Curing inflammatory diseases using phosphorous dendrimers.

Different types of water-soluble phosphorous dendrimers have been synthesized and display many different biological properties. It has been shown in particular that phosphorous dendrimers of first generation functionalized with azabisphosphonate terminal functions are able to stimulate the human immune system ex vivo. These dendrimers are internalized by monocytes within a few seconds, and induce their anti-inflammatory activation. The presence of the dendrimers induces also the inhibition of the differentiation of monocytes into osteoclasts, the maturation of dendritic cells, and inhibits the proliferation of the proinflammatory CD4+ T lymphocytes. Finally, after 2-3 weeks of culture of peripheral blood mononuclear cells, amplifications by several tens of natural killer cells is observed. In view of all these properties, the influence of these azabisphosphonate-dendrimers has been tested in vivo with several animal models, against different chronic or acute inflammatory diseases, such as multiple sclerosis, rheumatoid arthritis, uveitis, and psoriasis, but also against myeloid leukemia, a hematological cancer. The hematological safety has been demonstrated in mice, as there is no platelet aggregation, no hemolysis, and no disturbance in the hematological formula. The safety of the azabisphosphonate-dendrimer has been assessed also with non-human primates (cynomolgus monkeys) which received repeated injections, as a de-risking pre-clinical test. Biochemical, hematological, and all immunological parameters in peripheral blood remained within a normal physiological range throughout the study, and all survived well. Other phosphorous dendrimers also display anti-inflammatory properties in vivo, in particular dendrimers functionalized with mannose derivatives, which prevent acute lung diseases when given orally (per os) to mice. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease.

Repolarization of Unbalanced Macrophages: Unmet Medical Need in Chronic Inflammation and Cancer.

Monocytes and their tissue counterpart macrophages (MP) constitute the front line of the immune system. Indeed, they are able to rapidly and efficiently detect both external and internal danger signals, thereby activating the immune system to eradicate the disturbing biological, chemical, or physical agents. They are also in charge of the control of the immune response and account for the repair of the damaged tissues, eventually restoring tissue homeostasis. The balance between these dual activities must be thoroughly controlled in space and time. Any sustained unbalanced response of MP leads to pathological disorders, such as chronic inflammation, or favors cancer development and progression. In this review, we take advantage of our expertise in chronic inflammation, especially in rheumatoid arthritis, and in cancer, to highlight the pivotal role of MP in the physiopathology of these disorders and to emphasize the repolarization of unbalanced MP as a promising therapeutic strategy to control these diseases.

Supramolecular and Macromolecular Matrix Nanocarriers for Drug Delivery in Inflammation-Associated Skin Diseases.

Skin is our biggest organ. It interfaces our body with its environment. It is an efficient barrier to control the loss of water, the regulation of temperature, and infections by skin-resident and environmental pathogens. The barrier function of the skin is played by the stratum corneum (SC). It is a lipid barrier associating corneocytes (the terminally differentiated keratinocytes) and multilamellar lipid bilayers. This intricate association constitutes a very cohesive system, fully adapted to its role. One consequence of this efficient organization is the virtual impossibility for active pharmaceutical ingredients (API) to cross the SC to reach the inner layers of the skin after topical deposition. There are several ways to help a drug to cross the SC. Physical methods and chemical enhancers of permeation are a possibility. These are invasive and irritating methods. Vectorization of the drugs through nanocarriers is another way to circumvent the SC. This mini-review focuses on supramolecular and macromolecular matrices designed and implemented for skin permeation, excluding vesicular nanocarriers. Examples highlight the entrapment of anti-inflammatory API to treat inflammatory disorders of the skin.

An Anti-Inflammatory Poly(PhosphorHydrazone) Dendrimer Capped with AzaBisPhosphonate Groups to Treat Psoriasis.

Dendrimers are nanosized, arborescent macromolecules synthesized in a stepwise fashion with attractive degrees of functionality and structure definition. This is one of the reasons why they are widely used for biomedical applications. Previously, we have shown that a poly(phosphorhydrazone) (PPH) dendrimer capped with anionic azabisphosphonate groups (so-called ABP dendrimer) has immuno-modulatory and anti-inflammatory properties towards human immune cells in vitro. Thereafter, we have shown that the ABP dendrimer has a promising therapeutic efficacy to treat models of acute and chronic inflammatory disorders in animal models. In these models, the active pharmaceutical ingredient was administered systematically (intravenous and oral administrations), but also loco-regionally in the vitreous tissue. Herein, we assessed the therapeutic efficacy of the ABP dendrimer in the preclinical mouse model of psoriasis induced by imiquimod. The ABP dendrimer was administered in phosphate-buffered saline solution via either systemic injection or topical application. We show that the topical application enabled the control of both the clinical and histopathological scores, and the control of the infiltration of macrophages in the skin of treated mice.

