Serge Mignani
email: serge.mignani@parisdescartes.fr

Xiangyang Shi
email: xshi@dhu.edu.cn

Valentin Ceña

Dzmitry Shcharbin

Maria Bryszewska

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

In vivo therapeutic applications of phosphorus dendrimers: state of the art

His research interest is focused on the design and the properties of macromolecules such as phosphorus dendrimers and hyperbranched polymers. His main efforts are directed at the use of dendrimers in medicinal chemistry, nanotechnologies, material sciences and catalysis. He is a co-founder and scientific director of the start-up Dendris. He is a member of several scientific academies worldwide, has received 17 international awards and has authored more than 685 publications, seven books, 35 book chapters and 45 patents.(h index 72, over 19,000 citations).

Highlights

 In vivo therapeutic applications of phosphorus dendrimers as nanocarriers or drugs  Phosphorus dendrimers as in vivo anti-inflammatory agents  Phosphorus dendrimers as in vivo anticancer agents  Phosphorus dendrimers against infections in in vivo experiments  Phosphorus dendrimers for non-invasive in vivo imaging studies

Introduction

Lammers and Ferrari [START_REF] Lammers | The success of nanomedicine[END_REF] highlight the challenges faced by nanomedicine. High expectations for the field have been stymied by disappointing clinical trial results from BIND Therapeutics and Cerulean Pharma [START_REF] Ledford | The two directions of cancer nanomedicine[END_REF] (see Smarter Analyst, 'Here's Why Cerulean Pharma Inc (CERU) Shares Are Falling 65%', https://www.smarteranalyst.com/stock-news/heres-cerulean-pharma-inc-ceru-shares-falling-65/), as well as a reduction of funding from the US National Cancer Institute (NCI) for the Centers of Cancer Nanotechnology Excellence (CCNEs) [START_REF] Service | US cancer institute cancels nanotech research centers[END_REF]. Nevertheless, Lammers and Ferrari claim that nanomedicine will emerge as a next-generation platform in medicine on the basis of several important successes in both academia and the pharmaceutical industry.

Oncology is the main therapeutic realm in which nanoparticles (NPs) are used. The biggest issue in this field is developing NPs with good tumor penetration; indeed, an analysis found that on average only around 0.7% of an NP dose ends up in tumors [4], although this figure has been challenged [START_REF] Lammers | Cancer nanomedicine: is targeting our target?[END_REF]. However, more than 50 drug products containing nanomaterials have been approved by the US Food and Drug Administration (FDA) for clinical use, and more than a dozen of those were approved in the past decade [START_REF] Blomme | Toxicology strategies for drug discovery: present and future[END_REF].

Various NP types have been developed as nanocarriers of drugs in several therapeutic fields (mainly in oncology) in the form of liposomes, albumin-based particles, biodegradable polymer-drug composites, polyethylene glycol (PEG)ylated proteins, polymeric micelles, polymer-drug conjugate-based particles, inorganic particles, dendrons and dendrimers. A large variety of dendrimers exists [START_REF] Soares | Nanomedicine: principles, properties, and regulatory issues[END_REF]. These macromolecules, such as phosphorus dendrimers [START_REF]Phosphorus Dendrimers in Biology and Nanomedicine: Synthesis, characterization and Properties[END_REF], are a family of nanodevices, and they have been developed as drug-delivery systems, active drugs (in relation to the nature of the terminal groups on their surface) and contrast-bearing agents for imaging purposes. This contextual review describes the in vivo applications of phosphorus dendrimers as drugs in several therapeutic areas: as anti-inflammatory and anticancer agents; for treating neurodegenerative diseases, ocular hypertension and infections; and for non-invasive in vivo imaging. In silico and in vitro studies continue to refine the quality of the drugs; and in vivo preclinical assays remain the gold standard for assessing both human risk and efficiency.

Drug characteristics during the preclinical process

The process of drug discovery and development takes about 10-15 years and costs in excess of US$1 billion, depending on the therapeutic area (see Science Translational Medicine 'Drug Development Costs Revisited', https://blogs.sciencemag.org/pipeline/archives/2017/10/18/drug-development-costs-revisited). Nowadays, the timeline breaks down into six critical steps: (i) disease pathology study, (ii) target identification, (iii) lead identification and optimization (hit-to-lead phase) for investigational new drug (IND) filing, (iv) Phase I-III clinical trials for new drug application (NDA) filing, (v) FDA regulatory approval and (vi) post-market safety monitoring. After the identification and validation of a target (or targets), drug discovery involves several steps, including the discovery of hits (e.g., using phenotypic-and target-based screening or machine-learning approaches) and the optimization of hits using medicinal chemistry to find druggable leads, followed by the selection of the best clinical candidates. The preclinical stage encompasses the use of in vitro and in vivo studies to evaluate the efficacy and safety of the drug candidates up to the clinical-trial stage.

In 2019, 48 new medicines were approved by the FDA, versus 59 in 2018. Eleven of the drugs approved in 2019 were cancer treatments, and 44% of the total were treatments for rare diseases. In addition, 67% of these 2019 medicines were small molecules, 42% were first-in-class drugs and three were antibody-drug conjugates. Novartis added six drugs to its portfolio in 2019 [9].

The objective of the hit-to-lead stage is to produce more potent and selective compounds with adequate pharmacokinetic (PK) and pharmacodynamic (PD) profiles (PK/PD profiles) and to examine their efficacy in any in vivo models relevant to the considered disease. The main challenge of in vivo studies is choosing the animal model (e.g., mice, rats, dogs, cats, minipigs, rabbits, baboons or zebrafishes) that best represents the disease systems that drugs will affect in humans. In vivo studies are essential to evaluate the physiological characteristics and effects in a living organism; biochemical processes of biologically active compounds that are not present in in vitro studies might affect the release of the drug in vivo.

It is not clear whether in vitro toxicodynamic evaluation can predict the in vivo interactions of drugs with organs; furthermore, the absorption, distribution, metabolism, excretion and toxicity (ADMET) parameters cannot provide full quantitative interpretation in animal models and humans [START_REF] Zhang | Preclinical experimental models of drug metabolism and disposition in drug discovery and development[END_REF]. An important point is that in vitro studies have a positive impact on the selection of hits and early leads, helping to avoid failures in later steps of the development process. However, in vitro and in vivo results do not always correlate in terms of, for example, showing bioequivalency [START_REF] Janaszewska | Cytotoxicity of dendrimers[END_REF], toxic effects and the ideal dosage regimen for clinical trials. Consequently, in vivo research is necessary to select a drug that is safe and effective in humans. In vitro results can provide a snapshot of a particular aspect of the drug (e.g., ADMET), but multifactorial in vivo results help to show its combined effects, including its efficiency, PK/PD profile and toxicological effects.

Nanomedicine is an umbrella term, and it is defined as a specific branch of medicine that uses nanotechnology for the prevention, treatment and diagnosis of diseases, as well as for theranostics [START_REF] Tran | Cancer nanomedicine: a review of recent success in drug delivery[END_REF]. Nanodelivery platforms can help to improve the solubility and bioavailability of small molecules, genes and vaccines, as well as reduce their off-target effects. Several approved nanomedicine formulations have been described, encompassing liposomes (e.g., Abelect, a lipid amphotericin B formulation), PEGylated liposomes (e.g., Doxil, PEGylated liposomal doxorubicin), nanocrystals (e.g., Emend), NPs (e.g., Abraxane, paclitaxel albumin-bound particles) [START_REF] Patra | Nano based drug delivery systems: recent developments and future prospects[END_REF][START_REF] Lombardo | Smart nanoparticles for drug delivery application: development of versatile nanocarrier platforms in biotechnology and nanomedicine[END_REF][START_REF] Choi | Nanomedicines: current status and future perspectives in aspect of drug delivery and pharmacokinetics[END_REF] and dendrimers (e.g., VivaGel from Starpharma). Doxil was the first FDA-approved nanodrug in 1995, and recently the generic doxorubicin hydrochloride liposome injection Lipodox (Sun Pharmaceutical Industries) has been approved by the FDA.

Dendrimers in nanomedicine: a concise overview

Dendrimers are monodisperse, carefully assembled NPs of synthetic monomers that form branch-like structures. Examples include polyamidoamine (PAMAM) dendrimers, poly-L-lysine (PL) dendrimers, poly(propylene imine) (PPI) dendrimers, PEG polyester dendrimers, carbosilane dendrimers [16] and phosphorus dendrimers [START_REF]Phosphorus Dendrimers in Biology and Nanomedicine: Synthesis, characterization and Properties[END_REF]. As shown in Figure 1, phosphorus dendrimers enable hollow cavities to form within the branching structures; these cavities can be loaded with drugs, imaging agents, and so on. Both the size and the functionalized surface groups can be tightly regulated or modified. The addition of new generations (G1, G2, etc.) increases the dendrimer's diameter, as well the number of surface groups. Dendrimers can be used as nanocarriers or be active drugs themselves in nanomedicine [START_REF]Dendrimers and Other Dendritic Polymers[END_REF][18][19].

