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(The FASEB Journal. 2006;20:2339-2351.)
© 2006 FASEB

Design of phosphorylated dendritic architectures to promote human monocyte activation

Mary Poupot*,1, Laurent Griffe{dagger},1, Patrice Marchand{dagger}, Alexandrine Maraval{dagger}, Olivier Rolland{dagger}, Ludovic Martinet*, Fatima-Ezzahra L’Faqihi-Olive*, Cédric-Olivier Turrin{dagger}, Anne-Marie Caminade{dagger},2, Jean-Jacques Fournié*, Jean-Pierre Majoral{dagger},2 and Rémy Poupot*,2

* INSERM, U.563, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, F-31300 France; Université Paul-Sabatier, Toulouse, F-31400 France; and

{dagger} Laboratoire de Chimie de Coordination du CNRS, Toulouse cedex, France

2Correspondence: INSERM 563, Centre de Physiopathologie de Toulouse-Purpan, Hôpital Purpan, BP3028, 31024 Toulouse cedex 03, France. E-mail: remy.poupot{at}toulouse.inserm.fr; Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse cedex 04, France. E-mail: majoral{at}lcc-toulouse.fr; Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse cedex 04, France. E-mail: caminade{at}lcc-toulouse.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As first defensive line, monocytes are a pivotal cell population of innate immunity. Monocyte activation can be relevant to a range of immune conditions and responses. Here we present new insights into the activation of monocytes by a series of phosphonic acid-terminated, phosphorus-containing dendrimers. Various dendritic or subdendritic structures were synthesized and tested, revealing the basic structural requirements for monocyte activation. We showed that multivalent character and phosphonic acid capping of dendrimers are crucial for monocyte targeting and activation. Confocal videomicroscopy showed that a fluorescein-tagged dendrimer binds to isolated monocytes and gets internalized within a few seconds. We also found that dendrimers follow the phagolysosomial route during internalization by monocytes. Finally, we performed fluorescence resonance energy transfer (FRET) experiments between a specifically designed fluorescent dendrimer and phycoerythrin-coupled antibodies. We showed that the typical innate Toll-like receptor (TLR)-2 is clearly involved, but not alone, in the sensing of dendrimers by monocytes. In conclusion, phosphorus-containing dendrimers appear as precisely tunable nanobiotools able to target and activate human innate immunity and thus prove to be good candidates to develop new drugs for immunotherapies.—Poupot, M., Griffe, L., Marchand, P., Maraval, A., Rolland, O., Martinet, L., L’Faqihi-Olive, F.-E., Turrin, C.-O., Caminade, A.-M., Fournié, J.-J., Majoral, J.-P., Poupot, R. Design of phosphorylated dendritic architectures to promote human monocyte activation.


Key Words: cellular immunotherapy • targeting • phosphorus dendrimers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
COOPERATIVE ASSOCIATION ENERGIES, synergistic effects, or receptor clustering, among others, are often targeted properties when dealing with dendrimers interacting with biological entities. These interactions have been thoroughly studied during the last decade, bringing attention to the peculiar properties and biomedical potential of dendritic macromolecules (1 2 3) by virtue of well-defined structure, multivalency, or shape, and the possibility to precisely tune the degree of branching and the flexibility of the dendritic skeleton or the hydrophilicity of the whole dendritic object.

Cell surface-mediated immunoregulation has been shown to be highly relevant from the multivalency point of view. For instance, dendrimer glucosamine conjugates were safely used to prevent scar tissue formation via immunomodulatory and antiangiogenic properties (4) . Dendritic multiple antigenic peptides have also proved to be promising pivotal compounds for immune response modification or immunodiagnostics (5 , 6) . The proof of the multivalent concept applied to dendrimer science has also been illustrated by recent advances in the development of novel dendritic ligands for lethal bacterial toxins or promising antiviral dendritic-based materials (7) .

Phagocytes of the innate immune system provide a first line of defense against many common microorganisms and are essential for the control of common bacterial infections. Among phagocytes, monocytes are white blood mononuclear cells that are precursors to macrophages (8) . The cells of the innate immune system play a crucial part in the initiation and subsequent direction of adaptive immune responses. Moreover, because there is a delay of several days before the initial adaptive immune response takes effect, the innate immune response has a critical role in controlling infections during this period.

Macrophages and neutrophils have surface receptors that have evolved to recognize and bind common constituents of many bacterial surfaces (9) . Bacterial molecules binding to these receptors trigger the cells to engulf the bacterium and also induce the secretion of biologically active molecules by these phagocytes.

In this study we looked for interactions between phosphorus-containing dendrimers (10 , 11) and hematopoietic cells of the human immune system. Synthesis of a fluorescein isothiocyanate (FITC) -derived phosphorylated dendrimer enabled us to monitor these interactions. By using this probe we showed that, among peripheral blood mononuclear cells (PBMC), monocytes were the main population targeted by dendrimers in vitro. Later on we showed that these monocytes were activated by phosphorylated dendrimers. By synthesizing a series of phosphonic acid or carboxylic acid surfaced dendrimers, we identified that phosphonic acid groups as a major structural requirement for phosphorus-containing dendrimer bioactivity. The entire dendritic structure appeared as another structural requirement for this bioactivity, as branches or surface groups alone did not trigger monocyte activation. Finally, we showed that sensing of dendrimer by monocytes involved a typical receptor of innate immunity, namely the Toll-like receptor (TLR)-2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis procedures
All reactions were carried out in the absence of air using standard Schlenk techniques and vacuum line manipulations. Commercial samples were used as received. All solvents were dried before use. Thin-layer chromatography was carried out on Merck Kieselgel 60F254 precoated silicagel plates. Preparative flash chromatography was performed on Merck Kieselgel. Instrumentation: Bruker AC 200, AM 250, ARX 250, DPX 300, AMX 400, Avance 500 (1H, 13C, and 31P NMR). The number of atoms shown as a subscript refers to the generation; the superscript number and letter refer to the O-C6H4 (C1 linked to O) and C6H5 groups, respectively. Elemental analyses were performed by the Service d’Analyse du Laboratoire de Chimie de Coordination (Toulouse, France). The synthesis and characterization of the FITC derivative 1b-G1 (Fig. 1 ) and of compounds 2, 5, 7, 9, Xab-G1 (X=2, 5, 7, 10, 12) (Fig. 2 ) will be described elsewhere. The synthesis of compounds 11ab-Gn (n=0, 1, 2) was carried out as reported previously (12) from a cyclotriphosphazene core (13) . The synthesis and characterization of two representative compounds are given below.


