Julien Claudot

Estelle Soubeyrand-Lenoir

Guillaume Maurin

Competitive adsorption of water and chemical warfare agents on transition metal embedded graphene

Keywords: Metal impregnated carbons, Chemical Warfare Agents, Water, Competitive Adsorption, Density functional theory

Adsorption of three CWA molecules (soman, sarin and mustard gas) was systematically explored on transition metal (Chromium and Vanadium) embedded graphene models under dry and wet conditions. Density Functional Theory calculations first revealed that both metal@graphene substrates show a very high affinity for all CWAs under dry conditions, sarin being expected to be even more strongly coordinated since its associated adsorption energies above 43 kcal.mol -1 are higher than the values calculated for soman and mustard gas. Two different wet scenarios were further explored with the consideration of a prehumidification and a simultaneous competitive adsorption of water with CWAs. These calculations evidenced that the presence of water decreases the CWA adsorption energy for sarin and soman in the two embedded graphene models by maximum 20%. Interestingly, we demonstrated that this decrease is much less pronounced (3.3%) in the case of gas mustard adsorbed on chromium embedded graphene. Such metal embedded carbon substrates are thus predicted to maintain globally high level of CWA capture performances under humidity which corresponds to the most common operating conditions of protective gas masks. An Indepth analysis of the interactions between the CWAs and the metal embedded graphene in the presence of water was further conducted to gain insight into the origin of this energetic trend at the electronic-level.

Introduction

Chemical warfare agents (CWAs) are a major threat to human health owing to their potential usage as chemical weapons by terrorist groups. Sulfur mustard also known as mustard gas and nerve agents including sarin and soman are the most typical CWAs [START_REF] Chauhan | Chemical warfare agents[END_REF]. Sulfur mustard (C4H8Cl2S), discovered accidentally in 1822 [START_REF] Johnson | Historical perspective of chemical warfare agents[END_REF], is an alkylating agent capable of causing short and long term morbidity and was used during World War I [START_REF] Mangerich | Chemical warfare in the First World War: reflections 100 years later[END_REF]. The nerve agents are organophosphorus compounds containing P-X bonds (with X = Fluor in the case of sarin -C4H10FO2P and soman -C7H16FO2P) discovered by Gerhard Schrader in 1937 [START_REF] Nelson | Organophosphorus compounds; the past and the future[END_REF] and they are among the deadliest CWAs to date. These nerve agents were used during World War II [START_REF] Karalliedde | Organophosphorus insecticide poisoning[END_REF], as well as over the first Persian Gulf War [START_REF] Barnaby | Iran-Iraq War: the use of chemical weapons against the Kurds[END_REF][START_REF] Dingeman | Chemical warfare in the Iran-Iraq conflict[END_REF] and more recently in 1995 with the terrorist attacks in Tokyo [START_REF] Yokoyama | Clinical profiles of patients with sarin poisoning after the Tokyo subway attack[END_REF][START_REF] Okumura | Report on 640 victims of the Tokyo subway sarin attack[END_REF]. Despite the international ban on their uses [START_REF] Mangerich | Chemical warfare in the First World War: reflections 100 years later[END_REF], some of these CWAs are still considered as chemical weapons in modern warfare. Consequently, their detection and capture are still a vital concern. As a result, the use of porous materials, such as activated carbons owing to their large surface area and high chemical stability, has been envisaged as one of the most efficient strategies to adsorb CWAs [START_REF] Kim | Adsorption-desorption characteristics of VOCs over impregnated activated carbons[END_REF][START_REF] Kaplan | Degradation of adsorbed sarin on activated carbons: a 31P-MAS-NMR study[END_REF][START_REF] Wang | Volatile organic compounds in indoor environment and photocatalytic oxidation: state of the art[END_REF][START_REF] Dai | Gas adsorption on graphene doped with B, N, Al, and S: a theoretical study[END_REF][START_REF] Junkaew | Metal cluster-deposited graphene as an adsorptive material for m-xylene[END_REF][START_REF] Wang | The physical and surface chemical characteristics of activated carbons and the adsorption of methylene blue from wastewater[END_REF]. However, the standard activated carbons suffer from a relatively poor affinity for polar molecules. This encouraged the community to take advantage of defects present in the carbon-based structures to impregnate them with transition metal atom [START_REF] Pašti | Atomic adsorption on graphene with a single vacancy: systematic DFT study through the periodic table of elements[END_REF][START_REF] Gupta | Computational predictions of electronic properties of graphene with defects, adsorbed transition metal-oxides and water using density functional theory[END_REF][START_REF] Hu | Density functional calculation of transition metal adatom adsorption on graphene[END_REF][START_REF] Jungsuttiwong | Density functional theory study of elemental mercury adsorption on boron doped graphene surface decorated by transition metals[END_REF][START_REF] Wang | Doping monolayer graphene with single atom substitutions[END_REF][START_REF] Santos | First-principles study of substitutional metal impurities in graphene: structural, electronic and magnetic properties[END_REF][START_REF] Lu | Simultaneous removal of VOC and NO by activated carbon impregnated with transition metal catalysts in combustion flue gas[END_REF] with the objective to boost their affinities to target CWAs. Although such a strategy allows the enhancement of the interactions between the adsorbent and the molecules we wish to capture [START_REF] Kunaseth | A DFT study of arsine adsorption on palladium doped graphene: effects of palladium cluster size[END_REF][START_REF] Krasheninnikov | Embedding transition-metal atoms in graphene: structure, bonding, and magnetism[END_REF][START_REF] Ma | Hydrogen adsorption and storage on palladiumdecorated graphene with boron dopants and vacancy defects: a first-principles study[END_REF][START_REF] Lu | Metal-embedded graphene: a possible catalyst with high activity[END_REF][START_REF] Ramos-Castillo | Palladium clusters supported on graphene monovacancies for hydrogen storage[END_REF], it might lead to competitive adsorption with other polar molecules present under specific operating conditions. A humidified environment is a major concern regarding the efficiency of activated carbons, typically in regards of diverse polar molecules including a series of volatile organic compounds [START_REF] Slasli | Water adsorption by activated carbons in relation to their microporous structure[END_REF][START_REF] Mccallum | A molecular model for adsorption of water on activated carbon: comparison of simulation and experiment[END_REF][START_REF] Huang | Effect of moisture on H2S adsorption by copper impregnated activated carbon[END_REF][START_REF] Jing | Effect of relative humidity on adsorption of formaldehyde on modified activated carbons[END_REF][START_REF] Sitthikhankaew | Effects of humidity, O2, and CO2 on H2S adsorption onto upgraded and KOH impregnated activated carbons[END_REF][START_REF] Chiang | Effect of relative humidity on adsorption breakthrough of CO2 on activated carbon fibers[END_REF][START_REF] Qi | Predicting humidity effect on adsorption capacity of activated carbon for water-immiscible organic vapors[END_REF][START_REF] Brennan | Water in porous carbons[END_REF]. While carbon-based materials are mostly hydrophobic [START_REF] Müller | Molecular simulation study of hydrophilic and hydrophobic behavior of activated carbon surfaces[END_REF] implying that water is not expected to interfere with the capture of the target molecules, the impregnation of carbons with transition metal atoms might lead to an enhancement of the hydrophilicity of the resulting materials [START_REF] Müller | Molecular simulation study of hydrophilic and hydrophobic behavior of activated carbon surfaces[END_REF][START_REF] Fletcher | Adsorption and desorption kinetics for hydrophilic and hydrophobic vapors on activated carbon[END_REF][START_REF] Ahnert | A study of the influence of hydrophobicity of activated carbon on the adsorption equilibrium of aromatics in non-aqueous media[END_REF][START_REF] Xiang | Carbon nanotubes and activated carbons supported catalysts for phenol in situ hydrogenation: Hydrophobic/hydrophilic effect[END_REF]. Hence, the hydrophilic character of the impregnated carbons is potentially an obstacle to their efficient use for CWA capture. In the field of respiratory protection devices for civilian and military purposes [START_REF] Sharma | Chemical Protection Studies of Activated Carbon Spheres based Permeable Protective Clothing Against Sulfur Mustard, a Chemical Warfare Agent[END_REF], aging of activated carbons cartridges [START_REF] Amitay-Rosen | The Effects of Aging on the Dynamic Adsorption of Hazardous Organic Vapors on Impregnated Activated Carbon[END_REF] or their uses under high relative humidity are two potential drawbacks that might alter the protection of the users. In this field, most of the theoretical studies on the adsorption performance of carbons in the presence of humidity have been limited to the application of macroscopic models to assess the lifetime of the corresponding cartridges [START_REF] Wood | Correlations for High Humidity Corrections of Rate Coefficients for Adsorption of Organic Vapors and Gases on Activated Carbons in Air-Purifying Respirator Cartridges[END_REF][START_REF] Wood | Affinity coefficients of the Polanyi/Dubinin adsorption isotherm equations: A review with compilations and correlations[END_REF][START_REF] Lodewyckx | The influence of humidity on the overall mass transfer coefficient of the Wheeler-Jonas equation[END_REF][START_REF] Hall | Effects of adsorbed water vapor on the adsorption rate constant and the kinetic adsorption capacity of the Wheeler kinetic model[END_REF][START_REF] Fletcher | Role of surface functional groups in the adsorption kinetics of water vapor on microporous activated carbons[END_REF]. While the microscopic exploration of CWA adsorption on metal oxides surfaces in the presence of water [START_REF] Quintero | Molecular and dissociative adsorption of DMMP, Sarin and Soman on dry and wet TiO2 (110) using density functional theory[END_REF][START_REF] Wagner | Effect of adsorbed water and surface hydroxyls on the hydrolysis of VX, GD, and HD on titania materials: the development of selfdecontaminating paints[END_REF] is well documented, this has never been achieved so far for any CWA/carbon systems. To address this lack of microscopic insight, here we propose a computational exploration of the competitive adsorption of CWA and water on metal impregnated carbons. To that purpose, periodic Density Functional Theory calculations (DFT) were performed to predict the impact of water on the interactions between three representative CWA molecules i.e., sarin, soman and mustard gas, and a graphene layer embedding one transition metal atom either Chromium (Cr) or Vanadium (V). Such a microscopic carbon model has been already successfully used to gain insight into the interactions between diverse toxic molecules and impregnated carbons [START_REF] Dong | First principles study of HCN adsorption on graphene doped with 5d transition metal[END_REF][START_REF] Kunaseth | A DFT study of volatile organic compounds adsorption on transition metal deposited graphene[END_REF][START_REF] Jaiswal | Sarin chemisorbent based on cobalt-doped graphene[END_REF]. Both pre-humidification and simultaneous competitive adsorption of water with CWAs were considered and the resulting preferential arrangements and associated energetics were assessed. As a further stage, a careful analysis of the electronic features of the corresponding H2O/CWA/carbon systems was conducted to gain insight into the microscopic origin of the corresponding competitive adsorption phenomena on the carbon-substrates.