To date, few clinical successes in using dendrimers as nanocarriers or drugs have been reported in the literature. In 2012, Starpharma (Melbourne, Australia) started two pivotal Phase III trials with the G4 PL polyanionic dendron VivaGel (SPL7013 Gel, astodrimer sodium) for the treatment of bacterial vaginosis. This NP has 32 naphthalene disulfonate groups on its surface and demonstrated potent topical vaginal microbicidal activity. Recently, Starpharma and AstraZeneca have moved from Phase I to Phase II with a PL dendrimer-based nanocarrier encapsulating docetaxel (named DEP docetaxel), which shows higher anticancer activities against breast, prostate, lung and ovarian tumors than docetaxel alone.

Among around 100 types of dendrimer, those incorporating phosphorus groups represent a wonderful opportunity to extend the dendrimer functionalization space. The preparation of phosphorus dendrimers starts generally from commercially available hexachloro-cyclo(triphosphazene), and they are easy to synthesize in large-gram-scale and soft-reaction conditions, with high yields. All the by-products can be removed without wordly techniques [START_REF]Phosphorus Dendrimers in Biology and Nanomedicine: Synthesis, characterization and Properties[END_REF]. In addition, several types of AB3, AB5 and CD3 or CD5 monomers have been prepared, allowing the preparation of a variety of dendrimers (e.g., [START_REF] Caminade | Multicharged and/or water-soluble fluorescent dendrimers: properties and uses[END_REF]), as well as hybrid phosphorus-viologen dendrimers [START_REF] Katir | Viologen-based dendritic macromolecular asterisks: synthesis and interplay with gold nanoparticles[END_REF], onion peel dendritic structures [START_REF] Katir | Synthesis of onion-peel nanodendritic structures with sequential functional phosphorus diversity[END_REF] and Janus dendrimers [START_REF] Caminade | Janus" dendrimers: syntheses and properties[END_REF].

In drug discovery, one of the leading challenges is the identification of specific biologically active compounds with adequate therapeutic and pharmacological properties to become a medicinal product. In the nanomedicine domain, one important aspect for the development of NPs such as dendrimers is to consider these nanodevices as active drugs themselves. Thus far, the majority of engineered NPs have been developed for drug delivery, mainly in cancer therapy [16]. To explore the huge chemical space with dendrimer collections as nanocarriers and as drugs, we introduced the concept of dendrimer space as a druggable cluster [START_REF] Mignani | Dendrimer space concept for innovative nanomedicine: a futuristic vision for medicinal chemistry[END_REF]. Notably, the multidimensional limits of drug-dendrimer space can be associated with their biological and pharmacological profile. Within the huge dendrimer space, the development of phosphorus-based dendrimers has been outstandingly performed in several directions, encompassing, for instance, hybrid materials, catalysts, nanotubes for diagnosis, DNA chips, fiber formation and nanomedicine (e.g., [START_REF] Wasiak | Phosphorus dendrimers affect Alzheimer's (Aβ1-28) peptide and MAP-Tau protein aggregation[END_REF]).

Huge numbers of therapeutic areas have been well considered with phosphorus dendrimers, both as drugs and nanocarriers [e.g., small interfering RNAs (siRNAs)]. The majority of the PAMAM dendrimers have been used as nanocarriers, mainly in oncology (as depicted by several studies from Tomalia [START_REF] Tomalia | Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging[END_REF], Backer [27] and Newkome [START_REF] Zechel | From dendrimers to macrocycles: 80 Years George R. Newkome-milestones of a gentleman scientist[END_REF]), using Fréchet's polyester dendrimer-[poly(ethylene oxide)] ('bow-tie' dendrimer) [START_REF] Gillies | Designing macromolecules for therapeutic applications: polyester dendrimer-poly(ethylene oxide) "bow-tie" hybrids with tunable molecular weight and architecture[END_REF] and Simanek's triazine dendrimers [START_REF] Lim | Antitumor activity and molecular dynamics simulations of paclitaxel-laden triazine dendrimers[END_REF]. In addition, theranostic applications in cancer have been developed by Shi, who used non-invasive functionalized PAMAM-entrapped gold(0) NPs [START_REF] Xu | Targeted tumor SPECT/CT dual mode imaging using multifunctional RGD-modified low generation dendrimer-entrapped gold nanoparticles[END_REF], and Tomalia [32]. Few in vivo studies of antitumor agents using the dendrimer approach have been published [16].

A remarkable example of the architecture optimization of dendrimers has been reported by the Fréchet group [START_REF] Lee | A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas[END_REF]. They prepared an asymmetric, biocompatible, doxorubicin-functionalized bow-tie dendrimer that showed potent in vitro and in vivo antiproliferative activities against C-26 colon carcinoma cells. In addition, an innovative strategy against difficult cancers such as pancreatic cancer has been presented by Shi et al. [START_REF] Lin | UTMD-promoted co-delivery of gemcitabine and miR-21 inhibitor by dendrimerentrapped gold nanoparticles for pancreatic cancer therapy[END_REF]. 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) and miR-21 inhibitor (miR-21i) from PEGylated G5 PAMAM dendrimer-entrapped gold NPs (Gem-Au DENPs and miR-21i NPs).

Whereas most other types of dendrimer have chiefly been used in classical research experiments, phosphorus dendrimers have tended to be used as drugs, as well as to deliver siRNAs. In this review, we survey and analyze successes in the development of phosphorus dendrimers in vivo. These in vivo studies represent fundamental pharmacological knowledge in drug discovery and the development of phosphorus dendrimers in animal models, and ultimately in humans. Importantly, the main challenge in in vivo studies is choosing an animal model that best illustrates the human disease. The many physiological differences between animals and humans do not allow for completely similar results if the drug testing reaches the clinical-trial stage, even when using primates, which is strongly recommended by the regulatory agencies (the FDA and European Medicines Agency).

Crucial chemical parameters for preclinical therapeutic applications of dendrimers: general aspects

It is generally thought that the in vitro and in vivo therapeutic applications of dendrimers are mainly dependent on the nature of their surface chemical units (e.g., polycationic, polyanionic and neutral moieties). In this regard, several of us analyzed the different parameters to delineate new 'druggable' clusters within the chemical space (vide supra). These clusters can be delimited using 'drug-likeness' parameters, which are fully integrated into the practical drug-discovery process. Both in vitro and in vivo physicochemical parameters were evaluated for preclinical evaluation of biomedical dendrimers [START_REF] Mignani | Exploration of biomedical dendrimer space based on in-vitro physicochemical parameters: key factor analysis (Part 1)[END_REF][START_REF] Mignani | Exploration of biomedical dendrimer space based on in-vivo physicochemical parameters: key factor analysis (Part 2)[END_REF]. For instance, in the toxicity domain, there are two main strategies to tackle dendrimers' toxicity issues: the preparation of biodegradable and biocompatible scaffolds (for the core and branches), and/or surface modifications. Jain et al. proposed an interesting approach involving the development of non-toxic polypropyletherimine (PETIM) dendrimers [START_REF] Jain | Poly propyl ether imine (PETIM) dendrimer: a novel non-toxic dendrimer for sustained drug delivery[END_REF]. Also, to extend the usage scope of dendrimers in nanomedicine, Haag and colleagues have conducted an important analysis of the in vivo druggability requirements of multifunctional dendritic polymers [START_REF] Khandare | Multifunctional dendritic polymers in nanomedicine: opportunities and challenges[END_REF]. Figure 2 presents an overview of the physical characteristics of NPs, including dendrimers, in terms of in vivo biocompatibility profile.

Influence of the size and chemical structure of phosphorus dendrimers on therapeutic applications

It is widely known that, against various infection types, the activation of monocytes and the amplification of functional natural killer cells boost the innate immune system's first line of defense against 'assailants' [START_REF] Fruchon | Pro-inflammatory versus anti-inflammatory effects of dendrimers: the two faces of immuno-modulatory nanoparticles[END_REF].

Rolland and colleagues conducted a study on the influence of the end phosphonate groups of several phosphorus dendrimers on the cellular activation of human monocytes [40]. In a similar vein, an interesting study has been conducted by the Toulouse team on the key role of the scaffold in the efficiency of dendrimers [START_REF] Caminade | The key role of the scaffold on the efficiency of dendrimer nanodrugs[END_REF]. Thirteen dendrimers from seven different families were studied using a molecular dynamic simulation technique; the study included PAMAM, PPI, poly(carbosilane) and PL dendrimers along with three phosphorus dendrimer types. The dendrimers had different internal (core) structures but identical azabisphosphonic terminal groups (N[[CH 2 -P(O)(OH)ONa)] 2 ), which activate human monocytes. This study confirmed that the geometry of the scaffold had an important role in therapeutic efficiency. In a similar way to how the scaffold of a simple drug molecule interacts with, for instance, a receptor, the change in shape and the spatial distribution of the terminal moieties of the dendrimers influenced their biological activity. The most potent dendrimers have their surface functions assembled on one side to potentially maximize interactions with the biological target.

