding of the mechanisms of platinum‐induced radiosensitization and should have significant impact on the design of more efficient treatment protocols.
8.3. Interaction of LEEs with DNA bound to gold nanoparticles
So far in this section, we have shown that cancer cells can be made more sensitive to high‐energy radiation by chemically modifying their nuclear DNA with small molecules. These latter provide at least some of their radiosensitizing action, by increasing the interaction of LEEs with DNA, the products of DEA, and the resulting induced damage. Another approach consists of simply increasing the numbers of LEEs near the DNA of cancer cells. The best examples of this type of radiosensitization have been provided by the numerous fundamental, in vitro, and in vivo investigations of enhanced radiation absorption by gold nanoparticles (GNPs).
Both in vitro and in vivo experiments [189–204] have shown radiation enhancement effects due to the presence of GNPs. Several models have been developed to account for dose enhancement in cells by considering the increase in radiation energy deposition [205–211], due to additional energy absorption by the GNPs, as a function of their size. As expected, the energy of electrons emanating from the GNPs is inversely proportional to their diameter. Many models [206–211] take into account localized effects of Auger‐electron cascades. They consider the huge enhancement of energy deposited in the vicinity of GNPs, as arising from the considerable increase in photoelectric absorption cross section of gold in comparison to that of tissue [200, 208, 210, 211]. The increase in this cross section produces an additional local generation of photoelectrons, Auger electrons, and characteristic X‐rays [208, 212]. The major portion of the energy absorbed by the GNPs is converted into electrons, most of which escapes the GNPs with low energy (0–30 eV) [213–215].
The indirect effect of emitted electrons was investigated in water solutions containing GNPs, where the nanoparticle‐induced OH concentration from radiolysis was measured. Relevant literature and details can be found in the paper of Sicard‐Roselli et al. , who also proposed a new mechanism for hydroxyl radical production in irradiated GNP solutions.
The direct effect of high‐energy radiation on DNA, resulting from the presence of GNPs, was first investigated by Zheng and coworkers [35, 214–218]. Relatively thick (~0.3 and 2.9 μm) films of plasmid DNA with or without electrostatically bound GNPs were bombarded with 60 keV electrons. The probabilities of formation of SSBs and DSBs from the exposure of 1:1 and 2:1 GNP–plasmid mixtures to fast electrons increased by a factor of about 2.5, compared to DNA alone. It was suggested that the additional damage in the presence of GNPs was generated by LEEs escaping the nanoparticles. This hypothesis was later verified experimentally by the work of Xiao et al. . These authors investigated the radiosensitization efficiency in terms of DNA damage as a function of the length of a ligand bound at one end to the surface of the GNP and at the other to DNA. They used the same DNA film preparation as in the experiments of Zheng et al.  and measured the ratio of induced damage with GNPs to that without GNPs (i.e., the enhancement factor, EF) for different lengths of the ligand. As indicated in Figure 8 from their work, the corresponding EFs induced by 60 keV electrons on plasmid DNA bound to GNPs of various coatings range from 2.3 to 1.6 and 1.2, depending on the length of ligand separating the gold surface from the plasmid. This length ranged from 0 to 2.5 and 4 nm, respectively. The attenuation by the coating of short‐range LEEs emitted from the GNPs could explain the decrease in radiosensitization with increasing length of the ligand . Since the attenuation range of LEEs is shorter than about 10 nm, it is obvious that the emission of LEEs from the GNPs and LEE‐interaction with DNA plays a major role in the mechanism of GNP radiosensitization.
Later, similar DNA–GNP films were bombarded with electrons of energies below the ionization potential of DNA. In this case, essentially no secondary LEEs were emitted from the DNA and the gold surface, so that Yao et al.  could investigate the purely chemical radiosensitization induced by GNPs. They showed that even without the emission of photoelectrons, direct electrostatic binding of an average of 0.2–2 GNPs to DNA could increase sensitization to LEEs by factors varying from 1.5 to 4.