Computational details

The metal-embedded graphene models labelled metal@graphene were first built by substituting one carbon atom of a 5 × 5 graphene sheet (12.30 Å × 12.30 Å) by a transition metal atom i.e. either Chromium or Vanadium. A length of 20 Å along the z-direction was considered to avoid interactions between their periodically replicated images. 1 molecule of each CWA, i.e., sarin, soman and mustard gas was further incorporated in these models using different starting configurations. DFT geometry optimizations were further performed using the PBE functional [START_REF] Perdew | Accurate and simple analytic representation of the electron-gas correlation energy[END_REF] and the DFT+D2 [START_REF] Grimme | Semiempirical GGA-type density functional constructed with a long-range dispersion correction[END_REF] dispersion correction as implemented in the Quantum Espresso package [START_REF] Giannozzi | QUANTUM ESPRESSO: a modular and open-source software project for quantum simulations of materials[END_REF]. The D2 dispersion correction was demonstrated to accurately describe the interactions between polar molecules and impregnated carbons [START_REF] Jogender | Adsorption of methyl isocyanate on M-4 (M= Fe, Ni, and Cu) cluster-decorated graphene and vacancy graphene: a DFT-D2 study[END_REF]. Ultrasoft Vanderbilt pseudopotentials [START_REF] Vanderbilt | Soft self-consistent pseudopotentials in a generalized eigenvalue formalism[END_REF] were used to describe ion cores of atom. An energy cut-off of 80 Ry was applied in combination with a convergence criteria on the energy of 1 × 10 Ry. A Gaussian smearing with = 0.01 was employed for the two transition metals. Monkhorst-Pack grids of k-points replicating the graphene sheet dimensions was considered.

To further explore the impact of water, two scenarios were considered mimicking either a pre-humidification condition or a simultaneous H2O/CWA adsorption. In the first case, one water molecule was initially incorporated in the metal-embedded graphene models and then DFT-geometry optimized prior to introduce one CWA molecule. In the second scenario, both water and CWA molecules were simultaneously adsorbed and their geometries were further DFT-geometry optimized. In all these calculations, several starting configurations were considered corresponding to various positions of water and different orientations of the CWA molecules, i.e, parallel and perpendicular to the graphene sheet as well as with either F or O and S or Cl atoms pointing towards the metal sites for sarin/soman and mustard gas respectively, to ensure convergence towards minimum energy states.

The adsorption energies for the 3 CWAs were further calculated as follows:

(i) In the absence of water was obtained using equation 1:

= / @ -( + @ ) (1)
where

/ @
is the energy of the DFT-optimized molecule/metal@graphene complex while $ and @ correspond to the single point energy of the single constituents, i.e. CWA and metal/graphene respectively.

(ii) in the presence of water, % & ' was obtained using equation 2:

% & ' = / % & ' @ -( + % & ' @ ) (2)
where

/% & ' @
is the energy of the DFT-optimized molecule/H2Ometal@graphene complex while $ and % & ' @ are the single point energies of the single constituents, i.e. CWA and H2O -metal@graphene complex respectively. Such a definition of the adsorption energy for CWA allows the evaluation of the role of water on the strength of the CWA/substrate interactions.

Results and Discussion

Fig. 1 reports the DFT optimized structures for soman and mustard gas/metal@graphene models. We can observe in all cases an out-of-plane shift of the metal sites upon adsorption that become more exposed to CWAs (see Tables S1-S2). Soman was found to interact preferentially with the metal center via its oxygen atom in a similar way than sarin [START_REF] Claudot | Computational exploration of Sarin and simulants adsorption on a series of transition metal embedded graphene[END_REF]. Soman shows an equivalent adsorption behavior in the case of V@graphene (Fig. 1 (a)) and Cr@graphene (Fig. 1 (b)), with associated O(soman)-Metal interacting distance that only slightly varies from 2.06 to 2.00 Å respectively. The scenario differs for mustard gas which implies a preferential interaction between its central sulfur atom and the metal sites (Fig. 1 (c)) and Fig. 1 (d)). This observation was confirmed by considering different initial configurations where the chlorine atom of mustard gas was positioned in the vicinity of the metal sites (see Tab. S3). These calculations evidenced that the interacting S(mustard gas)-Metal distance is shorter for Chromium (2.34 Å) (Fig. 1 (d)) as compared to Vanadium (2.50Å) (Fig. 1 (c)).

The adsorption energies associated with the most stable DFT-optimized structures (Fig. 1) are reported in Tab. 1, while the corresponding data previously obtained for sarin are reminded for comparison [START_REF] Claudot | Computational exploration of Sarin and simulants adsorption on a series of transition metal embedded graphene[END_REF]. One observes that the adsorption energies for soman and mustard gas are similar for both V@graphene and Cr@graphene, this behavior confirming our previous finding that V and Cr enable very similar strength of interactions for a given CWA molecule [START_REF] Claudot | Computational exploration of Sarin and simulants adsorption on a series of transition metal embedded graphene[END_REF]. Furthermore, the adsorption energy sequence is as follows:

(sarin) > (mustard gas) > (soman), the corresponding values varying in the range of 39.71 -43.84 kcal.mol -1 , and 38.48 -43.00 kcal.mol -1 for Cr@graphene and V@graphene respectively. This trend highlights that both metal-impregnated carbons show a slightly higher affinity for sarin vs the other two CWAs. Fig. 2 reports the projected density of states (PDOS) for the soman/Cr@graphene and mustard gas/Cr@graphene complexes under dry conditions. Same analysis for the Vanalogues can be found in Fig. S1. The total density of states is projected in such a way to represent the contributions of the orbitals of each interacting atoms, i.e. p-orbitals of P, F and O atoms for soman, p-orbitals of S and Cl atoms for mustard gas, s-and p-orbitals of C atoms for graphene as well as s-, p-and d-orbitals of the metal atom. One first notes the presence of a peak at the Fermi level which corresponds to a contribution of the p-orbitals of the carbon atom of the graphene sheet and the d-orbitals of Cr atom. This results from a hybridization phenomenon due to a shift in position of the metal out of the graphene plane. More specifically, in the case of soman/Cr@graphene complex, one can note that the peak corresponding to p-orbitals of the O atoms of soman is shifted of around -1eV, towards lowest energy compared to that of the isolated molecule. The peaks corresponding to porbitals of S and Cl of mustard gas adsorbed on Cr@graphene are shifted to a lesser extend towards lowest energy compared to the isolated state, leading to a small overlap of these orbitals of CWA with d-orbitals of Cr atom. Nonetheless, same trend is observed for both complexes at high energy. In particular, the peak situated initially around 1 eV, corresponding to d-orbitals of Cr atom, is shifted at higher energy when both CWAs are adsorbed. This evidences that, even by considering CWAs of different chemical nature, specifically organophosphorus and sulfur-based compounds, a similar hybridization is observed between the d-orbitals of chromium atom and the orbitals of the adsorbed molecules.

In order to further explore the electronic features of the CWA/Cr@graphene complexes, Fig. 3 presents their charge density differences computed using a Bader charge analysis [START_REF] Tang | A grid-based Bader analysis algorithm without lattice bias[END_REF]. In both cases, a major accumulation of electron density is observed in the region situated between the Cr atom and the interacting atom of soman (Fig. 3 (a)) and mustard gas (Fig. 3 (b)) respectively. It can be observed that the expansion of this charge accumulation region is much more pronounced for mustard gas. This is attributed to the fact that in addition to the sulfur atom, the chlorine atom of the CWA also establishes side interactions with Cr atom while soman exclusively interacts through its oxygen atom.

As a further stage, we explored the impact of water on the adsorption of CWAs on metal@graphene. Fig. 4 reports the DFT-optimized structures for all CWAs on Cr@graphene for the pre-humidification scenario. The analogue for V@graphene is represented in Fig. S2. One can observe that H2O is strongly bounded to the metal sites in the presence of all CWAs with characteristic separating O(H2O)-Cr distances of 2.03-2.05 Å. H2O molecule establishes a strong hydrogen bond via its H atom with the oxygen atom of both soman and sarin (separating distances of 1.57-1.58 Å) while its two hydrogen atoms interact each with one chlorine atom of the mustard gas. Indeed, in this scenario, the interactions between CWA and the metal@graphene substrate are shielded by the water molecule that acts as a bridge between the two components.

We then explored the case of a simultaneous co-adsorption of water and CWAs. Fig. 5 and Fig S3 report the corresponding DFT-optimized structures for both H2O on Cr and on V@graphene, respectively. Table S4 indicates that the so-obtained configurations are significantly more energetically stable than those obtained for the pre-humidification scenario. Referring to soman molecule, a small elongation of the distance between the oxygen atom and the chromium atom is observed with respect to the dry state from 2.00 Å to 2.08 Å (Fig. 5 (a)) on Cr@graphene and from 2.06 Å to 2.13 Å (Fig. S3a) on V@graphene. Furthermore, the inclusion of water leads to a reduction of the adsorption energy for soman of -5.40 kcal.mol -1 , and -7.58 kcal.mol -1 in the cases of Cr@graphene and V@graphene, respectively (Tab. 1).

Regarding sarin, as compared to the dry states, the presence of water also leads to a slight elongation of the distance between the oxygen atom of the CWA and the metal site, i.e. from 2.00 Å [57] to 2.08 Å (Fig. 5 (b)) and from 2.06 Å [57] to 2.10 Å (Fig. S3 (b)) for Cr@graphene and V@graphene respectively. This translates into a decrease of the adsorption energy between sarin and the metal@graphene substrate for Cr (-8.91 kcal.mol -1 ) and V (-6.80 kcal.mol -1 ) once water is present as reported in Tab. 1. Finally in the case of gas mustard, the interacting distances between the sulfur atom and the metal atom vary from 2.34 Å to 2.51 Å (Cr@graphene -Fig. 5 (c)) and from 2.50 Å to 2.52 Å (V@graphene -Fig. S3 (c)) in the presence of water. In the case of Cr@graphene the significant elongation of the S-Cr distance is accompanied by a large reduction of the Cl-Cr distance from 3.28 Å to 2.72 Å, while the opposite is observed for V@graphene (2.69 Å to 3.16 Å). Nonetheless, as for soman and sarin, the presence of water leads to a reduction of the adsorption energies for mustard gas in the cases of Cr@graphene and V@graphene, however the decrease is of much lower magnitude, -1.37 kcal.mol -1 and -4.61 kcal.mol -1 respectively compared to the situation described above for sarin and soman (Tab. 1).