Phosphorus dendrimers as anti-inflammatory agents

Living beings respond to a variety of insults, such as the presence of pathogens or damaged cells, with an inflammatory response that generally includes activation of both the innate and adaptive responses. An inflammatory response is generally acute and resolves after the originating cause has disappeared. However, under certain circumstances, inflammation becomes chronic, causing damage in different organs [START_REF] Chen | Inflammatory responses and inflammation-associated diseases in organs[END_REF]. Autoimmune diseases are particular cases of chronic inflammation in which the recognition of the body's own cells and tissues as 'self' fails, and the organism generates an immune response against selfantigens, causing damage to tissues and organs. Inflammation has a key role in the pathophysiology of a number of diseases, including arthritis, asthma, multiple sclerosis and inflammatory bowel disease.

Recently, Posadas et al. reported that neutral and biocompatible G3 and G4 phosphorous dendrimers, with 48 and 96 terminal biphosphonate groups, respectively, behaved as potent anti-inflammatory agents (Figure 3a). Both compounds were not toxic, up to a concentration of 10 µM, for inflammatory cells such as mouse and human macrophages or lymphocytes, and had good water solubility and stability in aqueous solutions [START_REF] Posadas | Neutral high-generation phosphorus dendrimers inhibit macrophagemediated inflammatory response in vitro and in vivo[END_REF]. In mice, purified peritoneal macrophages collected after thioglycolate intraperitoneal injection were stimulated using lipopolysaccharide (LPS), inducing a robust inflammatory response characterized by activation of nuclear factor-κB (NF-κB) induction, inducible nitric oxide synthase (iNOS) expression, an increase in nitric oxide production and secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β). Moreover, LPS also induced the expression of CD86, a marker for the macrophage M1 pro-inflammatory population. The presence of either G3 or G4 phosphorous dendrimers markedly decreased all LPS-induced inflammatory markers and decreased CD86 levels, restoring levels of the macrophage M2 anti-inflammatory population marker CD163. Taken together, these results indicate that neutral phosphorous dendrimers can switch the macrophage population from the pro-inflammatory M1 subtype to the anti-inflammatory M2 subtype, helping to resolve inflammation.

Neutral phosphorous dendrimers also showed anti-inflammatory properties in vivo against a subchronic model of inflammation: the air pouch. Zymosan injection into the air pouch induced an inflammatory response with increased iNOS expression and nitrite levels in the infiltrating cells, as well as an increase in the macrophage M1 population marker CD86. Administration of G3 or G4 phosphorous dendrimers [10 mg/kg, intravenous (i.v.)] caused a decrease in inflammation markers in the surrounding tissue as well as a decrease in CD86 and an increase in CD163, the marker for the anti-inflammatory M2 macrophage population.

Taken together, these data indicate that neutral G3 and G4 phosphorous dendrimers have antiinflammatory properties both in vitro and in vivo, and are able to switch macrophages from a proinflammatory M1 population towards an anti-inflammatory M2 population, which would help to resolve inflammation In the realm of chronic inflammatory disease, and osteoclastogenesis, i.v. injection of the G1 anionic phosphorus dendrimer has been performed in two different murine animal models of autoimmune arthritis: IL-1ra -/-mice and serum transfer from K/BxN mice. These two animal models are most relevant to human rheumatoid arthritis [44]. In the IL-1ra -/-mice model, the animals spontaneously develop autoimmune-like arthritis at 4 weeks old, whereas in the K/BxN serum-transfer arthritis model, analyses of the immunological mechanisms occurring in rheumatoid arthritis and other arthritis types can be evaluated [45,46]. The decrease of disease severity was determined using three different biomarkers: (i) normal synovial membranes, (ii) reduced levels of inflammatory cytokines and (iii) absence of cartilage destruction and bone erosion. In this study, three dendrimer types were compared against inflammatory disease: G1 azabisphosphonate (compound 1 in Figure 3b), G1 azamonophosphonate (compound 2 in Figure 3b) and G1 bisphosphonate PPI dendrimer (compound 3 in Figure 3b).

Arguably, the authors demonstrated that compound 1 inhibited the expression of pro-inflammatory cytokines in monocytes in vitro and also activated monocytes, increasing IL-10 secretion by CD4 + T cells. At a dose of 10 mg/kg, only compound 1 demonstrated significant efficacy in the two animal models at 15 weeks of age, on the basis of hind-paw swelling and clinical arthritis score evaluation analysis. Interestingly, histopathological analysis of the ankle joints revealed near-normal synovial membranes versus hyperplasia, inflammation and eroded cartilage in untreated mice. In addition, no osteoclasts corresponding to the multinucleated cells responsible for bone resorption or cells expressing the arthritis-associated cytokine IL-17 were observed in the bone matrix in mice treated with compound 1. Using hematoxylin-eosin (a stain in histology allowing for the localization of nuclei and extracellular proteins), the histological scoring analysis showed a 50% and 80% reduction with compound 1 at 0.1 and 10 mg/kg, respectively. Also, antiosteoclastic activity on mouse and human cells has been demonstrated with this dendrimer. A recent review highlighted and analyzed the different immunomodulatory effects of compound 1 [START_REF] Fruchon | The ABP dendrimer, a drug-candidate against inflammatory diseases that triggers the activation of interleukin-10 producing immune cells[END_REF].

The potential use of compound 1 for the treatment of multiple sclerosis has recently been highlighted [48]. Multiple sclerosis is a chronic inflammatory diseases of the central nervous system. Compound 1 displayed potent efficiency in a mouse model of multiple sclerosis, in which myelin oligodendrocyte glycoprotein (MOG35-55) induces experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. The macromolecule compound 1 inhibited the progression of the neuroinflammation by promoting IL-10producing CD4 + T cells, which produce pro-inflammatory cytokines such as interferon-γ and IL-17. In this in vivo study, mice were treated with 10 mg/kg of compound 1 (i.v.) on day 1, day 3 and then every 3 days after that.

Compound 1 has also been tested in a rat model of acute uveitis induced by LPS. The authors showed that at a dose of 2 μg per eye, it was as efficient as 'standard' dexamethasone at 20 μg per eye. This antiinflammatory activity is mediated through an increase of production of the anti-inflammatory IL-10 cytokine [START_REF] Fruchon | An azabisphosphonate-capped poly(phosphorhydrazone) dendrimer for the treatment of endotoxin-induced uveitis[END_REF].

Repeated intravenous injections of compound 1 (10 mg/kg on days 0, 7, 14 and 21) in male cynomolgus macaques (Macaca fascicularis) were demonstrated to be safe, with no specific side effects. During the study, biochemical, hematological and immunological parameters remained within the normal physiological range. The administered dose was tenfold higher than the lowest active dose (1 mg/kg) identified in the mouse model of chronic inflammatory disorder rheumatoid arthritis [START_REF] Fruchon | Repeated intravenous injections in non-human primates demonstrate preclinical safety of an anti-inflammatory phosphorus-based dendrimer[END_REF].

Note that recently an interesting analysis of the pro-inflammatory versus anti-inflammatory effects of dendrimers has been conducted by Fruchon and Poupot [START_REF] Fruchon | Pro-inflammatory versus anti-inflammatory effects of dendrimers: the two faces of immuno-modulatory nanoparticles[END_REF].

For imaging purposes, two different G1 phosphorus dendrimer types were prepared, with 24 terminal azabisphosphonate groups (carried by five main branches) and one main branch bearing a fluorophore probe such as julolidine (green fluorescence) and a specific fluorophore probe emitting near-infrared fluorescence that does not overlap with the auto-fluorescence of tissues [START_REF] Fruchon | Biodistribution and biosafety of a poly(phosphorhydrazone) dendrimer, an anti-inflammatory drug-candidate[END_REF]. The fluorescent phosphorus dendrimers displayed the same biological profile as compound 1 (in vitro in human monocyte models and in vivo in a mouse model of arthritis). In mice, the dendrimers were mainly detected in the lungs and liver (10 mg/kg i.v., single injection) rather than the spleen and kidney. Importantly, no bioaccumulation of these macromolecules was detected, because the total fluorescence decreased slowly over time. After intraperitoneal and subcutaneous administration, no distribution was observed, and the dendrimers stayed at the site of injection for several days. The maximum tolerated dose, which defines the dose and schedule for Phase I clinical trials, of compound 1 is 100 mg/kg for a single injection and 60 mg/kg/day for repeated injections over seven consecutive days. These data indicate the favorable therapeutic window for compound 1. Generally speaking, a good safety profile was demonstrated, except in genotoxicity assays that showed potential mutagenicity, but at doses up to 100 times greater than the in vitro active dose against human immune cells.