Biodistribution and Biosafety of a Poly(Phosphorhydrazone) Dendrimer, an Anti-Inflammatory Drug-Candidate.

Dendrimers are nanosized, arborescent polymers of which size and structure are perfectly controlled. This is one reason why they are widely used for biomedical purposes. Previously, we showed that a phosphorus-based dendrimer capped with anionic azabisphosphonate groups (so-called ABP dendrimer) has immuno-modulatory and anti-inflammatory properties towards human immune cells in vitro. Thereafter, we have shown that the ABP dendrimer has a promising therapeutic efficacy to treat models of chronic inflammatory disorders. On the way to clinical translation, the biodistribution and the safety of this drug-candidate has to be thoroughly assessed. In this article, we present preliminary non-clinical data regarding biodistribution, hematological safety, genotoxicity, maximal tolerated doses, and early cardiac safety of the ABP dendrimer. One of the genotoxicity assays reveals a potential mutagen effect of the item at a concentration above 200 µM, i.e., up to 100 times the active dose in vitro on human immune cells. However, as the results obtained for all the other assays show that the ABP dendrimer has promising biodistribution and safety profiles, there is no red flag raised to hamper the regulatory pre-clinical development of the ABP dendrimer.

Multivalent nanosystems: targeting monocytes/macrophages.

Among all the cellular partners involved in inflammatory processes, monocytes and macrophages are the master regulators of inflammation. They are found in almost all the tissues and are nearly the only cells capable of performing each step of inflammation. Consequently, they stand as major relevant therapeutic targets to treat inflammatory disorders and diseases. The physiological phagocytic activity of macrophages prompts them to detect, to recognize, and eventually to engulf any nanosystem cruising in their neighborhood. Interestingly, nanosystems can be rationally engineered to afford multivalent, and multifunctional if needed, entities with multiplexed and/or reinforced biological activities. Indeed, engineered nanosystems bearing moieties specifically targeting macrophages, and loaded with or bound to drugs are promising candidates to modulate, or even eradicate, deleterious macrophages in vivo. In this review we highlight recent articles and concepts of multivalent nanosystems targeting monocytes and macrophages to treat inflammatory disorders.

The ABP Dendrimer, a Drug-Candidate against Inflammatory Diseases That Triggers the Activation of Interleukin-10 Producing Immune Cells.

The ABP dendrimer, which is built on a phosphorus-based scaffold and bears twelve azabisphosphonate groups at its surface, is one of the dendrimers that has been shown to display immuno-modulatory and anti-inflammatory effects towards the human immune system. Its anti-inflammatory properties have been successfully challenged in animal models of inflammatory disorders. In this review, we trace the discovery and the evaluation of the therapeutic effects of the ABP dendrimer in three different animal models of both acute and chronic inflammatory diseases. We emphasize that its therapeutic effects rely on the enhancement of the production of Interleukin-10, the paradigm of anti-inflammatory cytokines, by different subsets of immune cells, such as monocytes/macrophages and CD4+ T lymphocytes.

Three-Dimensional Directionality Is a Pivotal Structural Feature for the Bioactivity of Azabisphosphonate-Capped Poly(PhosphorHydrazone) Nanodrug Dendrimers.

Dendrimers are nanosized, nonlinear, hyperbranched polymers whose overall 3D shape is key for their biological activity. Poly(PhosphorHydrazone) (PPH) dendrimers capped with aza-bisphosphonate (ABP) end groups are known to have anti-inflammatory properties enabling the control of inflammatory diseases in different mouse models. Here we screen the anti-inflammatory activity of a series of PPH dendrimers bearing between 2 and 16 ABP end groups in a mouse model of arthritis and confront the biological results with atomistic simulations of the dendrimers. We show that only the PPH dendrimers capped with 10 and 12 ABP end groups can control the flare of the inflammatory disease. All-atom accelerated molecular dynamics simulations show that dendrimers with a low number of ABP end groups are directional but highly flexible/dynamic and have thereby limited efficiency in establishing multivalent interactions. The largest dendrimer appears as nondirectional, having 16 ABP end groups forming patches all over the dendrimer surface. Conversely, intermediate dendrimers having 10 or 12 ABP end groups reach the best compromise between the number of surface groups and their stable directional gathering, a real maximization of multivalency.