Figure 1
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  		Figure 1. Synthesis of the statistically fluo-tagged dendrimer 1b-G1.


Figure 2
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  		Figure 2. Dendrimer synthesis. Scheme 1) Synthesis of azabis(dimethyl)phosphonate armed phenols. a: (for tyramine) aqueous H2CO, HPO3Me2, THF, RT; (for tyrosine) aqueous H2CO, HPO3Me2, RT followed by MeOH, cat. PTSA, reflux. b: H2NR1, THF, MgSO4, RT; c: HPO3Me2; 50°C; d: aqueous H2CO, HPO3Me2, THF, RT; Scheme 2) Synthesis of phosphonic acid-capped dendrimers: G1 to Xa-G1 series (X=2, 4, 5, 7, 8): 12 eq. of phenol X and 24 eq. of Cs2CO3 for 1 eq. of dendrimer G1 in THF at RT; G1 to 9a-G1: 4-hydroxybenzaldehyde, Cs2CO3, THF, RT, then HPO3Me2, THF, RT; Xa-G1 series to Xb-G1 series (X=2, 4, 5, 7, 8, 9): BrSiMe3, CH3CN, RT; MeOH RT, 60 min; HONa.

To a solution of (D-L) tyrosine (2 g, 11.05 mmol) in THF (4 ml) was added a solution of formaldehyde (37% in water, 3 ml, and 30.8 mmol) and dimethylphosphite (3 ml, 32.7 mmol). The resulting mixture was stirred overnight at room temperature. The crude material was concentrated under reduced pressure and washed successively with 2 x 15 ml of ethyl acetate and 2 x 15 ml of dichloromethane. The solid was dried under reduced pressure to afford 3 as a white powder (yield: 85%). 1H NMR (CDCl3+ (CD)3OD): {delta} = 2.81 (dd, 2H, 2JHH=14.2 Hz, 3JHH=7.3 Hz, CHHCH); 3.04 (dd, 2H, 2JHH=14.2 Hz, 3JHH=7.3 Hz, CHHCH); 3.22–3.51 (m, 4H, PCH2); 3.70 (d, 6H, 3JHP=8.1 Hz, POMe); 3.76 (d, 6H, 3JHP=8.1 Hz, POMe); 4.14 (t, 3JHP=7.3 Hz, 1H, CH); 6.70 (d, 2H, 3JHH=8.5 Hz, C2-H); 7.13 (d, 2H, 3JHP=8.5 Hz, C3-H); 31P-{1H} NMR (CDCl3+CD3OD): {delta} = 31.04 (s, PO3Me2); 13C-{1H} NMR (cluster of differentiation3OD): {delta} = 35.05 (s, CH-CH2); 47.3 (dd, 1JCP=167.3 Hz, 3JCP=9.3 Hz, PCH2); 52.89 (d, 2JCP=7.0 Hz, POMe); 53.30 (d, 2JCP=7.0 Hz, POMe); 66.60 (t, 3JCP=6.9 Hz, CH); 115.39 (s, C2); 129.16 (s, C4); 130.87 (s, C3); 156.40 (s, C1); 173.76 (COOH) ppm. Anal. Calc. for C15H25NO9P2 (425.3 g.mol–1): C, 42.36, H, 5.92, N, 3.29; found: C, 42.48, H, 5.99, N, 3.17.

A solution of phenol 3 (760 mg, 1.79 mmol) in methanol (12 ml) containing a catalytic amount of paratoluenesulfonic acid was refluxed for 36 h. The mixture was then cooled down to room temperature and concentrated under reduced pressure. The resulting crude oil was washed with a mixture of pentane and diethylether. A sticky solid was obtained, dissolved in THF, and precipitated with a mixture of pentane and diethylether to afford 4 as a white powder that was purified by column chromatography (eluent: CHCl3/MeOH, 95/5) (yield: 90%). 1H NMR (CDCl3): {delta} = 2.83 and 2.96 (AB part of ABX syst, 2JHAHB=14.1 Hz, 3JHAHX=6.6 Hz, 3JHBHX=8.1 Hz, 2H, ArCHAHBCHX); 3.20 and 3.39 (AB part of ABX syst, 2JHAHB=2JHBHX=16.5 Hz, 2JHAHX=4.5 Hz, 4H, CHAHBPX); 3.63 (s, 3H, COOMe); 3.72 (d, 6H, 3JHP=10.5 Hz, POMe); 3.74 (d, 6H, 3JHP=10.8 Hz, POMe); 4.40 (t, 1H, 3JHH=7.2 Hz, CH); 6.79 (d, 2H, 3JHH=7.9 Hz, C2-H); 7.08 (d, 2H, 3JHH=7.9 Hz, C3-H); 7.28 (br s, OH); 31P-{1H} NMR (CDCl3): {delta} = 29.61 (s, PO3Me2); 13C-{1H} NMR (CDCl3): {delta} = 35.6 (s, CH2Ar); 47.6 (dd, 1JCP=166.8 Hz, 3JCP=9.8 Hz, PCH2); 51.8 (s, CO2Me); 53.9 (d, 3JCP=6.8 Hz, POMe); 53.8 (d, 3JCP=6.8 Hz, POMe); 66.5 (t, 3JCP=6.8 Hz, CH); 115.7 (s, C2); 128.2 (s, C4); 130,7 (s. C3); 156,2 (s. C1); 172,6 (s. CO2Me) ppm. Anal. Calc. for C16H27NO9P2 (439.3 g.mol–1): C, 43.74, H, 6.19, N, 3.19; found: C, 43.89, H, 6.26, N, 3.12.