An interesting study by Bohr and Fattal investigated the anti-inflammatory effect of anti-TNFα siRNA G3 cationic phosphorus dendrimers in a murine acute lung injury model after intranasal administration [START_REF] Bohr | Anti-inflammatory effect of anti-TNF-α siRNA cationic phosphorus dendrimer nanocomplexes administered intranasally in a murine acute lung injury model[END_REF]. Asthma, chronic obstructive pulmonary disease and acute lung injury are lung diseases that involve inflammation, and TNFα plays a key part in this inflammation process. In this study, two different phosphorus dendrimers were used as nanocarriers, bearing 48 protonated pyrrolidinium (named DP) or morpholinium (named DM) terminal groups (Figure 3c).

The dendriplexes had mean sizes of 120-190 nm, polydispersity indexes of 0.27-0.44, hydrodynamic diameters of around 3 nm and a positive zeta potential at N/P ratios of 5, 10 and 20. The biological studies were carried out using dendriplexes with N/P of 5 in order to maximize the amount of siRNA associated with the dendriplexes. In cytotoxicity studies against RAW 264.7 cells (monocyte/macrophage-like cells), the DM dendriplexes did not exhibit cellular viability reduction (IC 50 > 15 μM siRNA), unlike the DP dendriplexes, which showed a reduction of cellular viability with an IC 50 siRNA concentration of 470 nM (dendrimer concentration: 41 μg/ml). Using flow cytometry, cell-uptake studies demonstrated that DM dendriplexes showed low uptake over time, unlike DP dendriplexes, which displayed significant uptake of siRNA.

Interestingly, using RAW 264.7 cells, flow-cytometry data indicated that DP dendriplexes were mainly taken up by cells, whereas DM dendriplexes mainly adhered to the outer surface of the cell membrane as aggregates. Enhanced in vitro silencing efficiency of TNFα in the LPS-activated mouse macrophage cell line RAW264.7 was observed with DP dendriplexes versus DM dendriplexes. In an LPS-induced murine model related to a short-term acute lung injury, DP dendriplexes showed a stronger silencing effect than noncomplexed siRNA via nasal administration. Indeed, this small chemical surface change, the introduction of morpholinium (DM) or pyrrolidinium (DP) groups, induced strong in vitro and in vivo effects in several inflammatory assays. Isothermal titration calorimetry and molecular dynamics simulations were performed by Deriu and Danani to evaluate the molecular factors influencing the different efficiencies and activities of DP and DM [START_REF] Deriu | Elucidating the role of surface chemistry on cationic phosphorus dendrimer-siRNA complexation[END_REF]. Chemical surface modification drives the dendrimer-siRNA affinity, as well the flexibility/rigidity ratio. Rigid polycationic dendrimers reorganized their peripheral groups to enhance contacts with the nucleic acid units of siRNA. DP dendrimers displayed better efficiency than DM dendrimers based on: (i) a strong entropic contribution from the free-energy-binding DP-siRNA complex and (ii) a lower number of terminal groups of DP directly interacting with siRNA, inducing additional binding with another siRNA molecule.

Phosphorus dendrimers against transmissible spongiform encephalopathies

The infectious prion protein PrP Sc is the root cause of the transmissible spongiform encephalopathies Creutzfeldt-Jakob disease (CJD), Gerstmann-Straüssler-Scheinker syndrome and fatal familial insomnia in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE) in cows [START_REF] Prusiner | Novel proteinaceous infectious particles cause scrapie[END_REF].

Solassol and colleagues designed and developed cationic phosphorus-containing dendrimers, which strongly reduced prion replication both in cell culture and in mice infected with scrapie [START_REF] Solassol | Cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie[END_REF]. These G3-5 cationic phosphorus dendrimers have different numbers of diethyl amine end groups: namely, 48 (PD-G3), 96 (PD-G4) and 192 (PD-G5) (Figure 4). Interestingly, in scrapie-infected neuroblastoma N2a cell assays, PD-G3, PD-G4 and PD-G5 removed PrP Sc rapidly with IC 50 s of 600 nM, 75 nM and 45 nM, respectively. Importantly, no effect on growth rate or on the morphology of the cells was observed at the concentrations used, indicating that these phosphorus dendrimers are non-cytotoxic agents. PD-G3-G5 macromolecules bind to PrP, and they decreased the amount of pre-existing splenic PrP Sc and BSE prion strains. More importantly, in the C57BL/6 mice scrapie model, PD-G4 decreased PrP Sc accumulation in the spleen by up to 66% and 88% at doses of 50 μg and 100 μg, respectively. In this study, C506M3 scrapie brain homogenate was obtained from terminally ill mice and was intraperitoneally administrated in C57BL/6 mice. Intraperitoneal administration of PD-G4 occurred every 2 days from day 2 to day 30 post-inoculation, and mice were sacrificed 30 days after infection. These in vivo experiments demonstrated that the phosphorus dendrimers predominantly reached the spleen, which is infected after the brain.

Phosphorus dendrimers against ocular hypertension

Carteolol is a non-selective β-adrenoceptor antagonist with partial agonist activity. It is used topically to reduce elevated intraocular pressure in patients with glaucoma or ocular hypertension [START_REF] Chrisp | Ocular carteolol. A review of its pharmacological properties, and therapeutic use in glaucoma and ocular hypertension[END_REF]. An original monomer complex bearing three carboxylic groups and phosphorus dendrimers bearing 6 or 12 terminal carboxylic groups have been prepared to complex with carteolol through ionic interactions. [57]. These three complexes have been tested in vivo in a rabbit model, as (nano)carriers for the ocular delivery of carteolol with the following protocol: G0, G1 and G2 macromolecules were administered in the eyes of rabbits as a solution in Milli-Q water. Importantly, no irritation was observed from these three compounds even after several hours of treatment. Using the G2 dendrimer, the concentration of complexed carteolol that penetrated inside the eyes was 2.5 times higher than expected when compared with carteolol alone.

Phosphorus dendrimers against infections

Mannose-capped lipoarabinomannan (ManLAM) is present in all members of the Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium bovis bacillus Calmette-Guérin (BCG) complexes. It is a high-molecular-mass amphipathic lipoglycan with a defined, crucial role in mycobacterial survival during infection. ManLAM plays the part of both an immunogen and a modulator improving tuberculosis prevention. ManLAM inhibits the release of pro-inflammatory cytokines by LPS-stimulated human dendritic cells (DCs) by targeting the C-type lectin receptor (CLR), which binds to carbohydrates in a calciumdependent manner. DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is one unit of transmembrane CLRs, and it is involved in the recognition of several viruses [START_REF] Turner | Mannose-capped lipoarabinomannan in Mycobacterium tuberculosis pathogenesis[END_REF].

With the aim of mimicking the bioactive supramolecular structure of ManLAM, an interesting study has been performed by Blattes et al. concerning the design and development of several types of phosphorus dendrimers, namely: (i) G1, G2 and G3 phosphorus dendrimers with one mannose unit on the surface, corresponding to 12, 24 and 48 units per dendrimer, respectively; (ii) G1-4 phosphorus dendrimers with dimannose units on the surface, corresponding to 12, 24, 48 and 96 units per dendrimer, respectively; and (iii) a G3 phosphorus dendrimer with a trimannose unit, corresponding to 48 units per dendrimer [59]. Among this phosphorus-dendrimer library, the G3 phosphorus dendrimers grafted with 48 trimannoside caps (named 3T) and the G4 dendrimer bearing 96 dimannosides (named 4D) displayed the highest binding avidity for DC-SIGN. Also, these macromolecules inhibited pro-inflammatory TNFα cytokine production. Importantly, in an acute lung inflammation model in mice exposed to aerosolized LPS, oral administration (1 mg/kg) of a 3T phosphorus dendrimer, bearing 48 end trimannoside units, highly reduced neutrophil influx by targeting DC-SIGN.

Cationic phosphorus dendrimers for tackling cancer in vivo using gene therapy

Gene therapy is used to treat diseases caused by missing, defective or overexpressed genes. The key issue in this field is developing a method for introducing a therapeutic gene into target cells. Currently, two approaches are used. One is called gene therapy in vivo, wherein the gene of interest is inserted into a vector and then the vector is transferred directly to the patient. The other approach is called ex vivo, in which the vector is introduced into cultured cells (which can be taken from the patient), and subsequently these genetically engineered cells are transplanted into the patient.

In both cases, viral or non-viral vectors (such as liposomes, cationic polymers or dendrimers) are necessary for introducing the gene of interest into cells [START_REF] Nishikawa | Nonviral vectors in the new millennium: delivery barriers in gene transfer[END_REF][START_REF] Yin | Non-viral vectors for gene-based therapy[END_REF]. Usually, nucleic acid-based therapies using plasmid DNA (pDNA), siRNA or miRNA are employed, and picking the optimal plasmid or siRNA design is of critical importance to improve the efficiency, safety and therapeutic value of delivery. Nucleic acids are highly hydrophilic: therefore, they are unable to penetrate lipid cell membranes, and additionally they can be degraded by nucleases in the blood.