Nanoparticle-Based Strategies to Treat Neuro-Inflammation.

Neuro-inflammation is a pivotal physio-pathological feature of brain disorders, including neurodegenerative diseases. As such, it is a relevant therapeutic target against which drugs have to be proposed. Targeting neuro-inflammation implies crossing the Blood-Brain Barrier (BBB) to reach the Central Nervous System (CNS). Engineered nanoparticles (ENPs) are promising candidates to carry and deliver drugs to the CNS by crossing the BBB. There are several strategies to design ENPs intended for crossing through the BBB. Herein, we first put nanotechnologies back in their historical context and introduce neuro-inflammation and its consequences in terms of public health. In a second part, we explain how ENPs can get access to the brain and review this area by highlighting recent papers in the field. Finally, after pointing out potential guidelines for preclinical studies involving ENPs, we conclude by opening the debate on the questions of nanosafety and toxicity of these ENPs and in particular on ecotoxicity related to regulatory issues and public concerns.

Pro-Inflammatory Versus Anti-Inflammatory Effects of Dendrimers: The Two Faces of Immuno-Modulatory Nanoparticles.

Dendrimers are soft matter, hyperbranched, and multivalent nanoparticles whose synthesis theoretically affords monodisperse compounds. They are built from a core on which one or several successive series of branches are engrafted in an arborescent way. At the end of the synthesis, the tunable addition of surface groups gives birth to multivalent nano-objects which are generally intended for a specific use. For these reasons, dendrimers have received a lot of attention from biomedical researchers. In particular, some of us have demonstrated that dendrimers can be intrinsically drug-candidate for the treatment of inflammatory disorders, amongst others, using relevant preclinical animal models. These anti-inflammatory dendrimers are innovative in the pharmaceutical field. More recently, it has appeared that some dendrimers (even among those which have been described as anti-inflammatory) can promote inflammatory responses in non-diseased animals. The main corpus of this concise review is focused on the reports which describe anti-inflammatory properties of dendrimers in vivo, following which we review the few recent articles that show pro-inflammatory effects of our favorite molecules, to finally discuss this duality in immuno-modulation which has to be taken into account for the preclinical and clinical developments of dendrimers.

Poly(phosphorhydrazone) dendrimers: yin and yang of monocyte activation for human NK cell amplification applied to immunotherapy against multiple myeloma.

Human natural killer (NK) cells play a key role in anti-cancer and anti-viral immunity, but their selective amplification in vitro is extremely tedious to achieve and remains one of the most challenging problems to solve for efficient NK cell-based immuno-therapeutic treatments against malignant diseases. Here we report that, when added to ex vivo culture of peripheral blood mononuclear cells from healthy volunteers or from cancer patients with multiple myeloma, poly (phosphorhydrazone) dendrimers capped with amino-bis(methylene phosphonate) end groups enable the efficient proliferation of NK cells with anti-cancer cytotoxicity in vivo. We also show that the amplification of the NK population relies on the preliminary activation of monocytes in the framework of a multistep cross-talk between monocytes and NK cells before the proliferation thereof. Thus poly(phosphorhydrazone) dendrimers represent a novel class of extremely promising drugs to develop NK-cell based anti-cancer therapies.

Interaction studies reveal specific recognition of an anti-inflammatory polyphosphorhydrazone dendrimer by human monocytes.

Dendrimers are nano-materials with perfectly defined structure and size, and multivalency properties that confer substantial advantages for biomedical applications. Previous work has shown that phosphorus-based polyphosphorhydrazone (PPH) dendrimers capped with azabisphosphonate (ABP) end groups have immuno-modulatory and anti-inflammatory properties leading to efficient therapeutic control of inflammatory diseases in animal models. These properties are mainly prompted through activation of monocytes. Here, we disclose new insights into the molecular mechanisms underlying the anti-inflammatory activation of human monocytes by ABP-capped PPH dendrimers. Following an interdisciplinary approach, we have characterized the physicochemical and biological behavior of the lead ABP dendrimer with model and cell membranes, and compared this experimental set of data to predictive computational modelling studies. The behavior of the ABP dendrimer was compared to the one of an isosteric analog dendrimer capped with twelve azabiscarboxylate (ABC) end groups instead of twelve ABP end groups. The ABC dendrimer displayed no biological activity on human monocytes, therefore it was considered as a negative control. In detail, we show that the ABP dendrimer can bind both non-specifically and specifically to the membrane of human monocytes. The specific binding leads to the internalization of the ABP dendrimer by human monocytes. On the contrary, the ABC dendrimer only interacts non-specifically with human monocytes and is not internalized. These data indicate that the bioactive ABP dendrimer is recognized by specific receptor(s) at the surface of human monocytes.