To summarize, Tab. 1 evidences that the CWA/metal@graphene adsorption energy remains substantial even in the presence of water and this implies that the CWAs are expected to remain efficiently captured by the corresponding impregnated carbons under humidity. This is reflected by a decrease of maximum 20% of the adsorption energy, in the presence of water, for soman -sarin respectively in both metal@graphene. Interestingly, this conclusion is even more valid for mustard gas, its interaction with the impregnated carbons being much less affected by water as stated by the very low adsorption energy decrease (3.3%) in the case of Cr@graphene substrate. To understand the origin of this slightly different behavior, we further conducted a Density of states analysis (Fig. 6) on the soman/H2O -Cr@graphene and mustard gas/H2O -Cr@graphene complexes. DOS of soman and mustard gas on H2O -Cr@graphene plotted in Fig. 6, are projected in a similar way as in Fig. 2, to highlight the water-induced modifications of the electronic states, the same analysis for the V-analogue being reported in Fig. S4. By comparing Fig. 2 (a) and Fig. 6 (a), one can note that for soman, similarly as under dry conditions, below the Fermi level, an overlap between p-orbitals of the oxygen atom of soman and d-orbitals of Cr is reduced to a small contribution around -5 eV. Moreover, at higher energy (about 2.5 eV), a reduction of the overlap between d-orbitals of Cr and p-orbitals of O, P and F atoms of soman is observed. Since the interactions of soman on Cr@graphene take place mostly through its oxygen atom, these water-induced changes are associated with a weakening of the bonding established by the oxygen atom. On the other hand, by comparing Fig. 2 (b) and Fig. 6 (b) for mustard gas, one can note that the overlap between the p-orbitals of Cl and P (CWA) and the d-orbitals of Cr below the Fermi level is more pronounced and is present up to -3 eV. Furthermore, the intensity of d-orbitals of Cr increases above the Fermi level around 3 eV. This observation correlated with the geometry of the mustard gas molecule, where one Cl atom is in the vicinity of Cr (2.72 Å), is consistent with a rather small reduction of the adsorption energy for gas mustard on Cr@graphene in the presence of water. Regarding the V-analogue, the corresponding analysis reported Fig. S4 shows that at high energy, above the Fermi level, the overlap observed between the p-orbitals of Cr and the porbitals of Cl and S atoms of the CWA (around 3.5 eV) is not anymore present. This lack of overlap contributes to a more pronounced reduction of the resulting adsorption energy in the case of V@graphene (-4.61 kcal.mol -1 ) vs Cr@graphene (-1.37 kcal.mol -1 ) upon water adsorption. Fig. 7 and Fig. 8 report the charge differences for each CWA (soman, sarin and mustard gas)/H2O -Cr@graphene and V@graphene complexes respectively with an isovalue of ± 1.0 × 10 ) *. Å ) . The comparison of Fig. 7 with its analogue for the dry state (Fig. 3) reveals that the presence of water induces a deformation of the electronic density surrounding the interacting atom (oxygen for soman and sarin, sulfur for mustard gas) for the Cr@graphene complex. For each CWA, the depletion region on the metal is deformed towards the single water molecule leading to an elongation of the distance between the CWAs and the metal atom and therefore to a weakening of the interaction between CWAs and the substrate through their main interacting atom. Indeed, the interacting region between the H2O -substrate with the CWAs is composed mostly by a depletion region of electronic density whereas accumulation phenomenon arises along the closest contact between the interacting atom of CWAs and the water oxygen atom. It can be seen that the electronic density deformation is made of the depletion regions on both top of the metal and at the vicinity of the water molecule that merged. This observation positions the water molecule as an extension of the metal@graphene substrate. The particular geometry of mustard gas molecule allows a side interaction between its chlorine atom and the metal atom which is less impacted by the presence of water as compared to the main interaction between metal and sulfur. While the only contact between soman and sarin on Cr@graphene is screened by the water molecule, the particularity of mustard gas geometry allows it to bypass this screening effect and to maintain a stabilizing contact resulting in a smaller adsorption energy reduction on Cr@graphene.

As expected from the variation of the adsorption energy observed for sarin between dry and simultaneous co-adsorption on Cr (-8.91 kcal.mol -1 ) and V@graphene (-6.80 kcal.mol -1 ), Fig. 8 shows that similar electronic deformations are observed for both systems with the inclusion of simultaneous co-adsorbed water. The same observation about the electronic behavior can be made from Fig. 7 (a) and Fig. 8 (a) for soman even though the variation of adsorption energy is superior for V@graphene (-7.58 kcal.mol -1 ) vs Cr@graphene (-5.40 kcal.mol -1 ). Alternatively, the electronic charges differences comparison relative to mustard gas on Cr@graphene (Fig. 7 (c)) with V@graphene (Fig. 8 (c)) provides some highlights about the more pronounced reduction observed with V@graphene rather than Cr@graphene. The separating distance associated with the Cl-metal side interactions equal to 2.72 Å and 3.15 Å, for Cr and V@graphene complexes respectively, emphasizes the distinctive deformations of this specific depletion region observed for both systems. This distinct behavior is at the origin of the different adsorption energy variations reported for mustard gas/Cr@graphene and mustard gas/V@graphene between dry and wet conditions.

Summary

The adsorption of soman, sarin and gas mustard on Vanadium and Chromium embedded graphene models was computationally explored under dry and wet conditions. Our DFT calculations first demonstrated the highly attractive features of these impregnated carbons in terms of affinity with respect to the three considered CWAs. Both metal-impregnated carbons were predicted to show a slightly higher affinity for sarin vs the other two CWAs. As a further stage, we explored the impact of water on the physisorption of the three CWAs considering two scenarios, i.e. pre-humidification and simultaneous co-adsorption. The inclusion of water was shown to not dramatically alter the host/CWAs interactions, gas mustard being the less impacted molecule with an associated decrease of the adsorption energy by only 3.3% in Cr@graphene vs from 13 to 20% for sarin and soman. Indeed, the CWAs are expected to remain efficiently captured by the corresponding impregnated carbons under humidity. This microscopic investigation demonstrated the transition metal embedded@graphene systems to maintain a high level of performance for the capture of CWAs even in the presence of humidity, which corresponds to the most common operating conditions for protective gas masks.

Figure captions

Figure 1

Most stable DFT-optimized configurations of soman on (a) V@Graphene and (b) Cr@graphene as well as of mustard gas on (c) V@graphene (d) Cr@graphene under dry conditions.

Figure 2

Comparison of PDOS for the (a) Soman/Cr@graphene and (b) Mustard gas/Cr@graphene complexes under dry conditions.

Figure 3

Charge differences for (a) Soman/Cr@graphene and (b) Mustard gas/Cr@graphene presented with isovalue of ± 1.0 × 10 ) *. Å ) . Blue and red colors represent, respectively, the charge accumulation and depletion regions.

Figure 4

Most stable DFT-optimized configurations considering the pre-humidified scenario with one water molecule involved for (a) Soman/Cr@Graphene, (b) Sarin/Cr@graphene and (c) Mustard gas/Cr@graphene

Figure 5

Most stable DFT-optimized configurations considering a simultaneous coadsorption of water molecule and CWA for(a) Soman/H2O -Cr@graphene, (b) Sarin/H2O -Cr@graphene and (c) Mustard gas/H2O -Cr@graphene.

Figure 6

PDOS for the (a) Soman/H2O -Cr@graphene and (b) Mustard gas/H2O -Cr@graphene complexes in the case of the simultaneous co-adsorption scenario. For comparison, PDOS for the isolated CWA molecules and H2O -Cr@graphene complexes are also reported. Op (soman), Fp (soman), Pp (soman), Clp (mustard gas), Sp (mustard gas), Cs (CWA), Cp (CWA), Cs (graphene), Cs (graphene), total DOS of molecule, s-, p-, and d-states of metal are represented in orange, light green, violet, dark green, cyan, dashed blue, dashed red, blue, red, black, bold blue, bold red, bold black, respectively.

Figure 7

Charge differences considering the simultaneous co-adsorption scenario for (a) Soman/H2O -Cr@graphene (b) Sarin/H2O -Cr@graphene and (c) Mustard gas/H2O -Cr@graphene complexes presented with isovalue of ± 1.0 × 10 ) *. Å ) . Blue and red colors represent the charge accumulation and depletion regions respectively.

Figure 8

Charge differences considering the simultaneous co-adsorption scenario for (a) Sarin/H2O -V@graphene (b) Soman/H2O -V@graphene and (c) Mustard gas/H2O -V@graphene complexes presented with isovalue of ± 1.0 × 10 ) *. Å ) . Blue and red colors represent the charge accumulation and depletion regions respectively.

Table captions

Table 1 CWA dependence of the DFT calculated CWA adsorption energies (in kcal.mol - 1 ) for both V/Cr@graphene in the absence and in the presence of water. For comparison the CWA adsorption energies are also reported for the graphene without transition metal atom and without water.

  For each system panel I, panel II and panel III present PDOS of the isolated CWA, isolated metal@graphene and resulting complexes. Op (soman), Fp (soman), Pp (soman), Clp (mustard gas), Sp (mustard gas), Cs (CWA), Cp (CWA), Cs (graphene), Cs (graphene), total DOS of molecule, s-, p-, and d-states of metal are represented in orange, light green, violet, dark green, cyan, dashed blue, dashed red, blue, red, black, bold blue, bold red, bold black, respectively.

Acknowledgements

The research leading to these results has received funding from the DGA Maîtrise NRBC.


MD Dominique De Ziegler

Rene Frydman

Keywords:

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Dominique de Ziegler, MD 1,2 Rene F. Frydman, MD 1 RPL Introduction Nov 11, 2020. Page 2 of 5.

One arbitrarily considers that miscarriages are the spontaneous terminations of pregnancy occurring before 20 weeks of gestation. Conversely, pregnancy losses that take place later are referred to as premature births, which generally have different etiologies.

Practically, miscarriages are commonly referring to spontaneous termination of pregnancies occurring before the 12 th week of gestation. It is possibly the most common pathology encountered in medicine. It is generally admitted that 12-15% of all pregnancies spontaneously terminate through an early miscarriage, an incidence that is markedly influenced by age. The frequency with which miscarriages occur has been however extraordinarily stable over time. In the past decades, we have notably observed similar incidences of miscarriage in spontaneously occurring and ARTinduced pregnancies. In ART however, early β-hCG measurements add the very early pregnancy arrests -before any clinical manifestation -, which are counted as biochemical pregnancies. This latter parameter is generally not recognized in non-ART conditions.

On certain occasions, miscarriages occur repetitively. By definition, one generally speaks of recurrent pregnancy losses (RPL) after 3 episodes of miscarriages, a fact encountered in approximately 1% of cases in the general population. Today however, investigations for sorting possible causes of RPL are commonly initiated after 2 miscarriages already. In infertile women, the incidence of RPL is sensibly higher. Several causes of RPL have been identified sometimes leading to practical measures to overcome recurrence. These are reviewed in the present series of articles. with RPL, with 40% being identifiable by traditional karyotypes. PGTM may help in selecting the disease free embryos. Balanced translocations are found in 4-8% of couples with RPL, including in those who may have had one or more healthy children.