The delivery vehicle can be a cationic dendrimer that is capable of condensing genetic material containing multiple phosphate residues, forming dendriplexes. The first attempt to use phosphorus dendrimers for DNA separation in microarrays was made by Le Berre et al. in 2003 [START_REF] Le Berre | Dendrimeric coating of glass slides for sensitive DNA microarrays analysis[END_REF]. The authors proposed to use cationic phosphorus dendrimers for the functionalization of glass slides for more sensitive and reliable DNA microarrays. In the same year, Maksimenko et al. [START_REF] Maksimenko | Optimisation of dendrimer-mediated gene transfer by anionic oligomers[END_REF] proposed to use cationic phosphorus dendrimers for gene therapy to optimize dendrimer-mediated gene transfer. To understand the mechanisms of entry of dendrimers into cells, several in vitro studies have been performed, such as those by Ionov et al. [64,65] and Shcharbin and colleagues [START_REF] Shcharbin | Fourth generation phosphorus-containing dendrimers: prospective drug and gene delivery carrier[END_REF]. For instance, the use of phosphorus dendrimers to deliver anticancer siRNA cocktails to treat cancer cells has been depicted by Dzmitruk et al. [START_REF] Dzmitruk | Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action[END_REF], as well to deliver siRNAs in HIV-1 infected cells [START_REF] Briz | Validation of a generation 4 phosphorus-containing polycationic dendrimer for gene delivery against HIV-1[END_REF]. In addition, Shakhbazau et al. described the use of PAMAM and phosphorus dendrimers to modify human and rodent stem cells ex vivo so that they overexpress neurotrophic factors [START_REF] Shakhbazau | Non-viral engineering of skin precursor-derived Schwann cells for enhanced NT-3 production in adherent and microcarrier culture[END_REF][START_REF] Shakhbazau | Dendrimer-driven neurotrophin expression differs in temporal patterns between rodent and human stem cells[END_REF].

Recently, Liang and Shi et al. highlighted the development of original polycationic phosphorus dendrimers for the delivery of DNA plasmids to treat tumors [START_REF] Chen | Revisiting cationic phosphorus dendrimers as a nonviral vector for optimized gene delivery toward cancer therapy applications[END_REF]. G1-G3 cationic phosphorus dendrimers bearing 1-(2-aminoethyl) pyrrolidine, 1-(3-aminopropyl) piperidine or 1-(2-aminoethyl) piperidine groups on their surface were prepared via straightforward synthetic pathways (Figure 5a). These protonated polycationic dendrimers were complexed with negatively charged pDNA encoding both enhanced green fluorescence protein (EGFP) and p53. The pDNA-p53-dendrimer polyplexes were prepared with different N/P ratios. For the G1 dendrimers, bearing 12 positive charges on the surface, the DNA condensation capacity followed the order of 1-G1.HCl (N/P = 1 or higher) > 2-G1.HCl (N/P = 2 or higher) > 3-G1.HCl (N/P = 4 or higher). In addition, polyplexes with the pyrrolidinium group displayed a greater positive surface charge than those with the piperidinium group.

Based on a CCK8 viability assay against human cervical carcinoma HeLa cells, the pDNA-p53 polyplexes showed cell viability of at least more than 50%, with the highest concentration of polyplexes at 3000 nM with an N/P ratio of 20. The cell viability decreased in a concentration-dependent manner. The formation of polyplexes from polycationic dendrimers and plasmids decreased the positive charge of the nanosystems, and consequently the polyplexes displayed a lower cytotoxicity than the corresponding dendrimers.

The flow cytometry experiments showed that the best gene-transfection efficiency was achieved with an N/P ratio of 20. At the same N/P ratio, the polyplexes bearing pyrrolidinium termini increased the expression of EGFP more than those with piperidinium termini. Consequently, the 1-G1.HCl-pDNA-p53 polyplex was selected for further studies. The transfection of 1-G1.HCl-pDNA-p53 to HeLa cells induced significant p53 protein expression, as demonstrated by western blotting, and the expression of p53 protein was proven to induce apparent G1/S cell cycle arrest based on the regulation of p21 and cyclin-dependent kinase 4 (Cdk4)/cyclin D1 expression, causing cancer cell apoptosis. The development of the 1-G1.HCl-pDNA-p53 polyplex for anticancer therapies was fully validated based on in vivo experiments. As shown in Figure 5b, intratumoral injection of the 1-G1.HCl-pDNA-p53 polyplex into mice bearing xenografted cervical HeLa tumors did not cause systemic toxicity, and the tumor cells displayed increased expression of p53 and p21 protein, and decreased expression of the Cdk4 and cyclin D1, in full agreement with the in vitro transfection assay data.

Multivalent copper(II)-conjugate phosphorus dendrimers to take down in vivo cancers using UTMD

Recently, in the domain of precision theranostics in nanomedicine, Fan and Shi et al. developed a G3 phosphorus dendrimer capped with 48 CuCl 2 moieties (denoted 1G3-Cu, Figure 6) for non-invasive UTMDpromoted pancreatic tumor magnetic resonance (MR) imaging and chemotherapy [START_REF] Fan | Phosphorus dendrimer-based copper(II) complexes enable ultrasound-enhanced tumor theranostics[END_REF]. 1G3-Cu displayed an IC 50 of 1.24 µM to inhibit the growth of SW1990 pancreatic cancer cell line and an adequate r 1 relaxivity of 0.7024 mM -1 s -1 for T 1 -weighted MR imaging purposes. The chemotherapeutic effect of 1G3-Cu was proven by flow cytometry and western-blot assays. Experimental data revealed that the incubation of SW1990 cells with 1G3-Cu for 24 h activated the apoptotic process through upregulation of the Bax, p53 and phosphatase and tensin homolog (PTEN) proteins, downregulation of the protein Bcl-2, and a decrease in intracellular adenosine triphosphate (ATP).

Interest has grown in the UTMD technique, which increases the tumor penetration of drugs or NPs through the induced sonoporation effect [START_REF] Lin | UTMD-promoted co-delivery of gemcitabine and miR-21 inhibitor by dendrimerentrapped gold nanoparticles for pancreatic cancer therapy[END_REF]. In this study, under the application of UTMD, 1G3-Cu enabled enhanced MR imaging of tumors. Biodistribution studies demonstrated that in the absence of UTMD, the majority of the uptake of 1G3-Cu occurred in the liver, lung and kidney, whereas in the presence of UTMD, higher accumulation of 1G3-Cu was observed in SW1990 tumors. 1G3-Cu displayed good anticancer effects on day 14 by decreasing the tumor volume by around 62%, versus around 35% without UTMD. Importantly, 1G3-Cu did not induce a significant hemolysis effect in the concentration range of 0.5-10 µΜ, and it displayed a good safety profile, without affecting the major organs, blood parameters and bodyweight of mice.

Water-soluble phosphorus dendrimers as two-photon tracers for non-invasive in vivo imaging studies

Two-photon excited fluorescence (TPEF) microscopy, invented by Denk et al. in 1990 [73], represents a powerful diagnostic tool. The main advantages are (i) the reduction of photo damage, (ii) images with submicrometer resolution, (iii) high-sensibility imaging and (iv) the allowance of photochemical reactions within a sub-femtoliter volume inside cells and tissues [START_REF] So | Two-photon excitation fluorescence microscopy[END_REF].

Krishna and colleagues reported the design and the synthesis of water-soluble amphiphilic two-photon markers from lipophilic fluorophores [START_REF] Krishna | Water-soluble dendrimeric two-photon tracers for in vivo imaging[END_REF]. As shown in Figure 7a, three different phosphorus dendrimers were prepared bearing the chromophore Q as a branch. Interestingly, the phosphorus dendrimers, named G1, G2 and G3, showed strong one-photon absorption in the near-ultraviolet spectrum and strong emission in the blue-visible region (Table 1). Their photoluminescence capabilities were similar in ethanol and water, indicating that these water-soluble dendrimers could be used for biological imaging purposes with high photoluminescence efficiency. Krishna et al. performed two-photon imaging of the vascular network in the dorsal part of a rat olfactory bulb. The vessels were labeled after the intravenous injection of a small bolus of 500 µM of G2 dendrimer in water [START_REF] Krishna | Water-soluble dendrimeric two-photon tracers for in vivo imaging[END_REF]. Imaging of the blood vessels of tadpoles in two and three dimensions was also performed.

In addition, Shakhbazau and colleagues have demonstrated that a G2 fluorescent phosphorus dendrimer (compound I in Figure 7b) can be fully internalized into macrophages. This phosphorus dendrimer can be used as a cytoplasmic tracer both in vitro and in vivo (Figure 7b) [START_REF] Caminade | Multicharged and/or water-soluble fluorescent dendrimers: properties and uses[END_REF][START_REF] Shakhbazau | Fluorescent phosphorus dendrimer as a spectral nanosensor for macrophage polarization and fate tracking in spinal cord injury[END_REF].