4a-G1: To a solution of dendrimer G1 (143 mg, 78.3 µmol) in THF (5 µl) was added cesium carbonate (383 mg, 1.175 mmol) and phenol 4 (420 mg, 0.956 mmol). The reaction mixture was stirred for 36 h at room temperature, then centrifuged and filtered through a pad of celite. The resulting clear solution was concentrated under reduced pressure (~1 ml), then precipitated with a large excess of pentane. Unreacted phenol 4 was removed by dissolving the powder in 2 to 5 ml of THF and subsequent precipitation with an excess of ether to afford dendrimer 4a-G1 as a white powder (yield: 73%). 1H NMR (CDCl3): {delta} = 2.79–3.10 (m, 24H, CH2Ar); 3.10–3.50 (m, 66H, PCH2 and NCH3); 3.62 (d, 72H, 3JHP=10.6 Hz, POMe); 3.68 (d, 72H, 3JHP=10.6 Hz, POMe); 3.75 (s, 36H, COOMe); 4.43 (t, 12H, 3JHH=6.9 Hz, CH); 7.01–7.09 (m, 36H, C02-H and C12-H); 7.23–7.27 (m, 24H, C13-H); 7.59–7.63 (m, 18H, C13-H and CH=N); 31P-{1H} NMR (CDCl3): {delta} = 11.52 (s, N3P3); 29.42 (s, PO3Me2); 66.55 (s, PS); 13C-{1H} NMR (CDCl3): {delta} = 32.78 (d, 2JCP=11.9 Hz, NCH3); 35.24 (s, CHCH2Ar); 46.99 (dd, 1JCP=165.5, 3JCP=9.2 Hz, PCH2); 51.48 (s, CO2Me); 52.42 (d, 2JCP=7.4 Hz, POMe); 53.16 (d, 2JCP=6.3 Hz, POMe); 65.22 (s, CH); 121.05 (d, 3JCP=3.0 Hz, C0,12); 128.26 (s, C03); 130.55 (s, C13); 132.02 (s, C04); 134.61 (s, C14); 138.86 (d, 3JCP=14.0 Hz, CH=N); 149.14 (d, 2JCP=6.5 Hz, C11); 151.24 (s, C01); 171.85 (s, CO2Me) ppm. Anal. Calc. for C240H360N27O114P33S6 (6662 g.mol–1): C, 43.27; H, 5.44, N, 5.68; found: C, 43.39; H, 5.54, N, 5.62.

4b-G1: To a solution of dendrimer 4a-G1 (150 mg, 22.5 µmol) in acetonitrile (5 ml) maintained at 0°C was added dropwise trimethylsilylbromide (222 µl, 1.68 mmol). The reaction mixture was stirred at room temperature overnight, then evaporated to dryness under reduced pressure. The crude residue was dissolved with methanol (5 ml), vigorously stirred for 2 h at room temperature, and evaporated to dryness under reduced pressure. The resulting white solid was washed with methanol (10 ml), then dried under reduced pressure. The acidic-terminated dendrimer was then transformed into its sodium salt as follows: the dendrimer was suspended in water (1 ml/100 mg) and one equivalent of sodium hydroxide per terminal phosphonic acid was added. The resulting solution was filtered (micropore, 0.1 µ) and lyophilized to afford dendrimer 4b-G1 as a white powder (yield: 91%). 1H NMR (CD3CN/D2O): {delta} = 3.27–4.06 (m, 126H, Ph-CH2-CH, CH3-N-P1, N-CH2-P, OMe); 4.95 (s, 12H, CH); 7.36–8.22 (m, 78H, CH=N, Harom); 31P-{1H} NMR (CD3CN/D2O): {delta} = 12.04 (s, PO3HNa); 12.64 (s, N3P3); 66.32 (s, PS); 13C-{1H} NMR (CD3CN/D2O): {delta} = 31.85 (s, Ar-CH2-CH); 33.16 (s, NCH3); 50.95 (d, 1JCP=138.4 Hz, CH2-P); 51.42 (d, 1JCP=148.5 Hz, CH2-P); 52.63 (s, CO2Me); 68.57 (s, CH-CH2-N); 121.71 (s, C02, C1 (2) ); 128.87 (s, C03); 131.25 (s, C13); 132.29 (s, C0 (4) ); 134.88 (s, C14); 140.44 (br s, CH=N); 149.74 (d, 2JCP=6.5 Hz, C11); 151.13 (s, C01); 171.91 (s, CO2Me) ppm.

8: Methylamine (25 mmol, 3 ml of a 33% solution in absolute ethanol, 8 M) and 4-hydroxybenzaldehyde (20 mmol, 2.5 g) were stirred in a flask for 1 day at RT. The reaction mixture was concentrated under reduced pressure and dissolved in 5 ml of diethylether. A white solid was obtained upon precipitation with pentane. This imine (17 mmol, 2.3 g) was immediately reacted with dimethylphosphite (18.7 mmol, 1.7 ml) with one drop of triethylamine. The reaction mixture was evaporated to dryness under reduced pressure, and the resulting powder was flash chromatographied on a pad of silica (acetone) to afford an aza-monophosphorylated phenol (12.2 mmol, 3.0 g, {delta} [31P]=29.6 ppm), which was immediately reacted with aqueous formaldehyde (33%, 24.4 mmol, 2 ml) and dimethylphosphite (48.8l mmol, 5.5 ml) for 12 h at room temperature. The crude residue was evaporated to dryness, then purified by column chromatography (silica, ethyl acetate, Rf=0.35) to afford 8 in 55% overall yield.