The oocyte, sperm and embryos: coping with genetic causes

Finally, asymmetric inactivation of the X chromosome has been found more common in women with RPL, but no specific treatment is currently available.

Uterus congenital and acquired factors

The uterine causes of miscarriages and RPL are divided in congenitalmalformations -and acquired disorders. The mechanisms causing the endometrial alterations in case of endometriosis are linked to a prevailing inflammation in the eutopic endometrium. A variant of endometriosis -adenomyosis -is also susceptible to induce the same consequences and notably, RPL. This is primarily the case for internal adenomyosis.

Still another circumstance associated with endometrial inflammation is chronic endometritis (CE) found in 10-15% of infertile women and 40% of those suffering from endometriosis. Cicinelli's team conduct a systematic review of the incidence of miscarriages and RPL in case of CE in the same article of this series (3). Cicinelli et al. also assess the beneficial effects of specific treatment (3). This team emphasizes the difficulty in making the diagnosis of CE -thereby explaining some discrepancy in results -and emphasizes the need to perform both endometrial biopsies and hysteroscopy. The core message on the role of CE on RPL conveyed by Cicinelli's team is that it can be diagnosed and effectively treated thereby reducing if not preventing the risk of recurrence (3).

Immunological, thrombophilia and hormonal causes of RPL

The development of pregnancy implies an immune-tolerance for the development of the 'non-self' embryo. In this issue of Views and Reviews, Diana RPL Introduction Nov 11, 2020. Page 5 of 5.

Alecsandru et al. review the immunological mechanisms that permit a normal

development of the embryo and the possible impairments that may lead to miscarriages and RPL (4). Likewise, Alecsandru et al. review the metabolic -including diabetes mellitus -and autoimmune disorder that me be associated with RPL (4). In the same article Klimczak and Franasiak review the alterations of hemostasisthrombophilias -that may lead to miscarriages and RPLs (4). Reminding us that pregnancy itself is a prothrombotic state, these authors explain that therefore thrombophilias may increase the risk of complication.

It is debated whether inherited thrombophilias are associated with RPLs, with a majority of publication indicating that this is not the case (4). Conversely acquired thrombophilias may cause RPLs such as notably, the antiphospholipid antibody syndrome encountered in 5%-20% of cases with RPLs (4).

  Klimczak et al. review the causes of RPL stemming from disorders affecting the sperm, oocyte or embryo (1). These authors emphasize that sperm anomalies likely contribute to a fraction of the high incidence of genetic disorders encountered in products of conception ending in miscarriage. Various methods of assessing sperm reproductive potential are reviewed with emphasis on sperm DNA integrity assessment and chromosomal aberrations. Options tested or in development for sperm sorting are reviewed. Abnormalities of the oocyte -intrinsic likely present in all of a person's cohort of oocytes and functional disorders -are likely lethal to the developing conceptus. Structural rearrangements of the chromosome may be present in 12% of couples RPL Introduction Nov 11, 2020. Page 3 of 5.

  The former category amounts to various degrees of non-resorption of the septum separating the two Mullerian ducts at the time of their fusion in utero. As described by Carbonnel et al. (2), the anomaly most frequently associated with miscarriages and RPL is the septated uterus defined by a septum protruding >10 mm in the uterine cavity (2). Conversely, more and less important non resorption of the septum -double uteri and arcuated uterus (septum <10 mm) -are less prone to cause miscarriages. While a near consensus of opinion recommends surgical metroplasty in case of septate uteri with RPL, surgical abstention is generally recommended for the latter malformations -double uteri and arcuated uterus (2). Uterine fibromas and endometrial polyps constitute the bulk of the acquired anatomical anomalies together with intra-uterine adhesions. Uterine fibroids are classified according to their anatomical position in relation to the uterine cavity and uterine wall. Today the classification of the international federation of gynecologists (FIGO) is preferred over the descriptive localization that was used before. Fibroids classified as FIGO 0-2 protrude inside the uterine cavity. Common sense and widely accepted medical practices recommend surgical removal of such fibroids even if guidelines, such as those proposed by ESHRE, remain elusive about such indications. As underscored by Carbonnel et al., clear benefit from removing endometrial polyps is lacking despite a general practice that calls for endoscopic removal of such protruding structures (2).Intra-uterine adhesions and scar tissue is a lingering problems encountered in women who had uterine procedures and or infections. As discussed by Carbonnel et al. in this issue of views and reviews, the management is a lingering problems and matter of debates(2). In unmanageable cases -alterations beyond hope for repaircertain are proposing the use of stem cells and even at times, possible uterine transplantation or gestational surrogacy.

  The endometrium: Endometriosis, inflammation and chronic endometritis Embryo implantation -the necessary step of the initiation of pregnancyrequires a proper development of the embryo itself and an adequate receptivity of the endometrium. Embryo quality -notably, its euploidy status -stems from oocyte and sperm quality as discussed by Klimczak et al. (1) in an article of this series. An impairment of endometrial quality may affect implantation and placentation. This may affect proper embryo development and result in miscarriage and in certain circumstances, RPL, as reviewed by Pirtea et al. (3). Endometriosis has been demonstrated to affect the eutopic endometrium. Inflammation caused by endometriosis ultimately impairs the normal endometrial response to progesterone. As addressed by Pirtea et al. in a dedicated article of this series (3), endometriosis-led changes alter the endometrial response to progesterone and notably, a proper predecidual transformation of the endometrium. Endometriosis has indeed been associated with increased miscarriage risk possibly, leading to RPL.


Serge Mignani
email: serge.mignani@parisdescartes.fr

Xiangyang Shi
email: xshi@dhu.edu.cn

Valentin Ceña
email: valentin.cena@gmail.com

João Rodrigues

Helena Tomas

Jean-Pierre Pierre Majoral
email: majoral@lcc-toulouse.fr

Xangyang Shi

Engineered non-invasive functionalized dendrimer/dendron-entrapped/complexed gold nanoparticles as a novel class of theranostic (radio)pharmaceuticals in cancer therapy

Keywords: Dendrimers, dendrons, gold(0)/(III) nanoparticles, gene and drug delivery, cancer therapy, theranostic strategy

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In medicine, nanomedicine which is an interdisciplinary field of science have sparked an extraordinary growing interest to achieve medical benefit. Also, nanotechnology in nanomedicine can overcome biological obstacles to treat severe diseases like complex cancers, which are presently difficult to cure completely. The most widespread cancers are breast, colorectal, lung, prostate as well stomach and liver cancer. [START_REF] Farjadian | [END_REF] Importantly, over the last decade, remarkable steps forward have been made in advancing cancer diagnosis and treatment with a move toward increased sensitivity, speed, and cost-effectiveness. [2] Nanomedicine has made a very significant contribution to current cancer treatments, in addition to surgical intervention; importantly, radiation and chemotherapeutic agents unfortunately also kill normal cells by inducing highly deleterious and often life-threatening side effects in the patient. [3] Consequently, to overcome these problems, within the nanomedicine domain, the development of highly efficient platforms for cancer therapy based on engineered nanoparticles (NPs) for drug delivery has been investigated as so-called "magic bullets". [4] Indeed, in order to decrease the side effects of highly toxic anti-cancer drugs, the development of drug delivery systems for cancer chemotherapy have been employed.

Liposomes, micelles, polymeric nanoparticles, nanocrystals, solid lipid nanoparticles, nanocages, metal nanoparticles (e.g. gold, iron), nanocrystals, graphene oxide, carbon nanotubes, and dendrimers have been used extensively as nanocarriers, and several of them have been approved for clinical use. [5] The two NPs in clinic are the first FDA approved (1995) PEGylated liposome injections Doxil® and Caelyx® (doxorubicin (DOX) HCl (Adriamycin®)), and paclitaxel albumin-bound particles called Abraxane™. [6] Dendrimers are different in comparison to conventional polymers, and represent a unique class of macromolecules offering well-defined 3D nanoarchitectures. The spherical shape, the surface functionalities and size of which can be precisely controlled affording high degree of molecular uniformity. Size and surface characteristics are the most important features to be managed to develop safe and biocompatible dendrimers. Presently, a large variety of dendrimers have emerged as non-viral versatile nanocarriers. These radially symmetric monodisperse macromolecules are described as highly branched, star-shaped with nanometer size. Three major macromolecular architectural components can characterize dendrimers: a central core, interior dendritic structure including branches, and a peripheral surface with diverse functional groups. The drugs of interest can be entrapped by encapsulation within the cavities of dendrimers, or covalently conjugated with a cleavable linker sensitive to, for instance, acidic pH. [7] Dendrimers have been widely used to deliver anticancer drug by passive and active targeting strategies. Dendrimers represent ideal vectors for drugs for therapeutic purposes or imaging molecules for diagnostic use. Based on the different attributes of dendrimers including pharmacokinetic, pharmacodynamic and toxicological properties, the concept of dendrimer space has been presented and developed. [8] The main advantages of dendrimers are as follows: 1) strong functionalization of surface, 2) hydrophobic and hydrophilic drugs can be encapsulated, conjugated or complexed, 3) facile control of the synthesis as well degradation process, 4) high penetration into the cell membranes, 5) high structural homogenicity, 6) high water solubility with adequate surface functionalization, 7) meaningfully lower viscosity versus linear polymer, 8) higher ligand density on the surface than linear polymers, 9) multiple routes of administration: intravenous (iv), intraperitoneal (ip), ocular, transdermal, oral (po), intranasal, and pulmonary) [9] versus micelles and liposomes for which only the iv route is possible, 10) controlled biodistribution, 11) high drug loading capacity (local concentration effect), 12) enhanced permeability and retention (EPR) effect for the accumulation in tumor tissues, [10], [11] whereas the main disadvantages are 1) few examples in clinic due to translational issues including good manufacturing practices (GMP) production and quality control. This aspect is general for any nanoparticles in medicine [12], 2) high cost of production, and 3) challenged EPR effect: 'the EPR effect works in rodents but not in humans'. [13] All these advantages have contributed to the development of dendrimers in nanomedicine.

The historical applications of gold in art and ancient medicine dates back to the dawn of time. Naturally, the body does not have gold nanoparticles, but several formulations in biomedicine with gold molecule are generally non-toxic and non-immunogenic, and consequently suitable for in vivo studies [14,15]. Importantly, the tailored chemical (e.g. facile surface chemistry), and physical (e.g. comparable size relative to proteins) properties of gold nanoparticles allow their accumulation, for instance in tumor tissues and cells, and they can be detected and quantified with high sensitivity [16,17]. Importantly, based on the ability of gold nanoparticles to intensively absorb X-ray radiation, they can be used as in cancer radiation therapy, as well as imaging contrast agents in diagnostic CT (computed tomography) scans [18].