Conclusion and perspectives

Phosphorus dendrimers can be easily synthesized by the incorporation of phosphorus in various positions, such as in the core, internal branches and external surfaces. The construction of several generations of phosphorus dendrimers has allowed specific control of their size, molecular mass, chain types and surface units that show noteworthy therapeutic activities. Their structures can be easily prepared based on the strong versatility of organophosphorus chemistry. Polycationic, polyanionic and neutral phosphorus dendrimers can be prepared by simple chemical modification of the nature of their surface. A large number of synthetic pathways have provided diverse types of phosphorus dendrimers, including: (i) phosphorhydrazone linkages in the internal branches; (ii) viologen dendritic structures; (iii) P=N-P=S linkages in the internal branches; (iv) onion peel dendritic structures; and (v) Janus phosphorus dendrimers. Importantly, these syntheses occurred under mild reaction conditions and produced non-toxic by-products (e.g., HCl and NaCl) that can be removed without elaborate techniques.

Collectively, the major advantages of phosphorus dendrimers in comparison with the widely known PAMAM dendrimers are: (i) several routes of administration can be used; (ii) good manufacturing practices for clinical use can be developed; (iii) higher lot-to-lot chemical stability was observed; (iv) easily tunable surface synthesis, allowing the development of phosphorus dendrimers as active drugs per se; and (v) low generation of phosphorus dendrimers showing potent anticancer and antituberculosis activities.

In the preclinical process, in vivo studies are pivotal assets that can enable selection of the optimal drugs and dosages. They can provide relevant information (e.g., PK/PD characteristics, which are not available with in vitro studies) for making decisions as well as for avoiding potential hazards. The development of dendrimers in general (and phosphorus dendrimers in particular) is no exception to this rule. Phosphorus dendrimers have been developed as nanomedicines in several important and unmet therapeutic areas; they have been studied as anti-inflammatory agents, anticancer agents and drugs to treat neurodegenerative diseases, ocular hypertension and infections, as well for use in in vivo imaging. One of the challenges is the selection of animal models based on the physiological and biochemical similarities between the animal model and humans, including ADMET evaluation. Consequently, further studies are still needed to develop animal models that better represent human physiology.

In the dendrimer realm, few PK/PD studies have been published owing to the mandatory need for validated analytical methods in both preclinical and clinical PK studies. To the best of our knowledge, few preclinical data on phosphorus dendrimers have been published; for instance, there is a lack of data on chemical stability and long-term toxicity under good laboratory practices, as well as on the production of phosphorus dendrimers under good manufacturing practices. Strong recommendations for the design of dendrimers towards translation into nanotherapeutics were recently highlighted by several of the authors of this review [START_REF] Mignani | Dendrimers toward translational nanotherapeutics: concise key step analysis[END_REF]. In the dendrimer domain, the lack of robust and validated analytical methods for performing PK studies remains a key obstacle to medical translation. To ensure that we keep on making progress, it is crucial that public-health professionals work with researchers from industry to develop 'druggable' dendrimers as well as more-holistic approaches addressing real-world medical demands in a reliable manner. In addition, strategies should be developed to manage the risk, cost and therapeutic benefits of innovative NPs in general, and dendrimers in particular [START_REF] Kakkar | Dendrimers: A Themed Issue in Honor of Professor Donald A. Tomalia on the Occasion of His 80th Birthday[END_REF]. The development of dendrimers for human therapeutic applications must not remain such a struggle. Generally speaking, we agree with Antoine de Saint-Exupéry's viewpoint in The Little Prince: "The only things you learn are the things you tame." pDNA-p53 or 1-G1.HCl-pDNA-p53 polyplexes (20 μg pDNA/mouse for pDNA groups). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as an internal control. (d) Quantitative analysis of the G0/G1 phase-related protein expression level from the western blot data in vivo. Reproduced, with permission, from [START_REF] Chen | Revisiting cationic phosphorus dendrimers as a nonviral vector for optimized gene delivery toward cancer therapy applications[END_REF]. Copyright 2020 ACS Publications.

Figure 1 .

 1 Figure 1. 2D chemical structure of an example of a G1 phosphorus dendrimer.

Figure 2 .

 2 Figure 2. Overview of a schematic representation of physical characteristics of nanoparticles for in vivo biocompatibility profile. Adapted from [38].Figure 3. Phosphorus dendrimers as anti-inflammatory agents. (a) 2D chemical structure of neutral phosphorus dendrimers I (G3) and II (G4). (b) 2D chemical structure of G1 azabisphosphonate (compound 1), G1 azamonophosphonate (compound 2) and G1 bisphosphonate PPI dendrimer (compound 3). (c) 2D chemical structure of G3 phosphorus dendrimers DM and DP.

Figure 3 .

 3 Figure 2. Overview of a schematic representation of physical characteristics of nanoparticles for in vivo biocompatibility profile. Adapted from [38].Figure 3. Phosphorus dendrimers as anti-inflammatory agents. (a) 2D chemical structure of neutral phosphorus dendrimers I (G3) and II (G4). (b) 2D chemical structure of G1 azabisphosphonate (compound 1), G1 azamonophosphonate (compound 2) and G1 bisphosphonate PPI dendrimer (compound 3). (c) 2D chemical structure of G3 phosphorus dendrimers DM and DP.

Figure 4 .

 4 Figure 4. Chemical structure of PD-G3, PD-G4 and PD-G5.

Figure 5 .

 5 Figure 5. Cationic phosphorus dendrimers for gene therapy. (a) 2D chemical structure of 1-G1, 2-G1, 3-G1, 1-G2 and 1-G3 phosphorus dendrimers. (b) Formation of 1-G1.HCl-pDNA-p53 polyplexes for in vivo gene delivery experiments. (c) Western blot assay indicating the expression of the proteins related to G0/G1 phase in xenografted HeLa tumor cells at 4 days after treatment with normal saline (NS), 1-G1.HCl, free

Figure 6 .

 6 Figure 6. Schematic representation of the development of 1G3-Cu phosphorus dendrimers to tackle tumors using a UTMD-enhanced MR imaging strategy. Reproduced, with permission, from [72]. Copyright 2020 Elsevier.

Figure 7 .

 7 Figure 7. Phosphorus dendrimers as two-photon tracers for non-invasive in vivo imaging studies. (a) 2D chemical structure of phosphorus dendrimers G1, G2 and G3. (b) Chemical structure of G2 fluorescent phosphorus dendrimers (compound I).

  Figure 3c:

Table 1 . Photophysical properties of the chromophore Q and the dendrimers G1-G3

 1

	Compound Solvent	λ abs,max	ε max	λ em	Φ (fluorescence	σ 2 (GM) a
			(nm)	(10 4 M -1 cm -1 )	(nm)	quantum)
	Q	EtOH	379	7.80	435	0.79	155 (8 in water)
	G1	Water	377	7.27	442	0.53	104
	G2	Water	381	6.24	444	0.71	119
	G3	Water	383	6.31	443	0.66	127

a At 705 nm, 1 GM = 10 -50 cm 4 s photon -1 .

Acknowledgments

X.S., S.M. and J-P.M. thank the PRC NSFC-CNRS 2019 (21911530230 for X.S. and 199675 for S.M. and J-P.M.). S.M. and X.S. 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 M1420-01-0145-FEDER-000005-CQM+ (Madeira 14-20 Program). S.M., V.C. and J-P.M. acknowledge transnational EuroNanoMed III funded projects, including a proper acknowledgement of ERANET EuroNanoMed III and the respective funding partner organizations. V.C. acknowledges the support of the Spanish Ministerio de Economía y Competitividad (project SAF2017-89288-R from MINECO/AEI/FEDER/UE) and JCCM (project SBPLY/19/180501/000067). J-P.M. thanks CNRS (France) for financial support. This work was supported by the Polish National Agency for Academic Exchange, grant EUROPARTNER, No. PPI/APM/2018/1/00007/U/001; partially supported by the Belarusian Republican Foundation for Fundamental Research and State Committee of Science and Technology of Belarus, grants B19ARMG-002 and B20SLKG-002 (to D.S. and M.B.).


Gabrielle Sarlon-Bartoli
email: gabrielle.sarlon@ap-hm.fr

Quentin Michel

Emmanuelle Sarlon

M Carcopino-Tusoli

Pierre Suchon

Raphael Soler

Michel Alain Bartoli

Dominique Brunet

Pierre Morange

Aude Charbonnier

Magali Carcopino- Tusoli

Dr Sarlon-Bartoli Gabrielle

Ultrasound arterial anomalies in patients exposed to nilotinib therapy for chronic myeloid leukemia

Keywords: ultrasound arterial anomalies, nilotinib, chronic myeloid leukemia

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

INTRODUCTION

Chronic myeloid leukemia (CML) is defined by expansion of uncontrolled myeloid cells with BCR/ABL1 oncogene [START_REF] Faderl | The biology of chronic myeloid leukemia[END_REF]. The "gold standard" therapy is the BCR/ABL1 tyrosine kinase inhibitor (TKI) imatinib. However more effective second generation TKI nilotinib (and dasatinib in some countries) may be used at diagnosis. Second and third generation TKIs including nilotinib, dasatinib, bosutinib, and ponatinib, have been validated in case of resistance or intolerance in clinical trials and daily practice [START_REF] Jabbour | Resistance to tyrosine kinase inhibition therapy for chronic myelogenous leukemia: a clinical perspective and emerging treatment options[END_REF].