Phosphorus-Based Dendrimer ABP Treats Neuroinflammation by Promoting IL-10-Producing CD4(+) T Cells.

Dendrimers are polyfunctional nano-objects of perfectly defined structure that can provide innovative alternatives for the treatment of chronic inflammatory diseases, including multiple sclerosis (MS). To investigate the efficiency of a recently described amino-bis(methylene phosphonate)-capped ABP dendrimer as a potential drug candidate for MS, we used the classical mouse model of MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). Our study provides evidence that the ABP dendrimer prevents the development of EAE and inhibits the progression of established disease with a comparable therapeutic benefit as the approved treatment Fingolimod. We also show that the ABP dendrimer redirects the pathogenic myelin-specific CD4(+) T cell response toward IL-10 production.

The key role of the scaffold on the efficiency of dendrimer nanodrugs.

Dendrimers are well-defined macromolecules whose highly branched structure is reminiscent of many natural structures, such as trees, dendritic cells, neurons or the networks of kidneys and lungs. Nature has privileged such branched structures for increasing the efficiency of exchanges with the external medium; thus, the whole structure is of pivotal importance for these natural networks. On the contrary, it is generally believed that the properties of dendrimers are essentially related to their terminal groups, and that the internal structure plays the minor role of an 'innocent' scaffold. Here we show that such an assertion is misleading, using convergent information from biological data (human monocytes activation) and all-atom molecular dynamics simulations on seven families of dendrimers (13 compounds) that we have synthesized, possessing identical terminal groups, but different internal structures. This work demonstrates that the scaffold of nanodrugs strongly influences their properties, somewhat reminiscent of the backbone of proteins.

Repeated intravenous injections in non-human primates demonstrate preclinical safety of an anti-inflammatory phosphorus-based dendrimer.

Dendrimers are nanosized hyperbranched polymers synthesized through an iterative step-by-step process; their size and structure are perfectly controlled, and they are widely used for biomedical purposes. Previously, we showed that a phosphorous-based dendrimer capped with anionic AzaBisPhosphonate groups (so-called ABP dendrimer) has immunomodulatory and anti-inflammatory properties toward the human immune system. It dramatically inhibits the onset and development of experimental arthritis in a mouse model relevant for human rheumatoid arthritis, a chronic inflammatory disease of auto-immune origin. In this article, we demonstrate in an unprecedented study that cynomolgus macaques repeatedly injected with the ABP dendrimer displayed no adverse response. Indeed, biochemical, haematological, clotting and immunological parameters remained with a normal physiological range during the study. Moreover, quantification of serum cytokines and histopathological analyses failed to reveal any noticeable lesion or noteworthy non-physiological occurrence. These results strengthen the potential of the ABP dendrimer as an innovative drug-candidate for the treatment of inflammatory diseases and favor the regulatory preclinical development of the molecule.

Modulation of pro-inflammatory activation of monocytes and dendritic cells by aza-bis-phosphonate dendrimer as an experimental therapeutic agent.