Over the past decades, nanotheranostic nanoparticles (TNPs) that simultaneously tranmit diagnostic information and monitor the therapy process in situ have been developped mainly in nano-oncology realm. [19] These TNPs dispose of unique physical and chemical properties to target desired cells and tissues producing therapeutic and imaging action against the disease. [20] Multiple imaging approaches were used such as optical imaging, ultrasound (US), magnetic resonance imaging (MRI), computed tomography (CT), singlephoton computed tomography (SPECT) and positron emission tomography (PET). [21] Importantly, NPTs are able to increase the accumulation and delivery of encapsulated or conjugated biologically active compounds in tumours, which enhances therapeutic efficacy and reduces side effects on healthy tissue. [22] An interesting analysis to develop ideal TNPs was advocated by Chen et al. [23] The Table 1 shows few selected TNPS in oncology therapeutic domain, and consequently does not present exhaustive list of TNPS. To extend a panoramic view about the developpemnt of TNPS in the oncology field, il is important to note, that for example, mesoporous silica nanoparticles (MSN) in the theranostic strategy were developped with several imaging technique including photoacustical (near infrared radiation, NIR) [24], ultrasound [25], magnetic resonance (Table 1), optical (e.g. NIR, visible light) [26], and radionucleotide-based (NIR). [27] In vitro and in vivo advances in theranostic nanocarriers of doxorubicin based on iron oxide and gold nanoparticles were higlighted by Gautier et al. including hybrid and metallic nanocarriers. [28] Recently, hybrid mesoporous silica and hydroxyapatite nanoparticles nanocarriers for theranostic applications were described. [29,30]

Doxorubicin

Manganese oxide [39] Ultrasmall manganese oxide nanoparticles (USMO)

Doxorubicin

Manganese oxide [40] HA-CD Several examples of the design of targeted dendrimers and dendrons for theranostic applications in oncology were highlighted, and selected examples were depicted in Table 2.

In brief, using dendrimers or dendrons as stabilizers or templates, on one hand, the size of the in-situ formed Au NPs can be readily controlled by dendrimers/dendrons through a reducing reaction; on the other hand, with the combination of dendrimers/dendrons and Au NPs, both properties of dendrimers/dendrons and Au NPs can be rendered. Hence, there are many opportunities that can be explored for theranostic applications in cancer therapy based on the dendrimer/dendron-complexed Au NPs. [51] These are the biggest advantages of dendrimer/dendron-complexed Au NPs. The only disadvantage of the combination could be the high cost of commercially available such as PAMAM dendrimers, limiting their uses in some developing countries. G4.5 PAMAM Magnetic resonance imaging: Gd(III) and Mn(II) [56] G2 and G6 PAMAM Magnetic resonance imaging: Gd(III) [57] G4 PAMAM Magnetic resonance imaging: Gd(III) Folic acid [58] G6 PAMAM Magnetic resonance imaging: Gd(III) [59] Janus dendrimersome: Another interesting dendrimer Givosiran (Givlaari TM ) for the treatment of adults with acute hepatic porphyria, was developed by Alnylam. Givosiran bears a double-stranded siRNA containing 16 2'-F-ribonucleosides units and six thiophosphate linkages. [72] This review will describe the recent developments in the design, synthesis, functionalization, in biomedicine of engineered functionalized dendrimer-entrapped gold(0) and dendrimercomplexed gold(III) nanoparticles in cancer therapy. The therapeutic applications of dendrimer-stabilized AuNPs and AuNP-cored dendrimers are not within the scope of this review.

Hybrid gold nanoparticles entrapped in dendrimers for gene and drug delivery, and nuclear medicine applications to take down cancers

Dendrimers encapsulating gold(0) nanoparticles (Au DENPs): general principles

The molecular luminescent AuNPs are chemically inert NPs with a size from 0.3 to 2 nm, and generally showed high X-ray attenuation intensity, higher than the CT contrast media Omnipaque [73].

With the use of non-invasive theranostic functional nanoprobes [74], the major objective of specialized nuclear medicine is to 1) characterize and quantify biological processes involved, for instance, in the diagnosis of early cancer, 2) to stimulate specific drug delivery to tackle cancer, and 3) to track cellular evens. [75] Positron emission tomography (PET) and singlephoton emission computed tomography (SPECT) combined with dendrimer platforms have been investigated in nuclear medicine imaging applications to avoid common drawbacks which are associated with radioactive isotopes used in the clinic, including 1) short imaging time, 2) renal toxicity, and 3) non-specificity. [76] Radionuclides which emit positrons and gamma (γ) rays strategy can be used in nuclear medicine imaging with high sensitivity, whereas therapeutic radionuclides emitting alpha (α) or beta (β) rays represent applicable agents for radiotherapy (RT) applications. Instead of an external radiation source such as Xrays, ionizing radiation emitted from therapeutic radionuclides induces radiation within a short distance, thereby minimalizing side effects in normal tissues. However, although both PET and SPECT imaging technique show strong efficacity, they are limited by their spatial resolution and poor anatomical measurements. Structural imaging modes such as powerful computed tomography (CT) and magnetic resonance (MR) imaging techniques have become matched tools to PET and SPECT imaging technique [77]. Interestingly, the recent development of radioimmunotherapy (RIT) technique which specifically targets cancer cells using monoclonal antibodies (mAbs) has been highlighted [78].

For several decades, multimodal imaging contrast agents incorporated with simultaneously therapeutic radioisotopes for theranostic applications have been under development. [79] Taking advantage of the characteristics of dendrimers as multifunctional nanoplatforms for drug and gene delivery, multimodal imaging, and theranostics [80], two different strategies were developed for the use of radiolabeled dendrimer-based nanodevices: 1) conjugation of radiolabels on the surface of dendrimers and 2) encapsulation of radiolabels inside the free space (interior space) of dendrimers. For instance, the conjugated labels technetium-99m (99mTc) and Copper-64 radiopharmaceuticals were developed for SPECT and PET imaging with polyamidoamine (PAMAM) dendrimers [81]. Recently, the analysis of the different interactions between gold and phosphorus dendrimers have been presented as versatile ways to prepare hybrid organic-metallic macromolecules [82].

The most common strategy to prepare hybrid dendrimer-Au(0)NPs is to first mix higher generation dendrimers with tetrachloroauric acid trihydrate ([Au(III)Cl4] -) gold salt.

Complexation between [Au(III)Cl4] -and dendrimers as both templates and nanoreactor hosts is generally based on electrostatic interactions. Then, reduction of the gold salt using ascorbic acid, sodium citrate or sodium borohydride affords metallic gold (Scheme 1) with generally size distribution ranging between 1 and 2 nm with good chemical stability [83].

Scheme 1. Schematic preparation of dendrimer-entrapped gold nanoparticles

Recently, the easy preparation of chemically stable, and soluble in water, G0-G2 triazine dendrons bearing gold NPs has been advocated by Enciso and colleagues [84]. Reduction of diazonium tetrachloroaurate salts by sodium borohydride afforded corresponding triazine dendrons which are covered with gold nanoparticles (Scheme 2).

Scheme 2. Synthetic route for the synthesis of G0 triazine dendron-like-coated gold nanoparticles

In order to prepare non-toxic and non-immunogenic hybrids based on dendrimer-gold nanoparticles for therapeutic applications such as cancer imaging and photothermal therapy, the amino groups located on the surface of PAMAM dendrimers have been modified by the introduction of PEG chains. Consequently, the increase of the circulation time in the blood of these modified dendrimers was observed. Also, to reduce toxicity, a neutral surface of dendrimers can also obtained by the introduction of glycidol groups and the reaction of acetic anhydride [85].

Notably, a very interesting and promising direction is to develop stable and biocompatible non-invasive functionalized dendrimer which entraps gold nanoparticles (AuNPs). Two types of nanoparticles have been developed as templates: 1) dendrimer encapsulating AuNPs (Au DENPs) and 2) dendrimer stabilizing AuNPs (Au DSNPs) [86]. For Au DENPs, one AuNP is encapsulated within each dendrimer as the template macromolecule, and the size of the core of Au NPs are generally smaller than 5 nm, whereas for Au DSNPs, the Au core size is usually larger than 5 nm. In this case, dendrimers play the role of stabilizer. Both of these Au NPs have been used as contrast agents for CT imaging applications. Scheme 3 presents the preparation of one example of DENPs entrapping Au(0) from G5 PAMAM dendrimer and bearing folic acid (FA) binding with its corresponding receptors on the cancer cell membrane and the preparation of Au DSNPs. [87], [88] Importantly, hydrophilic PEGylation of highgeneration dendrimers (e.g., G5) increases the size of the dendrimer, allowing it to entrap more elemental Au within the dendrimer interior.

Hybrid Au DENPs for imaging and treatment of cancer cells

Combined gamma scintigraphy (SPECT) and computed tomography (CT) approaches

Regarding the development of entrapped radiopaque metal NPs, Shi and colleagues described the design and development of biocompatible G2 PAMAM dendrimers conjugated with PEGylated (polyethylene glycol)-arginine-glycine-aspartic peptide (RGD) as a targeting ligand, and NOTA as the chelator agent of labeled 99m Tc, with entrapped gold nanoparticles (Au DENPs); the nanoparticle was named DENPs (Scheme 4) [91]. Notably, the same team designed and developed a CT imaging agent based on the integration of both Au NPs and the SPECT imaging radioisotope 99m Tc within the PEGylated G5 PAMAM dendrimeric nanoplatform for SPECT/CT imaging applications. [92] As shown in Scheme 5, two types of nanoparticles were prepared from G5 PAMAM dendrimer I, i.e. 99m Tc-Au-Ac DENPs bearing on its surface N-acetylated groups using Ac2O for biocompatibility purpose and 99m Tc-Au-Gly DENPs with N-hydroxylated groups, for biocompatibility purpose, using glycidol on the remaining terminal amino groups of the G5 PAMAM dendrimer III. The chelating moiety of 99m Tc was DTPA. The number of DTPA ligands and hydrophilic mPEG moieties (polyethylene glycol monomethyl ether allowing good stability and desirable biocompatibility of the nanoparticles) attached to each G5 dendrimer was statistically 7.6 and 17.6, respectively. These two nanoparticles were colloidally stable, radiostable, and showed excellent hemocompatibility. Figure 6 shows the synthetic pathways for the preparation of 99m Tc-Au-Ac DENPs and 99m Tc-Au-Gly DENPs and their corresponding SPECT/CT images of the mouse lung, liver, and bladder at 0.5 h (a), 1 h (b), and 2 h (c) post-intravenous injection of 99m Tc-Au-Ac DENPs and 99m Tc-Au-Gly DENPs.