1H NMR (CDCl3): {delta} = 2.41 (s, 3H, NCH3); 2.61 (dd, 2JHH=15.3 Hz, 2JHP=6.3 Hz, 1H, CHH); 3.12 (dd, 2JHP=15.6 Hz, 2JHH=15.3 Hz, 1H, CHH); 3.32 (d, 3H, 3JHP=10.2 Hz, POMe); 3.60 (d, 3H, 3JHP=10.9 Hz, POMe); 3.67 (d, 3H, 3JHP=10.6 Hz, POMe); 3.73 (d, 3H, 3JHP=10.9 Hz, POMe); 4.05 (d, 2JHP=23.9 Hz, 1H, CH); 6.74 (d, 3JHH=7.8 Hz, 2H, Harom); 7.17 (d, 3JHH=7.8 Hz, 2H, Harom); 9.08 (br s, 1H, OH); 31P-{1H} NMR (CDCl3): {delta} = 28.1 (s, PO3Me2); 30.9 (s, PO3Me2); 13C-{1H} NMR (CDCl3): {delta} = 42.3 (t, 3JCP=6.3 Hz, N-CH3); 49.2 (dd, 1JCP=164.1 Hz, 3JCP=10.1 Hz, CH2); 52.58 (d, 2JCP=7.6 Hz, POMe); 52.97 (d, 2JCP=6.9 Hz, POMe); 53.08 (d, 2JCP=6.9 Hz, POMe); 53.45 (d, 2JCP=7.6 Hz, POMe); 65.2 (dd, 1JCP=161.7 Hz, 3JCP=13.5 Hz, CH); 115.4 (s, C2); 120.9 (d, 2JCP=3.5 Hz, C4); 131.8 (d, 3JCP=9.1 Hz, C3); 157.8 (s, C1) ppm. Anal. Calc. for C13H23NO7P2 (367.3 g.mol–1): C, 42.51, H, 6.31, N, 3.81; found: C, 42.62, H, 6.40, N, 3.62.

8a-G1: To a solution of dendrimer G1 (87 mg, 47 µmol) in THF (2 ml) was added cesium carbonate (390 mg, 1.2 mmol) and phenol 8 (220 mg, 0.6 mmol). The reaction mixture was stirred for 24 h at room temperature, then centrifuged and filtered through a pad of celite. The resulting clear solution was concentrated under reduced pressure (~1 ml), then precipitated with a large excess of pentane. Unreacted phenol 4 was removed by dissolving the powder in 2 to 5 ml of THF and subsequent precipitation with an excess of ether to afford dendrimer 8a-G1 as a white powder (yield: 75%). 1H NMR (CDCl3): {delta} = 2.46 (s, 36H, CH-N-CH3); 2.65 (dd, 2JHH=15.3 Hz, 2JHP=7.4 Hz, 12H, CH2); 3.12 (dd, 2JHP=15.5 Hz, 2JHH=15.3 Hz, 12H, CH2); 3.25 (d, 3JHP=10.1 Hz, 18H, CH3-N-P); 3.30–3.90 (m, 144H, OMe); 4.2 (d, 2JHP=23.4 Hz, 12H, CH); 6.7–7.6 (m, 78H, Harom, CH=N); 31P-{1H} NMR (CDCl3): {delta} = 11.4 (s, N3P3); 27,5 (s, PO3Me2); 30.4 (s, PO3Me2); 65.4 (s, PS); 13C-{1H} NMR (CDCl3): {delta} = 32.8 (d, 2JCP=12.3 Hz, CH3-N-P); 42.2 ("t", 3JCP=6.8 Hz, N-CH3); 49.3 (dd, 1JCP=164.0 Hz, 3JCP=9.9 Hz, CH2); 52.3 (d, 2JCP=5.8 Hz, OMe); 52.8 (d, 2JCP=7.2 Hz, OMe); 53.4 (d, 2JCP=7.0 Hz, OMe); 64.9 (dd, 1JCP=138.1 Hz, 3JCP=11.9 Hz, CH); 121,1 (br s, C02, C12); 128.2 (s, C03); 128.4 (d, 2JCP=3.1 Hz, C14); 131.8 (s, C04); 131.8 (d, 3JCP=8.2 Hz, C13); 139.0 (d, 3JCP=14.5 Hz, CH=N); 150.6 (d, 2JCP=6.9 Hz, C11); 151.2 (s, C01) ppm. Anal. Calc. for C204H312N27O90P33S6 (5797 g.mol–1): C, 42.26; H, 5.42, N, 6.52; found: C, 42.41; H, 5.51, N, 6.40.

8b-G1: To a solution of dendrimer 8a-G1 (3.97.10–2 mmol, 230 mg) at 0°C in acetonitrile (5 ml) was added dropwise trimethylsilylbromide (2.1 mmol, 280 µl). The reaction mixture was stirred at room temperature overnight, then evaporated to dryness under reduced pressure. The crude residue was dissolved with methanol (5 ml), vigorously stirred for 2 h at room temperature, and evaporated to dryness under reduced pressure. The resulting white solid was washed with methanol (10 ml), then dried under reduced pressure. The acidic-terminated dendrimer was then transformed into its sodium salt as follows: the dendrimer was suspended in water (1 ml/100 mg) and one equivalent of sodium hydroxide per terminal phosphonic acid was added. The resulting solution was filtered (micropore, 0.1 µ) and lyophilized to afford dendrimer 8b-G1 as a white powder (yield: 88%). 1H NMR (CD3CN/D2O): {delta} = 2.5–3.8 (m, 90H, CH3-N-P, CH-N-CH3, CH2, CH); 6.5–8.0 (m, 78H, Harom, CH=N); 31P-{1H} NMR (CD3CN/D2O): {delta} = 11.2 (s, PO3HNa); 14.1 (s, P0); 66.09 (s, PS); 13C-{1H} NMR (CD3CN/D2O): {delta} = 35.5 (br s, CH3-N-P); 44.8 (s, CH-N-CH3); 54.5 (d, 1JCP=132.5 Hz, CH2); 70.5 (d, 1JCP=129.4 Hz, CH); 124.4 (br s, C02, C12); 129.4 (s, C03); 131.4 (br s, C14); 135.7 (s, C04); 136.2 (br s, C13); 142.9 (m, CH=N); 153.9 (s, C01); 154.1 (s, C11) ppm.