INTRODUCTION: Our objective was to assess the capacity of dendrimer aza-bis-phosphonate (ABP) to modulate phenotype of monocytes (Mo) and monocytes derived dendritic cells (MoDC) activated in response to toll-like receptor 4 (TLR4) and interferon γ (IFN- γ) stimulation. METHODS: Mo (n = 12) and MoDC (n = 11) from peripheral blood of healthy donors were prepared. Cells were preincubated or not for 1 hour with dendrimer ABP, then incubated with lipopolysaccharide (LPS; as a TLR4 ligand) and (IFN-γ) for 38 hours. Secretion of tumor necrosis factor α (TNFα), interleukin (IL) -1, IL-6, IL-12, IL-10 and IL-23 in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA) and Cytokine Bead Array. Differentiation and subsequent maturation of MoDC from nine donors in the presence of LPS were analyzed by flow cytometry using CD80, CD86, CD83 and CD1a surface expression as markers. RESULTS: Mo and MoDC were orientated to a pro-inflammatory state. In activated Mo, TNFα, IL-1ß and IL-23 levels were significantly lower after prior incubation with dendrimer ABP. In activated MoDC, dendrimer ABP promoted IL-10 secretion while decreasing dramatically the level of IL-12. TNFα and IL-6 secretion were significantly lower in the presence of dendrimer ABP. LPS driven maturation of MoDC was impaired by dendrimer ABP treatment, as attested by the significantly lower expression of CD80 and CD86. CONCLUSION: Our data indicate that dendrimer ABP possesses immunomodulatory properties on human Mo and MoDC, in TLR4 + IFN-γ stimulation model, by inducing M2 alternative activation of Mo and promoting tolerogenic MoDC.

An azabisphosphonate-capped poly(phosphorhydrazone) dendrimer for the treatment of endotoxin-induced uveitis.

Over the last decade, different types of dendrimers have shown anti-inflammatory properties in their own right. In particular, we have shown that poly(phosphorhydrazone) (PPH) dendrimers are able to foster an efficient anti-inflammatory response in human monocytes and can resolve the main physiopathological features of chronic arthritis in mice at 1 mg/kg. Here we afford new insights into the therapeutic potential of an azabisphosphonate-capped dendrimer (dendrimer ABP). We have challenged its anti-inflammatory and immuno-modulatory properties in a robust rat model of acute uveitis induced by lipopolysaccharide (LPS). We show that dendrimer ABP at 2 µg/eye is as efficient as the "gold standard" dexamethasone at 20 µg/eye. We have demonstrated that the effect of dendrimer ABP is mediated at least through an increase of the production of the anti-inflammatory Interleukin(IL)-10 cytokine.

Targeting monocytes/macrophages in the treatment of rheumatoid arthritis.

Biotherapies have revolutionized the treatment of RA. However, much work is needed to understand all the mechanisms of these biotherapies, and alternatives are needed to circumvent adverse effects and the high cost of these long-lasting treatments. In this article we outline some of the approaches we have used to target monocytes/macrophages as major components of inflammation and bone homeostasis. We also discuss how anti-TNF-α antibodies target monocytes/macrophages in the complex mechanisms contributing to inhibition of inflammation.

The gene expression profile of phosphoantigen-specific human γδ T lymphocytes is a blend of αß T-cell and NK-cell signatures.

Global transcriptional technologies have revolutionised the study of lymphoid cell populations, but human γδ T lymphocytes specific for phosphoantigens remain far less deeply characterised by these methods despite the great therapeutic potential of these cells. Here we analyse the transcriptome of circulating TCRVγ(+) γδ T cells isolated from healthy individuals, and their relation with those from other lymphoid cell subsets. We report that the gene signature of phosphoantigen-specific TCRVγ(+) γδ T cells is a hybrid of those from αß T and NK cells, with more 'NK-cell' genes than αß T cells have and more 'T-cell' genes than NK cells. The expression profile of TCRVγ(+) γδ T cells stimulated with phosphoantigen recapitulates their immediate physiological functions: Th1 cytokine, chemokine and cytotoxic activities reflect their high mitotic activity at later time points and do not indicate antigen-presenting functions. Finally, such hallmarks make the transcriptome of γδ T cells, whether resting or clonally expanding, clearly distinctive from that of NK/T or peripheral T-cell lymphomas of the γδ subtype.

Anti-inflammatory properties of dendrimers per se.

Dendrimers are polybranched and polyfunctionalized tree-like polymers. Unlike linear polymers, they have perfectly defined structure and molecular weight, due to their iterative step-by-step synthesis. Their multivalent structure and supramolecular properties have made them attractive nanotools for applications, particularly in biology and medicine. Among the different biological and medical properties of dendrimers that have been developed over the past decades, the anti-inflammatory properties of several groups of dendrimers are the most recently discovered. Thereof, dendrimers emerge as promising, although heretical, drug candidates for the treatment of still-uncured chronic inflammatory disorders. This mini-review is based on the five main scientific articles giving an overview of what can be the spectrum of anti-inflammatory characteristics displayed by dendrimers.