Since GNPs increase the local density of LEEs and cisplatin enhances LEE interactions with DNA and damage to the molecule, it seemed likely that binding GNPs to a cisplatin–DNA complex would further boost radiosensitization and DNA damage induced by cisplatin . This hypothesis was verified by irradiating with 60 keV electrons, GNPs electrostatically bound to a cisplatin–DNA complex . Dry films of bare plasmid DNA and DNA–cisplatin, DNA–GNP, and DNA–cisplatin–GNP complexes were irradiated . The yields of SSBs and DSBs were measured as described in the protocol established by Zheng et al. . When the ratio of GNP to DNA was 1:1 and that for cisplatin to DNA was 2:1, the EFs for SSBs were between 2 and 2.5. With a cisplatin to GNP to plasmid ratio of 2:1:1, the EF increased to 3. This small increase could only be additive and unrelated to the interaction of additional LEEs with cisplatin. For DSB formation, however, the binding of both GNPs and cisplatin to a DNA molecule produced an impressive increase in the EF, that is, DSBs were increased by a factor of 7.5 with respect to pure DNA. It appeared quite obvious that the additional DSBs in the cisplatin–DNA–GNP complex arose from the generation of additional secondary electrons from the GNPs. The synergy between GNPs and cisplatin could arise from a number of basic phenomena, including the possibility of two or multiple event processes triggered by the interaction of a single 60 keV electron with a GNP. Within 10 nm of its location, a single gold atom increases the density of LEEs by a large factor [207, 212], and hence, a GNP that contains thousands of gold atoms is expected to generate a dramatic increase in this density . Combined with the fact that cisplatin considerably lowers the energy threshold for DSB formation, a single or multiple LEE interactions on opposite strands within a distance of 10 base pairs could increase considerably the number of DSBs formed in GNP–cisplatin–DNA complexes.
As shown by Zheng et al., only one GNP per DNA molecule is on average necessary to increase DNA damage considerably . Thus, as long as the nanoparticles reach the DNA of cancer cells, the amount to be administered to patients to obtain significant radiosensitization should be at most the same as that of the Pt‐drugs routinely administered in chemotherapy [176, 177]. In recent in vitro experiments, GNPs were targeted to the DNA in the cell nucleus by linking peptides to the gold surface [197, 202]. Such vectored GNPs, targeting the DNA of cancer cells, should be applicable in the clinic and may accordingly offer a new approach to radiotherapy treatments. However, this type of radiotherapy is expected to be limited to superficial tumors, owing to the requirement for that low‐energy (<100 keV) X‐rays be used to optimize LEE production and hence radiosensitization by the photoelectric effect. To treat deep tumors, a radioactive source may have to be encapsulated inside a gold nanoparticle (i.e., in a gold nanocage) . Furthermore, if DNA specificity cannot be achieved in patients, successful treatment may still be possible by intratumor injection of GNPs, as recently shown by Shi et al.  and Bobyk et al. .
9. Summary and Conclusions
The experimental and theoretical results of LEE impact on single‐ and double‐stranded DNA, its basic constituents, protein subunits, as well as radiosensitizers and chemotherapeutic agents alone or bound to DNA were reviewed. Experimental details of LEE interactions with these biomolecules were obtained in both the condensed and gas phases. The condensed‐phase experiments were conducted in UHV and at atmospheric pressure under environments closer to those of the cell. From these studies, which provide a fundamental comprehension of the role of TMAs in irradiated biological systems, we can arrive with considerable certainty at the following conclusions on LEE‐induced damage to biomolecules. In the low‐energy range (i.e., below the threshold for dipolar dissociation (~15 eV)), bond rupture in biomolecules occurs essentially via the formation of TMAs that decay either via autoionization with the accompanied production of dissociative electronically excited states, or into the DEA channel. The induced damage depends on a large number of factors, including electron energy, the environment and topology of the molecule, and the electrostatic or chemical binding of small radiosensitizing molecules. Such factors inevitably modify the lifetime and decay channels of transient anions, which often increase the damage cross sections.
Since secondary electrons of low energy possess a large portion of the energy deposited by high‐energy radiation, any modification of how their energy deposits at crucial cellular sites is expected to have a strong radioprotective or radiosensitizing action. With DNA being the main target in radiotherapy, parameters that affect LEE‐induced DNA damage are necessarily of relevance to radiosensitivity, and the mechanisms involved must be well understood to control and modulate the biological effects of ionizing radiation.
Many of these mechanisms are now well established as seen from the experiments and theoretical treatments reviewed in this chapter. Moreover, it has been shown that applying fundamental principles of action of LEEs to radiosensitizers or chemotherapeutic agents can lead to new strategies on how to improve radiotherapy outcomes. In particular, the role of LEEs in radiation damage was related to enhancement of the destruction of cancer cells by Pt‐drugs and gold nanoparticles. LEEs were found to play an important role in providing guidelines in chemoradiation cancer treatment, as well as in the development of more efficient clinical protocols. Such applications point out the need for multidisciplinary studies in this field, where LEE–biomolecule interactions have become an area of intensive investigation that encompasses many aspects of cancer therapy.
The authors gratefully express thanks for helpful comments of Dr. Andrew D. Bass and Dr. Darel J. Hunting. LS acknowledges the financial support from Canadian Institute of Health Research (CIHR, via MOP 81356 and 86676) and from Natural Sciences and Engineering Research Council of Canada (NSERC). S.P. acknowledges the financial support from the U.S. Department of Energy Office of Science, Office of Basic Energy Sciences under award number DE‐FC02‐04ER15533.