Blood samples, cells, and cell cultures
Fresh blood samples were collected from healthy adult donors, and PBMC were prepared on a Ficoll-Paque density gradient (Amersham Biosciences AB, Uppsala, Sweden) by centrifugation (800 g, 30 min at room temperature). Collected PBMC were washed twice and finally diluted at 1.5 million cells/ml in complete RPMI 1640 medium, i.e., supplemented with penicillin and streptomycin, both at 100 U/ml (Cambrex Bio Science, Verviers, Belgium), 1 mM sodium pyruvate, and 10% heat-inactivated fetal calf serum (both from Invitrogen Corporation, Paisley, UK).

Monocyte purification and culture
Highly pure CD14+ monocytes (>98%, as checked by flow cytometry) were positively selected from PBMC by magnetic cell sorting on LS Separation Column (CD14 Microbeads, Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instruction manual. Three million purified monocytes were cultured in 3 ml of complete RPMI 1640 medium in 6-well plates. Sterile filtered solutions of the specified dendrimers were added to cultures at a final concentration of 20 µM.

Labeling experiment with the FITC-derived phosphorus dendrimer and competition experiments
Purified monocytes were incubated (30 min at 4°C) with either nonfluorescent dendrimer 2b-G1 (or dendrimer 12b-G1) in a range of concentrations between 9 nM and 50 µM. Then a solution of FITC-dendrimer 1b-G1 (at 5 µM) was added without prior rinsing. After 30 min of incubation at 4°C, monocytes were rinsed with PBS and analyzed by flow cytometry. The mean fluorescence intensity (mfi) corresponding to the FITC fluorescence was reported in relation to the concentration range of the nonfluorescent dendrimer 2b-G1.

Flow cytometry and microscopy
Flow cytometry was performed on a LSR-II cytometer (BD Biosciences, San Jose, CA, USA). All cell stainings were done using fluorochrome-conjugated monoclonal antibodies mAb from BD Biosciences (San Jose, CA, USA): clone UCHT1 for anti-CD3; clone G46–2.6 for anti-human leukocyte antigen-A,B,C; clone 2331 (FUN-1) for anti-CD86; clone B159 for anti-CD56; clone 3G8 for anti-CD16; clone WM15 for anti-CD13; clone HI98 for anti-CD15; clone WM53 for anti-CD33; clone Tü36 for anti-human leukocyte antigen-DR; clone B-ly6 for anti-CD11c; clone ICRF44 for anti-CD11b and clone M5E2 for anti-CD14. Clone TL2.1 for anti-TLR2 was from BioLegend (San Diego, CA, USA). To compare the surface densities of various molecules among different monocyte populations, we calculated the mean fluorescence intensity ratio (mfi-R), i.e., the ratio between the mfi of cells stained with the selected mAb and that of cells stained with the isotype control (negative control) (14) .

Apoptotic cells were detected as annexin-V positive cells (Apoptosis Detection Kit I, BD Biosciences, San Jose, CA, USA). Analyses were based on acquisitions of 105 cells per sample, and results were presented using the FACSDiva (BD Biosciences, San Jose, CA, USA) or WinMDI software.

For confocal microscopy, monocytes were stained with dendrimer 1b-G1 (20 µM, 30 min at 37°C), then samples were prepared as already described (15) and examined using a LSM 510 confocal microscope (Carl Zeiss, Iena, Germany). For colocation experiments, purified monocytes were first stained with red LysoTracker® (DND-99, 1 µM, 45 min at 37°C) and with TOTO-3 (TOTO-3 iodide 642/660, 1 µM, 15 min at 37°C), both from Molecular Probes (Eugene, OR, USA). After rinsing, monocytes were incubated with dendrimer 1b-G1 (20 µM) for 15 min at 37°C. Then samples were prepared as described (15) and analyzed with a LSM 510 confocal microscope. Fluorescence curves for LysoTracker®, TOTO-3, and dendrimer 1b-G1 were reported for a cross section of one monocyte with AIM software.

Phagocytosis
Phagocytic activity of monocytes was measured by internalization of Mycobacterium bovis BCG genetically modified to express Green Fluorescent Protein (GFP) (from B. Gicquel, Institut Pasteur, Paris). Monocytes and bacteria were coincubated with a multiplicity of infection of 200, 1 h at 37°C. Then cells were washed and analyzed by flow cytometry to detect GFP inside monocytes.

Nuclear Factor-{kappa}B (NF-{kappa}B) nuclear translocation
Nuclear extracts of purified monocytes treated 4 h at 37°C with dendrimer 2b-G1 (20 µM), peptidoglycan (5 µg ml1, Invitrogen, San Diego, CA, USA), or not treated were prepared ("Nuclear Extract Kit," Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions manual. Quantification of p50/p50 and p50/p65 NF-{kappa}B in nuclear extracts was achieved using the "TransAM NF-{kappa}B p50 Chemi" kit (Active Motif) according to the manufacturer’s instruction manual.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Synthesis of a FITC-derived phosphorus dendrimer to monitor dendrimer-PBMC interactions
Phosphorus dendrimers used in this work (Table 1 ) were built from a cyclotriphosphazene core P3N3 via reiteration of a sequence of two reactions involving nucleophilic substitution and condensation reactions for constituting OC6H4CHNN(Me)P(S) branches (10) . Then the surface of these phosphorus dendrimers could be decorated with a given number of phosphonic acid groups, either mono- (dendrimer 9b-G1), azamono- (dendrimer 5b-G1), symmetrical azabis- (dendrimers 2b-G1 and 4b-G1), or unsymmetrical azabis- (dendrimers 7b-G1 and 8b-G1) phosphonic groups or carboxylate groups we described earlier (dendrimers 11b-G0, 11b-G1, and 11b-G2) (12) . To screen by flow cytometry interactions between phosphorus dendrimers and cells of the human immune system, we synthesized and characterized a FITC derivative of this category of dendrimers. In this fluorescent dendrimer 1b-G1, a FITC group replaced statistically one of the 12 azabisphosphonic groups of the key compound 2b-G1. This tagged compound 1b-G1 was designed through an original synthesis involving orthogonal reactivity (Fig. 1) : the statistical reaction of one equivalent of G1, a first-generation dendrimer capped with six PSCl2 functions (13) , and one equivalent of the sodium salt of 4-hydroxybenzaldehyde in THF led to a dendrimer bearing one aromatic aldehyde and 11 reactive chlorine atoms. The nucleophilic substitution of these chlorine atoms by an azabisphosphonate derived from tyramine (compound 2) was realized under basic mild conditions in the presence of cesium carbonate in THF. The aldehydic proton could be detected by means of 1H NMR, ensuring the orthogonality of this method even in the last step, which involved the transformation of dimethylphosphonate groups to phosphonic acid termini in the presence of trimethylsilylbromide and subsequent methanolysis. Finally, the fluo-tagged fluorescein-5-thiosemicarbazide was reacted with the remaining aldehyde function in water to afford 1b-G1.