Scheme 5. Synthetic pathways for the preparation of 99mTc-Au-Ac DENPs and 99mTc-Au-Gly DENPs and their respective SPECT/CT images. Modified reproduction with permission from Ref. [92] Reproduced by permission of The Royal Society of Chemistry

The Au core NPs for both 99m Tc-Au-Ac DENPs and 99m Tc-Au-Gly DENPs have similar size and crystalline lattice, but they displayed different surface plasma resonance (SPR) band locations (Au-Ac DENPs at 530 nm and Au-Gly DENPs at 510 nm, respectively) due to their varying aggregation behaviors in water with a hydrodynamic size of 96 nm for the former and 50 nm for the latter, respectively. These studies show that the surface modification (acetylation versus hydroxylation) availed them of varying biodistribution tendencies, this allowing for preferential imaging of lung using the 99m Tc-Au-Ac DENPs and blood vessels using the 99m Tc-Au-Gly DENPs. Interestingly, these two nanoplatforms were also used for SPECT/CT imaging of the lymph node of rabbits. Taken together, these studies demonstrate that SPECT/CT imaging of preferential organs can be realized through the chemical modifications of dendrimer surface, in this case hydrophobic acetyl groups over hydrophilic hydroxyl groups.

Further work by the same team reported on the development of G5 PEG-PAMAM dendrimer-entrapped gold nanoparticles showing good CT imaging in New Zealand rabbits [93].

Notably, Li et al. deepicted the development of multifunctional G2 PEG-PAMAM dendrimer entrapping gold NPs (Au DENPs), and bearing 99m Tc for SPECT/CT imaging of tumors. [94] Thus, Au-DENPs were functionalized by the introduction of 1) folic acid (FA) as targeting ligand through the PEG chain, and 2) cyclic diethylenetriamine penta acetic anhydride (cDTPAA) as the chelator of 99m Tc. The obtained {(Au 0 )6-G2-DTPA( 99m Tc)-PEG-FA} DENPs, were used for SPECT/CT dual-mode imaging studies of HeLa cancer cells overexpressing FA receptors. Both FA-targeted and non-targeted NPs were cleared by the liver and spleen reticuloendothelial system (RES), and metabolized through the kidney. In the tumor, the concentration of the targeted Au-NPs was two times higher than non-targeted Au-NPs due to the FA-mediated targeting process.

Interestingly, the same team designed and developed original 99m Tc-labeled Au DENP-based G5 dendrimers for early SPECT/CT detection of chemotherapy-induced tumor apoptosis, which is an important aspect for the evaluation of the therapeutic efficiency. Spherical shape with a uniform size distribution (diameters: 2.2-5.9 nm, and hydrodynamic sizes: 287.9 -327.0 nm) of the Au core of these NPs were observed using TEM image analysis. The remaining amino-terminal groups were acetylated to give Au-TOS-RGD DENPs [98]. α-TOS is the most effective form of vitamin E and results in the inhibition of proliferation and apoptosis of cancer cells (Scheme 7) [99]. Several complementary assays have been performed to evaluate the therapeutic efficiency of this nanoprobe, such as cell viability assays and cell morphological observations as well the evaluation of intracellular reactive oxygen species (ROS) activity and apoptosis pathway. The RGD-rendered targeting specificity of these NPs was confirmed using flow cytometry and confocal microscopy to check their cellular uptake, and using cytotoxicity assay to examine the enhanced therapy efficiency.

Likewise, the developed Au-TOS-RGD DENPs showed greater X-ray absorption properties than Omnipaque that is currently used in clinic and shows considerable drawbacks including fast metabolism, toxicity to kidney, and non-specificity to tumors [100]. Arguably, the use of low-generation PAMAM dendrimers (e.g., G2) to create partially PEGylated Au DENPs, named {(Au 0 )8-G2-mPEG} DENPs, have been described by Liu et al. for CT imaging applications [102]. The Au DENPs displayed a core size of 2.8 nm as revealed by TEM and a hydrodynamic size of 276 nm as examined by dynamic light scattering. The {(Au 0 )8-G2-mPEG} DENPs displayed good cytocompatibility, greater X-ray absorption coefficient than Omnipaque, and ability to CT image the mouse heart (major uptake observed), liver, spleen, lung, kidney, and bladder and the xenografted mouse KB tumor resulting from their extended blood circulation time and the well-known enhanced permeability and retention (EPR) effect [11]. Thus, on the basis of these interesting results, the same team starting from PEGylated G2 PAMAM dendrimers to develop an FA-or chemokine receptor 4 (CXCR4)-targeted 99m Tc-labeled Au DENP-based multifunctional nanoprobe for dual modal SPECT/CT imaging of tumors [94,103].

Recently, in the immunotherapeutic domain, multifunctional Au DENPs have been developed to label and track T cells using dual-mode CT/fluorescence imaging [104]. PEGylated G5 PAMAM dendrimers were conjugated with Fluo-4 and entrapped with Au NPs (Scheme 8). The remaining dendrimer surface amino groups were acetylated for biocompatibility purposes, affording the desired final {(Au 0 )25-G5.NHAc-(PEG)14-(Fluo-4)2}.

After up taken by T cells, Fluo-4 was released through the cleavage of the ester bond between Fluo-4 and the PEG hydroxyl group by intracellular esterases, inducing its binding with calcium ions and finally generating fluorescence emission.

Ultrasound and radiotherapy approaches

An important innovative strategy in oncology against difficult cancers such as pancreatic cancer (PaCa), associated with low drug permeability, poor vascularization, a dense stroma, and inherent or acquired drug resistance by classical chemotherapy, has been presented by Lin, Shi et al. [109] This strategy was based on the use of the ultrasound-targeted microbubble destruction (UTMD) technique, which promotes the co-delivery (synergistic effect) of gemcitabine (Gem), which is used in the treatment against advanced or metastatic PaCa, and miR-21 inhibitor (miR-21i) from PEGylated G5 PAMAM dendrimer-entrapped gold nanoparticles affording Gem-Au DENPs/miR-21i NPs (Figure 1). Clearly, these studies showed that Gem-Au DENPs/miR-21i NPs can be taken up by SW1990 cancer cells, and cellular uptake profile was facilitated by UTMD technique using an ultrasound power of 0.4 W/cm 2 , to boost the permeability of cells. Importantly, the enhanced tumor inhibition efficacy in vivo in the xenograft SW1990 tumor model in mice was also presented. The volume of tumors of treated mice were ample smaller than those of the Gem alone.

The construction of G4-PAMAM dendrimers encapsulating Au NPs, bearing 177 Lu, and linked with FA and bombesin as two different ligands has been advocated by Mendoza-Nava and colleagues (Figure 2). [110] The objective of this interesting radiotherapy study was to evaluate the radiopharmaceutical effect (RT), as well the contemporaneous evaluation of the expression of folate receptor (FR) and gastrin-releasing peptide receptor (GRPR) in breast cancer cells. The 177 Lu labeling moiety DOTA was linked to the dendrimer surface, and the incorporation of Au(0) was obtained through the incorporation of HAuCl4 followed by its reduction by NaBH4 (Figure 2). Cellular uptake showed that the 177 Lu-DenAuNP-folate-bombesin could be specifically taken up by T47D breast cancer cells. After intratumoral injection, the 177 Lu-DenAuNP-folatebombesin remained at the tumor site as long as 96 h as demonstrated by in vivo imaging. Consequently, the 177 Lu dendrimer (AuNP)-folate-bombesin could be regarded as an effective imaging agent for nuclear imaging and targeted radiotherapy of GRPR-and FRexpressing breast tumors. The same authors described also the preparation and development of 177 Lu-DOTA-PAMAM-AuNP-folate-bombesin. [111] Table 3

G4-PAMAM Inhibition of tumor growth of breast cancer cells

The toxicity of 198 Au/PAMAMG4 is higher against cancerous cells compared to normal cells [114]

Au DENPs: gene delivery

It is well-described that polycationic polymers in general and dendrimers in particular can be used as vectors for gene delivery, for instance of small interfering DNA (siRNA) by anionic DNA interactions with polycationic nanomaterials. [115] Qiu, Shi and colleagues [116] developed safe and effective β-cyclodextrin(β-CD)-modified Au DENPs (Au DENPs-β-CD) for the delivery of two different siRNAs to U87MG glioblastoma cells (Figure 3). Beta-cell lymphoma/leukemia-2 (Bcl-2) siRNA and vascular endothelial growth factor (VEGF) siRNA were selected. Once complexed with the siRNA, the AuNPs displayed good cytocompatibility. Flow cytometry and confocal microscopic observations revealed that these two siRNAs were able to be respectively transfected at the vector/siRNA polyplex N/P ratio (molar ratio of dendrimer terminal amine/siRNA phosphate) of 5:1. Further Western blot assays demonstrated that the Bcl-2 and VEGF protein expression in the transfected cancer cells were able to be significantly knocked down. In the same direction, an original approach based on the serum gene delivery [118] of zwitterionic dendrimers encapsulating gold nanoparticles (Au DENPs) to inhibit in vitro cancer cell metastasis, has been published by Xiong et al. [119] In order to avoid the interactions between serum protein and cationic vector and gene polyplexes inducing a significant reduction in cellular uptake efficiency, novel systems were developed. As shown in Scheme 10, G5 PAMAM dendrimers have been functionalized, on its surface, with both zwitterionic carboxybetaine acrylamide (CBAA) and morpholine (Mor) as lysosome-targeting agent. The final biocompatible nanodevice {(Au 0 )25-G5.NH2-PEG-Mor} NPs with a N/P value of 0.5 or above, displayed effective delivery of HIC1 gene which inhibits cancer cell metastasis. The MUC-1 aptamer was conjugated to the dendrimer to target human colon adenocarcinoma HT29 cells, mouse colon adenocarcinoma C26 cells, and Chinese hamster ovary (CHO cells) in vitro and in vivo. [123] Both AuNP and curcumin (CUR), as natural polyphenol anticancer agent, were entrapped in the G5 PAMAM dendrimer. This nanoplatform avoided for CUR the low water solubility, biodisponibility, and strong global clearance. The selectivity of the gold effect of curcumin delivery was ensured by the presence of the attached MUC-1 DNA aptamer with the sequence: 5'-thiol-C6 linker-GCAGTTGATCCTTTGGATACCCTGGTTTTTTTTTT-3, which targets MUC-1 receptors overexpressed in several tumor cells including colorectal, breast, prostate carcinoma, and lymphocytic leukemia. [124] The unmodified G5 PAMAM dendrimer had a zeta potential of 37.6 mV, whereas the PEGylated G5 PAMAM dendrimer showed a zeta potential of 8.2 mV in the presence of AuNPs over 4.6 mV in the presence of the conjugated MUC-1 DNA aptamer. Cellular uptake assays using flow cytometry showed higher cellular uptake of the MUC-1-PEG-AuPAMAM-CUR formulation versus PEGAuPAMAM-CUR formulation in HT29 cells. This effect resulted in the high affinity of the MUC-1 DNA aptamer for the MUC-1 protein on the surface of HT29 cells. In contrast, the MUC-1-PEG-AuPAMAM-CUR and PEGAuPAMAM-CUR formulations showed the same uptake levels in the CHO cell line without MUC-1 expression. Cytoxicity studies of Apt-PEG-AuPAMAM-CUR, PEGAuPAMAM-CUR and curcumin were determined in two MUC-1 positive cell lines (HT29 and C26) and a MUC-1 negative cell line (CHO). Curcumin induced greater cytotoxicity than PEG-AuPAMAM-CUR in the HT29 and C26 cell lines. Apt-PEG-AuPAMAM-CUR formulation induced more cytotoxicity in the HT29 and C26 cell lines than PEG-AuPAMAM-CUR formulation without targeting moieties, demonstrating the crucial role of the MUC-1 DNA targeting effect. PEG-AuPAMAM-CUR and Apt-PEGAuPAMAM-CUR did not displayed significant cytotoxicity in MUC-1 negative CHO cell line. In addition, an in vivo study was performed by the subcutaneous implantation of C26 mouse colorectal adenocarcinoma in Balb/C mice. The analysis of tumor growth inhibition, at a dose of 5 mg/kg iv administration twice during 30 days, showed greater efficacy of Apt-PEG-AuPAMAM-CUR over PEG-AuPAMAM-CUR, as well as greater efficacy than curcumin alone or PEG-Au-PAMAM. EPR effect can explain the higher therapeutic profile of PEG-AuPAMAM-CUR versus CUR alone.