No significant vascular adverse events (VAE) have been reported in initial clinical trials testing nilotinib (<1%) [START_REF] Kantarjian | Nilotinib is effective in patients with chronic myeloid leukemia in chronic phase after imatinib resistance or intolerance: 24-month follow-up results[END_REF][START_REF] Ema | Tasigna: Annex I. Summary of Product Characteristics[END_REF]. However, during the past few years several works suggest a higher prevalence of about 30% of arterial diseases during nilotinib therapy like peripheral arterial disease (PAD), renal artery stenosis, cerebral ischemia and myocardial infarction [START_REF] Aichberger | Progressive peripheral arterial occlusive disease and other vascular events during nilotinib therapy in CML[END_REF][START_REF] Coutre | Severe peripheral arterial disease during nilotinib therapy[END_REF][START_REF] Kim | Peripheral occlusive disease in chronic phase chronic myeloid leukemia patients treated with nilotinib or imatinib[END_REF][START_REF] Giles | Rates of peripheral arterial occlusive disease in patients with chronic myeloid leukemia in the chronic phase treated with imatinib, nilotinib, or nontyrosine kinase therapy: a retrospective cohort analysis[END_REF][START_REF] Levato | Progressive peripheral arterial occlusive disease and other vascular events during nilotinib therapy in chronic myeloid leukemia: a single institution study[END_REF][START_REF] Mirault | Rapid onset of peripheral artery disease in a chronic myeloid leukemia patient without prior arterial disorder: direct relationship with nilotinib exposure and clinical outcome[END_REF][START_REF] Bondon-Guitton | Cardiovascular risk profile of patients with peripheral arterial occlusive disease during nilotinib therapy[END_REF]. This prevalence is higher than that expected in general population: for example PAD prevalence based on ankle brachial index measurement (ABI) has been evaluated in several epidemiologic studies with a range of 3% to 10% before 65 years, increasing maximum to 15% to 20% in persons over 65 years [START_REF] Norgren | Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II)[END_REF].

Predisposing factors seemed to be: high doses of nilotinib [START_REF] Hochhaus | Long-term benefits and risks of frontline nilotinib vs imatinib for chronic myeloid leukemia in chronic phase: 5-year update of the randomized ENESTnd trial[END_REF], long exposure to nilotinib around 3 years exposure and pre-existing cardiovascular risk factors [START_REF] Bondon-Guitton | Cardiovascular risk profile of patients with peripheral arterial occlusive disease during nilotinib therapy[END_REF]. However sometimes PAD occurred also in younger patients without risk factors [START_REF] Stève-Dumont | Are nilotinibassociated vascular adverse events an under-estimated problem?[END_REF].

In 2016 the France Intergroupe des Leucémies Myéloïdes Chroniques provided an overview of nilotinib efficacy and cardiovascular safety profile and proposed practical recommendations with the goal to minimize the risk and severity of cardiovascular events in nilotinib-treated patients [START_REF] Rea | Management of the cardiovascular disease risk during nilotinib treatment in chronic myeloid leukemia: 2015 recommendations from the [France Intergroupe des Leucémies Myéloïdes Chroniques[END_REF]. An initial evaluation with calculation of the Systematic COronary Risk Evaluation (SCORE) from ESC 2012 guidelines is strongly recommended [START_REF] Perk | European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice[END_REF]. A low cardiovascular risk is not associated with systematic vascular imaging. A moderate risk encourages screening coronary artery with electrocardiogram and echocardiogram, carotid and leg arteries with duplex ultrasound. Patient with high or very high risk should benefit of a multidisciplinary discussion before nilotinib prescription.

Few months later, European recommendations for the management and avoidance of adverse events of treatment in CML were published and strongly suggested performing either the ankle-brachial index (ABI) or duplex ultrasonography to assess asymptomatic PAD in all newly diagnosed patients with CML aged over 65 years and in younger patients in the presence of cardiovascular risk factors or claudication symptoms [START_REF] Steegmann | European LeukemiaNet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia[END_REF].

Ultrasound screening was thus recently widely recommended in patients treated with nilotinib for CML but prevalence of asymptomatic ultrasound lesions and their characteristics are unknown.

Our objective was to describe prevalence and characteristics of ultrasound anomalies during the screening of patients exposed to nilotinib for CML.

METHOD Patients

Seventy-four patients treated with nilotinib for CML and followed up in oncohematology department of the Paoli-Calmettes Institute, Marseille, from 2006 to 2015 were retrospectively included. From 2010, patients already treated by nilotinib had an arterial duplex ultrasonography. The newly nilotinib treated patients had this exam three months after nilotinib initiation. Demographic, hematologic and cardiovascular parameters were collected after informed consent was obtained and procedures approved by the local human ethics committee.

Hematologic evaluation

All patients had a three months onco-hematologic survey. Nilotinib indications were: first line therapy or resistance to others therapies or side effects. Nilotinib dose were: 300mg twice a day or 400mg twice day for resistant patients.

Vascular examination

Patients had clinical, biological and arterial duplex ultrasonography first evaluation with experimented vascular doctors. Clinical examination consisted with cardiovascular risk evaluation, BP measurement, cardiovascular auscultation and palpation, ABI measurement. Biological parameters were: fasting glucose, HbA1c, lipids (cholesterol, low-and high-density lipoprotein (LDL and HDL) and triglycerides), and creatinine. Vascular duplex ultrasonography evaluation was exhaustive: extracranial carotid arteries, extracranial vertebral arteries and their ostia, subclavian arteries, abdominal aorta, digestive and renal arteries, legs arteries including hypogastric arteries with their ostia. Ultrasound aspect of arterial lesion was described: presence of plaque and its echogenicity, presence of stenosis and its degree, occlusion or not. Description of plaque echolucency was qualitative: no echo pattern = echolucent plaque, less echogenicity than media-adventice interface = hypoechogenic plaque, same echogenicity than adjacent bone = hyperechogenic plaque. A specific follow-up was determinate after the first vascular exam.

Definition of arterial anomaly

Vascular arterial anomaly was defined by: clinical and/or ultrasound anomalies. Clinical symptoms were: PAD, stroke, myocardial infarction, renovascular hypertension. Ultrasound anomalies were: arterial plaque, stenosis or occlusion, without symptoms.

Statistical analyses

Population characteristics were described and compared at baseline according to the presence of VAA. Results were expressed as mean ±SD or median [min-max] with interquartile range (IQR). Bivariate analyses were conducted with all appropriate continuous variables using Student's t-test or a Mann-Whitney test. Categorical variables were analyzed by the chi-squared or Fisher's exact test. The statistical analysis was considered to be significant for p <0.05. Multivariate analysis was carried out by elaborating a logistic regression model to estimate the probability of a VAA according to the relevant parameters.

The selection of variables was made according to literature data and the results of bivariate analyses (variables associated with p <0.05): age at nilotinib introduction, pre-existent hypertension, and arterial stenosis. Analysis was performed using R for Windows, version 2.11.1.

RESULTS

Hematological characteristics

A total of 74 patients (38 men and 36 women) were included with a mean age of 54.5 ±16.2 years. Seventy-one patients were in chronic phase, two in acute phase and one in blastic

Arterial duplex ultrasonography

Arterial ultrasound anomalies were screened at the first exam for 25 patients (33.8%; n=74). 18 patients had more than one arterial territory involved (72%, n=25). Involved arterial territories were (n=25): carotid bulb (44%), iliac (28%), superficial femoral artery (28%), common femoral artery (20%), renal artery (12%), vertebral ostium (12%), legs artery (12%), mediacalcosis (12%) abdominal aorta (4%) and popliteal artery (4%). First arterial ultrasound showed 22 patients with plaques (88%, n=25): 16 were echolucent or hypoechogenic (72.7%) (Figure 1) and 6 (27.3%) were hyperechogenic. Eleven patients had arterial stenosis >50% (44%; n=25). Three patients (12%) had arterial occlusion (one superficial femoral artery and two patients with occlusion of legs arteries).