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  		Table 1. List of structures screened for monocyte activation

Targeting of FITC-derived dendrimer to monocytes in PBMC
We incubated the FITC-derived phosphorus dendrimer 1b-G1 (20 µM) for 30 min with human PBMC freshly isolated from an healthy donor. Flow cytometry revealed that a CD3– CD56– CD14+ population, thus corresponding to monocytes was the only hematopoietic population labeled with dendrimer 1b-G1 (Fig. 3 A). To further characterize the interaction between the fluorescent dendrimer 1b-G1 and monocytes, we magnetically purified human monocytes and filmed by confocal video microscopy their interaction with dendrimer 1b-G1. Sequential images showed that dendrimer 1b-G1 rapidly bound within a few seconds to monocyte surface (Fig. 3B ) and was progressively internalized within a few minutes and for hours (Fig. 3C ). To distinguish the intracellular route of dendrimers after their internalization by monocytes, we performed confocal microscopy experiments using dendrimer 1b-G1 and fluorescent specific molecular probes. Red LysoTracker® is a specific fluorescent probe for intracellular acidic vesicles corresponding to phagolysosomes in monocytes. Confocal microscopy showed colocation of LysoTracker® and dendrimer 1b-G1 (Fig. 3D ), and depicted the phagolysosomial route as the main uptake mechanism of dendrimer by monocytes. Of interest in this colocation experiment was the strong decrease of the FITC fluorescence in the acidic environment created by the fusion of phagosomes with lysosomes (16) . However, using a specific fluorescent probe for DNA (TOTO3), we could not detect any location of dendrimers in the nucleus.


Figure 3
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  		Figure 3. A) After a short incubation of PBMC with dendrimer 1b-G1, only CD3–CD56–CD14+ cells (i.e., monocytes) are labeled. B) Sequential images (first 64 seconds) from confocal videomicroscopy of purified monocytes (cytoplasmic labeling with orange 5-(-6)-(4-chloromethyl(benzoyl)amino) tetramethylrhodamine [CMTMR]) incubated with dendrimer 1b-G1 emitting green fluorescence (white bar in the lower right image indicates 10 µm). C) Membranous and internal location at 15 min but only intracellular location at 120 min of dendrimer 1b-G1 (white arrows) seen in confocal microscopy (white bar in the 120 min image indicates 10 µm). D) Fluorescent labeling of purified monocytes with TOTO-3 (nuclear labeling in blue), LysoTracker® (phagolysosomial labeling in red), and dendrimer 1b-G1 (in green). Left image is phase contrast microscopy; central image is confocal microscopy, and the right graph reports fluorescence curves along the white arrow in central image.

Synthesis of a series of phosphorus-containing dendrimers
To decipher the structural requirements of phosphorus-containing dendrimers to activate monocytes in culture, we synthesized a set of variously surfaced dendrimers with either phosphonic (mono-, azamono-, and azabisphosphonic acids) (Fig. 2) or carboxylic acids. We also synthesized a subdendritic structure: a phosphorus-containing branch (10b-G1) used for the dendritic outgrowth of dendrimers, ending with azabisphosphonic acids. These compounds are all listed in Table 1 .

First, we synthesized nonfluorescent dendritic structures bearing different phosphonic derivatives on their surface following a standardized procedure. This method involved the nucleophilic substitution of the 12 chlorine atoms of G1 by various phenol compounds equipped with variously functionalized phosphonate derivatives, the latter being transformed into their phosphonic acid derivatives after the grafting on dendrimers. Thus, our main synthetic efforts aimed at designing these phenols (Fig. 2) according to two different pathways. Symmetrical azabis(dimethyl)phosphonate phenolic derivatives like 2 and 3 were obtained from a catalyst free Kabachnik-Fields reaction involving aqueous formaldehyde, tyrosine, and dimethylphosphite. The acidic function of 3 was routinely converted into its methyl ester. Other phenol derivatives with unsymmetrical azabis(dimethyl)phosphonate tweezers like 7 and 8 were generated in three steps. A 4-hydroxy-imine was generated and hydrophosphinylated with dimethylphosphite to afford azamono(dimethyl)phosphonate intermediates, the latter being converted to azabisphosphonates 7 and 8 by a Kabachnik-Fields procedure. Nucleophilic substitution of phenols 2, 4, 5, 7, and 8 on G1 was achieved in nearly quantitative yields in the presence of cesium carbonate in THF, and the phosphonic acid-terminated dendrimers were finally obtained by treatment with trimethylsilylbromide in acetonitrile and subsequent methanolysis/salification process to afford dendrimers 2b-G1, 4b-G1, 5b-G1, 7b-G1, and 8b-G1, respectively. Dendrimer 9b-G1 was obtained by a direct Abramov-Pudovik reaction of dimethylphosphite on a dendrimer capped with aromatic aldehyde functions (Fig. 2) . Other dendrimers capped with carboxylate groups (series 11b-Gn generation n ranging from 0 to 2) were obtained from cinnamic acid-terminated dendrimers by addition of stoichiometric amounts of aqueous sodium hydroxide. A substructure of compounds 1b-G1 and 2b-G1, namely 10b-G1, was obtained from paramethoxybenzaldehyde following a routine dendritic outgrowth and surface function derivatization, as described for its parent compound. Starting from a fluorescent core bearing five aldehyde groups, a fluorescent dendritic tool was also synthesized following the same procedure (Fig. 4 ). This dendrimer 12b-G1 possessed fluorescence properties that permit FRET experiments with phycoerythrin (PE) -tagged targets.