A noticeable study has been described by Wang et al. about the design and development of G3-G5 hydroxyl-terminated PAMAM AuNPs for glutathione(GSH)-responsive thiolated DOX and cisplatin drug delivery nanosystems. [125] Also, based on the same strategy, captopril (an antihypertensive drug) and 6-mercaptopurine (an antileukemic drug) were covalently linked to AuNPs (Figure 4). In this system, GSH played the role of a specific trigger (Scheme 13). The drug molecules were covalently linked with Au through Au-S linkages within the voided space of the dendrimer. The shape of G5 HO-PAMAM:Au NPs was globular form with a size around 3 nm. In summary, Doxorubicin, doxorubicin-SH, curcumin, 6-mercaptopurine, captopril and cisplatin SH were delivered using Au DENPs. Besides the use of Au DENPs for CT imaging, attempts have also been directed to the use of Au DENPs for photothermal therapy. For instance, Kono et al. [126] used poly(ethylene glycol) (PEG)-modified generation 4 (G4) poly(amidoamine) (PAMAM) dendrimers to entrap Au NPs, and found the formed Au DENPs possessed light-induced heat generation ability. However, due to the small Au core size (2 nm), the Au DENPs only displayed limited photothermal conversion efficiency, which is inadequate for further photothermal therapy (PTT) applications. In order to overcome this issue, dendrimer-stabilized Au NPs with a size around 30 nm [127] or dendrimer-stabilized Au nanorods [128] have been used for PTT of tumors. However, in this case, the dendrimer-associated Au NPs no longer belong to the category of Au DENPs.

Dendrimer/dendron-complexed gold(III) nanoparticles: technologies to take down cancer cells

Majoral and Mignani were pioneers in the development of dendrimer-complexed gold(III) nanoparticles to take down tumor cells. Biocompatible phosphorus dendrimers were selected as templates. In the cancer chemotherapy field, cisplatin, oxaliplatin, carboplatin, and Pt(IV) complexes as prodrugs are usually used, but with main drawbacks encompassing resistance and severe toxicity. In addition, other metal complexes to tackle tumors have been developed including, Ru(II/III)-, Au(I/III)-, Ga(III)-, Ti(IV)-, V(IV/V)-, Cu(I/II)-, Fe(II/III)-, and Zn(II)-based complexes. [129] Firstly, Majoral et al. reported the drawing and synthesis of biocompatible first-in-class multivalent Cu(II)-conjugated phosphorus dendrimers with a cyclotriphosphazene ring as core and Cu(II) chelating agents such as N-(pyridin-2-ylmethylene)ethanamine (called NN), N-(di(pyridin-2-yl)methylene) ethanamine, and 2-(2-methylenehydrazinyl)pyridine, with several generations (1-3). Based on the phenotypic approach, the phosphorus dendrimer of generation 3, with an N-(pyridin-2-ylmethylene)ethanamine chelating group complexing Cu(II) (called 1G3-Cu, with 48 metallic moieties on the surface; Figure 5) displayed the highest anti-proliferative efficiency against the epidermal carcinoma KB and leukemia, promyelocytic HL60 tumor cell lines with IC50s ~0.5-2 µM. Note that the G3-phosphorus dendrimer with the N,N chelating group without Cu(II) (called 1G3) also showed interesting antiproliferative activity but with higher IC50s ~0.4-1.3 µM. [130], [START_REF] Mignani | Phosphorus dedrimers in biology and nanomedicine[END_REF] 1G3-Cu and 1G3 showed similar antiproliferative activities versus a panel of tumor cells such as HCT 116 (human colon cancer), OVCAR8 (ovarian carcinoma), MCF7 (hormone-responsive breast cancer), and U87 (human glioblastoma astrocytoma epithelia-like) with IC50s ~0.3-0.8 µM). Remarkably, the complexation with Cu(II) of the terminal ligands NN (1G3-Cu) strongly intensified the IC50 values (~4-10-fold) in normal cell lines such as EPC (endothelial progenitor, Cyprinus carpio) and MCR5 (proliferative human lung fibroblasts) referred to as "safety" cell lines. Investigation of the mechanism of action of 1G3-Cu and 1G3 showed that they induced cell death through the activation of the apoptosis process. Very interestingly, the pro-apoptotic Bax protein was highly activated by 1G3-Cu and translocated to the mitochondrial compartment. Then the release of apoptosis inducing factor (AIF), and finally the activation of the caspase-independent apoptosis process were observed.

Then, based on these very interesting results, the same team turned to the development of original G3 phosphorus dendrimer-complexed gold(III) nanoparticles (called 1G3-Au(III)) as anticancer agents. The anti-proliferative activities of 1G3-Cu(II) (1G3-[CuCl2]48) and 1G3-Au(III) (1G3-[Au48][AuCl4]48; Figure 18) as well the safety ratio are presented in Figure 6. [73,74] The exact structure of the gold(III) complex on the surface of G3 phosphorus dendrimers was determined by the X-ray structure of the Au(III)-monomer complex 1 (Figure 7). The complexation of the G3 phosphorus dendrimer with Au(III) in place of Cu(II) strongly increased the antiproliferative activity against both the KB and HL-60 tumoral cell lines, showing IC50s in the low nanomolar range. Gold(III) conjugated phosphorus dendrimers showed low activity on the quiescent cell line EPC versus its potent antiproliferative activity against actively dividing cells, indicating a good safety ratio. The same results were obtained but to a lesser degree against the MRC5 cell line. Based on the strong collaboration between Majoral, Mignani, and Shi, the development of potent anticancer G1 phosphorus dendrons, bearing Cu(II) and Au(III) units, has been achieved (Figure 10). [133] These metaled dendrons exhibited significant antiproliferative activity versus aggressive breast cancer cell lines such as 4T1 mouse breast adenocarcinoma cells 4T1 which are highly tumorigenic and invasive tumor cell lines, and MCF-7 human breast adenocarcinoma cells. The safety aspect of these dendrons was evaluated using fibroblast NIH-3T3 cells and human fetal lung fibroblast MRC5 cells (Figure 11). As shown in Figure 11, the antiproliferative activity of 1GC11-Cu(II) was better than that of 1GC17-Cu(II) against both 4T1 and MCF-7 cells with IC50s ~0.6-1.5 µM over ~1-2.7 µM, respectively. These two metaled dendrons displayed safety ratios between 2.5 and 4.

Conclusion and future directions

There are many types of cancer treatments, and nanomedicine is part of this strategy. [134] One of the main challenges in nanomedicine is to increase delivery efficiency to the tumor, which is typically less than 1%. [135] Within the nanotechnology domain, a theranostic strategy has been developed to treat cancers. Promising theranostic agents/tools include metal nanoparticles, such as iron, gold, silver, zinc, and titanium, as these play crucial dual roles as 1) diagnostic and 2) active therapeutic agents to take down cancers. This original review highlights the development of functionalized dendrimer encapsulating gold nanoparticles as a novel class of theranostic (radio)pharmaceutical in cancer therapy. Several techniques were described and analyzed including 1) dendrimer-entrapped gold(0) hybrid nanoparticles for targeted imaging and treatment, 2) Au DENPs for gene and drug delivery applications, 3) dendrimer-entrapped gold radioactive nanoparticles for radiotherapy, and 4) dendrimer-complexed gold(III) nanoparticles as technologies to attack cancer cells. One of interesting and promising directions in the theranostic field is to develop stable and biocompatible non-invasive functionalized dendrimer-entrapped gold nanoparticles (AuNPs). Dendrimers have been used as both templates and nanoreactor hosts to engender Au(0) inside the cavities of dendrimers. Specific functionalizations (e.g. PEG chains, acetylation of terminal amino groups, introduction of a targeting ligand, and labeled/contrast agents (e.g. 99m Tc, 177 Lu) to dendrimers have allowed the development of non-toxic, non-immunogenic, and tumor-specific Au(0) nanoparticles for dual mode SPECT/CT imaging of tumor cells in vitro and in vivo. SPECT and CT techniques are fully integrated and translated into clinical practice. This domain is innovative, for instance by the use of the ultrasound-targeted microbubble destruction (UTMD) technique to promote the simultaneous delivery of anticancer agents and genes. [109] The majority of studies used high generation of PAMAM dendrimers (e.g. G4-G5). Importantly, the development of low generation of PAMAM dendrimers (e.g. G2) has also been described to solve several pitfalls observed with high PAMAM generation, such as decreasing the cost of development and increasing the stability as well as the chemistry versatility for GMP grade production. [91] Au DENPs are usually prepared using amine-terminated high-generation (G4 or above) PAMAM dendrimers as templates under appropriate Au salts/dendrimer molar ratio. [136] For biomedical applications, it is often required to modify the dendrimer terminal amines with acetyl groups. [137] However, in order to further improve the Au loading within the dendrimers and to improve the biocompatibility of Au without compromising their colloidal stability, the Au DENPs can always be prepared with dendrimers partially modified with PEG [138]or zwitterions, [139] followed by acetylation of the remaining dendrimer terminal amines. -FA, -Bombesin, -Gastrin-releasing peptide receptor (GRPR) [111] *Ratio of the different moieties not indicated One of the hopes of the development of nanoparticles in oncology was to develop therapeutic nanoparticles with targeting agents to specifically target tumor cells. This has led to a plethora of publications but unfortunately, to date, no clinically approved drugs despite at least 30 years of research by many laboratories and pharmaceutical companies. The cost of the development of this type of dendrimer with targeting ligands remains an important issue. Consequently, the development of low generation of dendrimers could be an interesting approach, including the development of non-invasive functionalized Au DENPs in the theranostic field.