Cardiovascular characteristics

They were resumed in Table I. VAA was present at the first vascular examination for 25 patients (33.8%, n=74). In <65 years patients, 20 had VAE (n=55; 36.3%), in ≥65 years 13 patients had VAE (n=19; 68.4%). In patient with no cardiovascular risk factor, 3 had VAE (n=24; 12.5%). Fifteen patients were symptomatic (n=74; 20.3%): 9 PAD, 2 strokes, 2 myocardial infarctions and 2 renal artery stenosis. Twelve patients had statins, 14 had antiplatelet agent, 4 had previous lower limb revascularization, 2 had previous coronary stenting, 2 had previous renal artery stenting. Five patients from VAA-group had antiplatelets for primary prevention. Baseline characteristics were compared for patients with and without VAA and have been reported in Table I. Patients with VAA were significantly older (64.9 vs 49.3; p<0.001) and older at nilotinib initiation (60.8 vs 46.5; p<0.001). Patients with VAA had significantly more hypertension (10 vs 6; p=0.01) and more cardiovascular risk factors (p=0.03). In multivariate analysis age at nilotinib initiation appeared to be an independent factor to VAA (p=0.03).

DISCUSSION

We described for the first-time the aspect of arterial ultrasound lesion of CML patients during nilotinib therapy. CML patients with nilotinib presented mostly with echolucent or hypoechogenic plaques (22%, n=74). In literature, these plaques were associated with instable atheroma: the carotid plaques with low grayscale median were associated with a twofold increase in stroke [START_REF] Mathiesen | Echolucent plaques are associated with high risk of ischemic cerebrovascular events in carotid stenosis. The Tromsø Study[END_REF][START_REF] Gupta | Plaque echolucency and stroke risk in asymptomatic carotid stenosis: a systematic review and meta-analysis[END_REF]. Echolucent plaque reflects lipid core of atheroma and it is associated to unstable plaque [START_REF] Stary | A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association[END_REF]. No prior data are available in literature but figures of Mirault et al. showed ever clearly hypodense stenosis and occlusion on CT angiography [START_REF] Mirault | Rapid onset of peripheral artery disease in a chronic myeloid leukemia patient without prior arterial disorder: direct relationship with nilotinib exposure and clinical outcome[END_REF].

Hypodense lesion on CT correspond to hypoechogenic lesion on ultrasonography. We also emphasize that these echolucent lesions are sometimes difficult to detect during ultrasonography because echogenicity is similar to the circulating blood. Rea et al. studied plasma lipid profile from 27 patients treated by nilotinib: data from a minimum 1-year follow up showed that nilotinib significantly increased total, low-and high-density lipoprotein cholesterol within three months [START_REF] Rea | Early onset hypercholesterolemia induced by the 2nd-generation tyrosine kinase inhibitor nilotinib in patients with chronic phase-chronic myeloid leukemia[END_REF]. The mechanisms of action of nilotinib on various nonhematopoietic cells that could explain this potential adverse effect are poorly understood.

Nilotinib binds to a limited number of key kinase targets. One of the more interesting kinasetargets in terms of vascular cells and events is the discoidin domain receptor 1 (DDR1). This receptor has recently been implicated in plaque formation in atherosclerosis [START_REF] Franco | Discoidin domain receptor 1 on bone marrow-derived cells promotes macrophage accumulation during atherogenesis[END_REF].

Our study shows that adding ultrasound anomalies to define VAA, ranges arterial anomaly prevalence from 20% (symptomatic patients) to 34% (symptomatic and asymptomatic patients). Screening asymptomatic patients may lead to prevent events and adapt hematological treatment. VAA defined by clinical symptoms and/or ultrasound anomalies is significantly and independently associated to nilotinib exposure time. Le Coutre et al. [START_REF] Coutre | Severe peripheral arterial disease during nilotinib therapy[END_REF] first described PAD during nilotinib therapy for 179 patients and relation with exposure time: the mean time from initiation of nilotinib to the first PAD event was 26 months. This relation between VAA and exposure time was also suggested by Levato et al. [START_REF] Levato | Progressive peripheral arterial occlusive disease and other vascular events during nilotinib therapy in chronic myeloid leukemia: a single institution study[END_REF]: VAA appeared after a median time of exposition to nilotinib of 24 months. European leukemia recommendations consider that the vast majority of documented cases of PAD occurred within the first 48 months of therapy, but some have been reported after only 4 months, or as late as 5 years [START_REF] Steegmann | European LeukemiaNet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia[END_REF]. We choose to take also into account ultrasound asymptomatic anomalies because patients had instable-like lesion with echolucent plaques. These data are in line with literature that shows high prevalence of VAA: after 2 years, the numbers of CML patients developing VAE during nilotinib ranged between 1% and 29% [START_REF] Valent | Vascular safety issues in CML patients treated with BCR/ABL1 kinase inhibitors[END_REF].

We find that 36% patients without or with only one cardiovascular risk factor had VAA, suggesting the importance for ultrasound follow-up in all patients treated by nilotinib. These results do not totally agree with Breccia et al. that used a systematic coronary risk evaluation chart (SCORE) to identify chronic myeloid leukemia patients at risk of cardiovascular diseases during nilotinib treatment [START_REF] Breccia | Application of systematic coronary risk evaluation chart to identify chronic myeloid leukemia patients at risk of cardiovascular diseases during nilotinib treatment[END_REF]. They obtained low cumulative incidence of atherosclerotic events at 48 months (8.5%) but highlighted a correlation between incident events and cardiovascular risk factors: none of the low-risk patients according to the SCORE chart experienced atherosclerotic events compared to 10 % in the moderate-risk category and 29 % in the high-risk. We obtained higher prevalence of cardiovascular anomalies in patients probably because we used ultrasound anomalies and clinical anomalies in our definition. We show also that hypertension is significantly associated to VAA; it was also the predominant cardiovascular risk factor (63.6%) for Le Coutre et al. [START_REF] Coutre | Severe peripheral arterial disease during nilotinib therapy[END_REF] and 35% for Breccia et al. [START_REF] Breccia | Application of systematic coronary risk evaluation chart to identify chronic myeloid leukemia patients at risk of cardiovascular diseases during nilotinib treatment[END_REF].

Study limitations are firstly that vascular examination is nonsystematic at nilotinib initiation. It is explained by the absence of knowledge of VAA during nilotinib therapy at the beginning of the drug use. It may explain why some patients were symptomatic at the initial vascular evaluation. Secondly, we chose VAA definition with clinical or ultrasound anomalies which can increase the prevalence of VAA but we think that echolucent lesions are instable-like lesions and of poor prognosis. We think that detection of asymptomatic lesions should be taken into account for cardiovascular prevention and may sometimes adapt hematologic treatment.

CONCLUSION

Treatment with nilotinib seems to be associated to atherosclerosis. The prevalence of ultrasound VAA is high, especially with nilotinib exposure, in older patients and in the presence of several cardiovascular risk factors. Of note, VAA may be present in patients without cardiovascular risk factors. Ultrasound characteristics are high frequency of echolucent plaques which reflect instable lesion. We believe that the initiation of nilotinib therapy should systematically be associated with a cardiovascular evaluation in all patients, even in the absence of cardiovascular risk factors. We also believe in the importance of close vascular follow-up in these patients.

  transformation. Two patients were allografted: one for blastic transformation and one because diagnosis was made before 2000. Seventeen patients were at first line therapy, 17 had side effects of others TKI, 25 patients had resistant to others TKI and 12 had lack of molecular response level 3 or 4 (3 missing data). Median exposure time to nilotinib was 22 months [1-84].

Figure 1 :

 1 Figure 1: Ultrasound picture of carotid bulb with hypoechogenic plaque in a 40-year-old patient without cardiovascular risk factors

Table I :

 I Baseline characteristics of the study population CV: Cardiovascular; VAA: Vascular Arterial Anomaly

		Population	Age <65 years	VAA +	VAA -
		n=74 (%)	n=51 (%)	n=25 (%)	n=49 (%)	p (bivariate)	p (multivariate)
	Sex M	38 (51.4)	28 (54.9)	10 (40)	26 (53)	0.41
	Age (mean)	54.5	46.0	64.9	49.3	<0.001
	Age at nilotinib initiation	51.3	43.2	60.8	46.5	<0.001	0.03
	Dyslipidemia	10 (14)		5 (20)	5 (10)	0.42
	Hypertension	16 (22)		10 (40)	6 (12)	0.01	0.77
	Tabacco	27 (36)		9 (36)	18 (37)	0.41
	Diabete	4 (5)		3 (12)	1 (2)
	Heredity	11 (15)		7 (28)	4 (8)	0.05
	CV score					0.03
	0 CV factor	24 (32)		3 (12)	21 (43)
	1 CV factor	19 (26)		6 (24)	13 (27)
	2 CV factor	18 (24)		8 (32)	10 (20)
	3 CV factor	9 (12)		6 (24)	3 (6)
	4 CV factor	4 (5)		2 (8)	2 (4)
	VAE	25 (34)
	Statin	12 (16)		7 (28)	5 (10)	0.08
	Antiplatelets	14 (19)		9 (36)	5 (10)	0.01
	Symptoms	15 (20)		15 (60)	0 (0)	<0.01
	Echolucent or hypoechogenic plaque	24 (32)		16 (64)	6 (12)	<0.01
	Stenosis	11 (15)		11 (44)	0 (0)	<0.01	0.99

Disclosure of interest

The authors declare that they have no competing interest.