Figure 4
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  		Figure 4. Dendritic FRET tool (12b-G1) synthesis. a/ pHO(C6H4)CHO, Cs2CO3, RT, THF; b/ H2NN(Me)P(S)Cl2, RT; c/ phenol 2, Cs2CO3, THF, RT; d/ BrSiMe3, CH3CN, RT; e/ MeOH RT, 60 min; HONa.

Azabisphosphonic phosphorus-containing dendrimers promote monocyte activation
At first we checked that dendrimer 2b-G1 bound to human monocytes. We assessed this point by displacing the binding of the fluorescent dendrimer 1b-G1 to the monocyte cell surface by competing with nonfluorescent dendrimer 2b-G1 (Fig. 5 A). Then we cultured purified human monocytes with dendrimer 2b-G1 over a few weeks and compared them to control nonstimulated monocytes. Within 3 to 6 days of culture, monocytes in culture with dendrimer 2b-G1 underwent morphological changes (Fig. 5B ); they also remained viable over longer periods than control monocytes (Fig. 5C ). Among early events during culture of monocytes with dendrimer 2b-G1, cells underwent phenotypic changes. Expression of a series of monocyte receptors and markers was analyzed by flow cytometry. CD14 and human leukocyte antigen (HLA)-DR were down-regulated as well as other markers when monocytes were cultured with dendrimer 2b-G1 (Fig. 6 A). As an additional proof for monocyte activation, we measured nuclear relocation of the NF-{kappa}B transcription factor. Despite their high level of basal NF-{kappa}B activation, purified monocytes cultured from 4 to 8 h with dendrimer 2b-G1 had increased nuclear relocation of NF-{kappa}B (Fig. 6B ). We also observed that within 3 to 6 days of culture, monocytes in culture with dendrimer 2b-G1 exhibited increased phagocytic activity toward Mycobacterium bovis BCG genetically modified to express GFP-M.b.BCG (Fig. 6C ). Moreover, neither morphological nor phenotypical criteria indicated that monocytes cultured with dendrimer 2b-G1 matured toward dendritic cells. Taken together, these results and observations indicated that monocytes were activated by dendrimer 2b-G1.


Figure 5
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  		Figure 5. A) Labeling of monocytes by dendrimer 1b-G1 can be displaced by competing with dendrimer 2b-G1 (IC50=45 µM). B) Monocyte activation is checked by microscopy: left image = unstimulated monocytes, right image = dendrimer 2b-G1 activated monocytes (cultures at day 3). These observations are confirmed by flow cytometry by morphological changes (increase in size and granularity). C) In culture of monocytes with dendrimer 2b-G1 (filled circles), % of apoptotic cells (annexin-V+) decreases (C, left), enabling a longer survey (C, right), comparatively to culture of monocytes without dendrimer (open circles).


Figure 6
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  		Figure 6. A) Phenotypical changes of monocyte activated by dendrimer 2b-G1 (filled bars, untreated monocytes: open bars). B) Monocytes are activated by dendrimer 2b-G1 as shown by NK-{kappa}B p50 nuclear relocation in a chemiluminescent readout (RLU: relative luminescent unit) (basal level: open bars; with dendrimer 2b-G1: filled bars; positive control: hatched bar, monocyte activation by peptidoglycan). Nuclear extracts of stimulated Jurkat cells serve as kit positive control (crossed bars). C) Monocyte activation is also checked in flow cytometry by an increase of phagocytosis detected by internalization of GFP-M.b.BCG. D) Screening of the bioactivity of various dendritic and subdendritic structures on human monocytes (mfi-R for HLA-DR and CD14 markers). Open circles: carboxylic acid capped dendrimers (11b-Gn, n from 0 to 2), open squares: monophosphonic acid capped dendrimers (5b-G1, 9b-G1) and monomer 5, open circles: bisphosphonic acid-capped dendrimers (2b-G1, 4b-G1, 7b-G1, 8b-G1), phosphorus-containing branch (10b-G1) and monomers 4, 8.

Dendritic structural requirements for human monocyte activation
Bioactivities of these dendritic and subdendritic structures and monomers were quantified by flow cytometry on morphological changes of monocytes (data not shown) and down-regulation of CD14 and HLA-DR surface markers on these monocytes (Fig. 6D ). This down-regulation was quantified by the mfi-R (see Materials and Methods): a low mfi-R indicated a strong activation of monocyte, and vice versa. This study showed that monomers could not elicit monocyte activation as dendrimer 2b-G1 did. The phosphorus-containing branch capped with an azabisphosphonic group (compound 10b-G1) promoted a better activation of monocyte in culture than monomers, but lower than that of dendrimer 2b-G1. Among phosphorus-containing dendrimers, molecules ending with carboxylic acid groups (dendrimers 11b-G0, 11b-G1, and 11b-G2) enabled lower activation of monocytes than molecules ending with phosphonic acid groups (dendrimers 2b-G1, 4b-G1, 5b-G1, 7b-G1, 8b-G1, and 9b-G1).