Another important point in the oncology domain is to improve the treatment of aggressive cancers encompassing triple negative breast cancers, glioblastoma, and pancreatic cancers, as developed by Shi et al. using ultrasound-targeted microbubble destruction (UTMD). [109] The treatment of metastatic cancers like breast cancers remains a challenge to be addressed by nanoparticle-based treatment. The development of first-in-class dendrimer/dendroncomplexed gold(III) nanoparticles opens new avenues in the oncology domain, as highlighted by several original studies. [START_REF] Mignani | [END_REF], [133] Finally, in the nanomedicine domain, the translational pathway through the preclinical stage to clinical trials and the commercialization of dendrimers/dendrons included in the dendrimer space concept [8] as drugs or nanocarriers needs to be boosted in order to cross the "valley of death" between promising research and pharmaceutical industry resources for clinical trials. [140] A specific 2018 issue in honor of Prof. D. Tomalia advocated the recent advances in the field of properties and applications of dendrimers.

[141] As so, aspects, like safety, polydispersity (or as recommended by IUPAC only dispersity) in different solvents, long term colloidal stability and lab scale-up, among, others cannot be forgotten in the development of the nanotherapeutics drugs in general and particularly in theranostic (radio)pharmaceuticals for cancer therapy.

Scheme 12. Illustration of the synthesis for the pH responsive theranostic nanoplatforms with Au encapsulating {(Au0)50-G5.NHAc-FA-DOX} (in short DOX-Au DENPs) Modified reproduction with permission from Ref. [122] Copyright 2020. ACS publications.

Scheme 13. Schematic preparation of G5 HO-PAMAM:Au NPs and drug release principle using GSH Modified reproduction with permission from Ref. [125] Copyright 2020. ACS publications.

  groth factor, b: N-(2-hydroxypropyl)methacrylamide, c: Arg-Gly-Asp peptide, d: poly(lactic-co-glycolic acid), e: superparamagnetic iron-oxide nanoparticles, f: polylactic acid, g: polyacrylic acid, h: 1,1-dioctadecyl-3,3,3,3tetramethylindocarbocyanine perchlorate, i: per-O-methyl-cyclodextrin-grafted-hyaluronic acid, j: poly(Nisopropylacrylamide), k: herpes simplex virus thymidine kinase, l : ganciclovir, m : poly-methacrylic acid (PMAA), polyacrylic acid (PAA)

Scheme 3 .

 3 Scheme 3. Examples of the preparation of Au DENPs and Au DSNPs. Modified reproduction with permission from Ref. [86] Reproduced by permission of The Royal Society of Chemistry

Scheme 4 .

 4 Scheme 4. (a) Schematic preparation of {(Au0)6-G2-NOTA( 99m Tc)-PEG-RGD} DENPs and (b-d) in vivo SPECT/CT imaging of tumors using {(Au0)6-G2-NOTA( 99m Tc)-PEG-RGD} DENPs and nontargeted {(Au0)6-G2-NOTA( 99m Tc)-mPEG } DENPs. Modified reproduction with permission from Ref. [91]. Reproduced by permission of The Royal Society of Chemistry

Scheme 6 .

 6 Scheme 6. Synthetic pathways for the preparation of {(Au 0 )200-G5.NHAc-DOTA-FI-mPEG-(PEG-duramycin)} DENPs ( 99m Tc-duramycin-Au DENPs).

  Flow cytometry and confocal microscopy studies with {(Au 0 )200-G5.NHAc-DOTA-FI-mPEG-(PEG-duramycin)} DENPs against C6 cancer cells showed reinforced fluorescence signals versus the control Au DENPs. In addition, SPECT/CT imaging of C6 cells using in vitro and subcutaneous in vivo xenografted tumor model in mice showed few accumulation of the

Scheme 7 .

 7 Scheme 7. Synthetic pathway for the preparation of Au-TOS-RGD DENPs

Scheme 8 .Scheme 9 .

 89 Scheme 8. Synthetic pathways for the preparation of {(Au 0 )25-G5.NHAc-(PEG)14-(Fluo-4)2} nanoprobe (a), and dual-mode CT/fluorescence images of T cells in vivo (b,c) Modified reproduction with permission from Ref. [104] Copyright 2020 ACS publications

Figure 1 .

 1 Figure 1. An ultrasound-enhanced drug and gene co-delivery system based on Au DENPs. Adapted reproduction with permission from Ref. [109] Copyright 2020. Open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license

Figure 2 .

 2 Figure 2. 2D representation of 177 Lu-DenAuNP-folate-bombesin nanoparticle. Ref. Modified reproduction with permission from Ref. [110] Copyright 2020. Open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license

Figure 3 .

 3 Figure 3. Global view of the cellular delivery process of genes using Au DENPs-β-CD/siRNA polyplexes. Modified reproduction with permission from Ref.[116] Copyright 2020. Open access article MDPI

Scheme 10 .Scheme 11 . 2 . 4 .Scheme 12 .

 10112412 Scheme 10. Schematic description of the preparation of {(Au 0 )25-G5-CBAA-PEG-Mor} DENPs. For the abbreviations, see text. Reproduced with permission from Ref. [119] Copyright 2020. Elsevier.

Figure 4 .

 4 Figure 4. Representation of chemical Structures of thiol-containing (captopril and 6mercaptopurine) and thiolated drugs (DOX and cisplatin)

Figure 5 .

 5 Figure 5. 2D chemical structure of 1G3-Cu(II) and 1G3-Au(III)

Figure 6 .Figure 7 .

 67 Figure 6. The anti-proliferative activities and safety ratio of 1G3-Cu(II) and 1G3-Au(III)

Figure 8 .

 8 Figure 8. Metal surface distribution of prepared metaled and non-metaled phosphorus dendrimers

Figure 9 .

 9 Figure 9. Antiproliferative activies against KB and HL60 of prepared metaled and nonmetaled phosphorous dendrimers

Figure 10 .Figure 11 .

 1011 Figure 10. 2D chemical structure of 1GC11-Cu(II), 1GC17-Cu(II), 1GC11-Au(III) and 1GC17-Au(III)

  PEG, -Folic acid (FA), -NH2, 99m TC[94] PAMAM -PEG, -NH2, -Duramycin, 99m TC, Fluoresceine isothiocynate (FI)[87] -NH2, -FI, -PEG-Tos (PEGylated alpha-

Figure 1 .

 1 Figure 1. An ultrasound-enhanced drug and gene co-delivery system based on Au DENPs. Adapted reproduction with permission from Ref. [105] Copyright 2020. ACS publications.

Figure 2 .

 2 Figure 2. 2D representation of 177 Lu-DenAuNP-folate-bombesin nanoparticle. Ref. Modified reproduction with permission from Ref. [110] Copyright 2020. Hindawi.

Figure 3 .

 3 Figure 3. Global view of the cellular delivery process of genes using Au DENPs-β-CD/siRNA polyplexes. Modified reproduction with permission from Ref. [54] Copyright 2020. MDPI.

Figure 4 .

 4 Figure 4. Representation of chemical Structures of thiol-containing (captopril and 6mercaptopurine) and thiolated drugs (DOX and cisplatin).

Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .Figure 10 .Figure 11 .

 567891011 Figure 5. 2D chemical structure of 1G3-Cu(II) and 1G3-Au(III)

Table 1 .

 1 Table2depicts selected therapeutic applications of theranostic principle with dendrimers in oncoly except gold NPs which is the aim of this review.[31] Selected TNPS in oncology therapeutic domain

	TNPs types	Materials involved	Anticancer	Contast agenst	Targeting	Ref.
			agents loaded		groups
	Carbon	Carbon	Cisplatin	Quantum dots	EGFa	[32]
	nanotubes

Table 2 .

 2 Selected examples of dendrimers and dendrons for theranostic applications in oncology

	Dendrimer/dendron	Contrast agent	Anticancer agent	Targeting moieties	Ref.
	types
	G5 PAMAM		Fluorecein isothiocyanate		FGFR a	[52]
	G5 PAMAM		Photochemical mechanism	Doxorubicin	Folic acid	[53]
	PEG-G3.5 PAMAM		CdSe/ZnS quantum dots		Folic acid	[54]
	PEG-lipoic	acid	Cy5.5 (NIR probe)	Pt(II) derivatives		[55]
	amphiphilic dendron

  Tc-duramycin-Au DENPs or 99m Tc-Au DENPs in the tumors before DOX treatment, but, interestingly, these NPs were well internalized in tumors in vivo after DOX treatment. Among diverse strategies developed in the oncology field, immunotherapeutic treatments that help immune cells to take down cancers, represent a hopeful strategy. Within this strategy, adoptive cell transfer therapy (ACT) using T lymphocytes from patients has recently gained increasing attention.[96],[97] Interesting work performed by Shi et al. concerns the use of G5 PAMAM dendrimer entrapping gold nanoparticles (Au DENPs) functionalized with fluorescein isothiocyanate (FI), PEGylated alpha-tocopheryl succinate (TOS) and RGD peptide through a PEG chain for chemotherapy targeting and computed tomography (CT) imaging purposes of tumor cells.

	2.2.2.	Computed tomography (CT) approach

99m

Table 3 .

 3 presents the additional studies regarding the development of dendrimer-entrapped radioactive gold nanoparticles. Recently, a review highlighting the use of radiolabeled dendrimers for nuclear medicine applications was published by Zhao and colleagues.[112] Examples of development of dendrimer-entrapped gold radioactive nanoparticles

	Therapeutic area	Radioactive	Radiation	Dendrimers	Results	Reference
		elements	types	used
	Nanobrachytherapy	98 Au(0) γ-radiation	G5-PAMAM Inhibition of tumor	[113]
	of mouse melanoma tumors		and neutron radiation		growth (45%) after intratumoral injection of NPs

Table 4 .

 4 Table 4 presents a selection of chemical surface modifications of Au DENPs Selection of chemical surface modifications of Au DENPs

	Approach	Dendrimer types	Chemical surface moieties*	Ref.
	Combined
	SPECT and
	TC

Table 1 .

 1 Selected TNPS in oncology therapeutic domain

Table 2 .

 2 Selected examples of dendrimers and dendrons for theranostic applications in oncology

Acknowledgements

X. Shi, S. Mignani and J-P. Majoral thank the PRC NSFC-CNRS 2019 (21911530230 for X.S. and 199675 for S.M. and J-P.M). J. Rodrigues, H. Tomas S. Mignani and X. Shi acknowledge the support of FCT-Fundação para a Ciência e a Tecnologia (Base Fund UIDB/00674/2020 and Programmatic Fund UIDP/00674/2020, Portuguese Government Funds) and ARDITI-Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação through the project

Conflit of Interest

The authors declare no conflict of interest.