Molecular elements for monocyte activation by azabisphosphonic dendrimers
Finally, using a novel fluorescent dendritic nanobiotool (dendrimer 12b-G1), we searched for a potential monocyte receptor for 2b-G1-like dendrimers. In dendrimer 12b-G1, one of the six dendritic branches was replaced by a fluorescent 3,4-diphenylmaleimide group (Table 1) . We tested the ability of dendrimer 12b-G1 to activate monocytes in vitro. Despite lacking one branch over six, we showed that this dendrimer activated human monocytes in the same way as dendrimer 2b-G1 (Fig. 7 A). Dendrimer 12b-G1 was designed so that its fluorescence characteristics allowed FRET with PE, a commonly described fluorochrome for flow cytometry (Fig. 7B ). After monocyte labeling with dendrimer 12b-G1 and PE-coupled mAb against various typical monocytes receptor, PE fluorescence emission was achieved by FRET if dendrimer 12b-G1 was sufficiently close to the PE-coupled mAb. Among the tested PE-coupled mAbs, only the mAb against TLR2, but not the mAb against CD14 (data not shown), could be stimulated to emit fluorescence by FRET from dendrimer 12b-G1 (Fig. 7C ). This indicated that this typical innate receptor was somehow involved in dendrimer sensing by monocytes.


Figure 7
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  		Figure 7. A) Bioactivity of dendrimer 12b-G1 measured by down-regulation of HLA-DR and CD14 markers (filled bars) compared with the effect of dendrimer 2b-G1 (gray bars) and to untreated monocytes (open bars) after a 5 day culture. B) Spectral properties of dendrimer 12b-G1 compared with spectral properties of PE: dendrimer 12b-G1 achieves FRET on PE. Dotted lines: excitation spectra, full lines: emission spectra. C) The involvement of TLR2 in the sensing of phosphorus-containing dendrimers evidenced by FRET experiment (immunoglobulin control is a mouse immunglobulin isotype control).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Dendrimers are monodisperse polyfunctionalized hyperbranched polymers whose nanometer size, topology, perfectly defined structures, multivalent character, and molecular weight can be rigorously controlled during synthesis. The high density of functionalities on their surface as well as their globular shape confer to these special polymers versatile and unique properties that have been exploited in biology and more intensively in material science (17) . Among these nano-objects, phosphorus-containing dendrimers occupy a special space due to the reactivity of phosphorus, which allows preparation of a large variety of dendritic and multidendritic macromolecules (18) . The biocompatibility of some phosphorus dendrimers bearing anionic charges on the end groups has been tested (19 , 20) . In this study we took advantage of the versatility of dendrimers in general and the reactivity of phosphorus in particular to synthesize a variety of phosphorus dendrimers. Using the fluorescent nano-biotool dendrimer 1b-G1, we showed that this family of dendrimers selectively targets monocytes among human PBMC. Blood monocytes and their tissue counterpart macrophages are mononuclear phagocytes. On the one hand, they are capable of engulfing endogenous matters, such as cellular apoptotic debris and injured or dead cells. On the other hand, they can also scavenge foreign substances such as whole infectious microorganisms or insoluble nano-objects; among these, phosphorus dendrimers as reported here. Moreover, we also realized that they are internalized via the phagolysosomial route.

Normally, microorganisms internalized by macrophages are to be killed when lysosomal content is delivered in phagosomes. In some cases, however, microorganisms can survive and even multiply within the macrophages: they are pathogens (e.g., Mycobacteria, Salmonella, Leishmania, dengue virus... ). Thus, targeting drug delivery to macrophages to fight against parasitic infections is an attractive therapeutic strategy (21) . With this aim, the two major points that have to be addressed are selective targeting and internal drug delivery to macrophages. Besides liposomes, nanoparticles, and microspheres, phosphorus-containing dendrimers are new nano-scale candidates. First, their versatility enables chemical binding of different active drugs with specific antimicrobial activities on one (or more) branch in place of phosphonic acid groups of the dendritic structure. Second, they specifically target monocytes/macrophages among PBMC. Third, they are internalized by monocytes/macrophages. Fourth, drugs will be delivered by the lytic content of lysosomes. To our knowledge, only poly(amidoamine) dendrimers have been proposed so far for controlled site-specific drug delivery (22) .

Studying human monocytes/macrophages in culture with azabisphosphonic acid capped phosphorus dendrimer 2b-G1 over a few days, we documented activation features of these cells. The bioactivity of several phosphorus-containing dendrimers, surfaced with phosphonic acid or carboxylic acid groups, was screened for their ability to activate monocytes. We demonstrated that surface phosphonic groups constitute an important determinant for the bioactivity, since phosphorus-containing dendrimers capped with carboxylic acid groups were much less active. We also prepared and tested a single phosphorus-containing branch (10b-G1) along with tyramine- or tyrosine-derived monomers used to cap dendrimers for monocyte activation. These subdendritic compounds were poor activators. Taken together, these results indicate a strong requirement for all the 3-dimensional scaffold of phosphonic acid surfacing groups framed around the P3N3 core of phosphorus-containing dendrimers.

Monocyte/macrophage can take several aspects (23) . Besides the early described classical activation pathway, an alternative activation mechanism emerged in 1992 in the mouse model (24) . The classical activation of monocytes/macrophages is mediated by IFN-{gamma} as primer, then tumor necrosis factor (TNF) or microbial trigger, whereas alternative activation is mediated by interleukin (IL) -4, IL-13, and glucocorticoids (23 , 25) . The classical activation pathway of monocytes evolves toward an inflammatory immune response (secretion of high levels of IL-1, IL-6, IL-12, and TNF-{alpha}) while the alternative pathway evolves toward an anti-inflammatory response (secretion of high amounts of IL-10 and IL-1 receptor antagonist) (25) . With a transcriptomic approach, we will further assess the type of activation that monocytes/macrophages stimulated with phosphonic acid group-ended, phosphorus-containing dendrimers undergo.

Using the fluorescent nano-biotool dendrimer 12b-G1 especially designed to enable FRET with PE-conjugated partners, we showed that the typical innate receptor TLR2 is involved in the sensing of phosphorus-containing dendrimers. Nevertheless, these dendrimers were not recognized and sensed by TLR2-transfected HEK293 eukaryotic cells or by combined TLR2/TLR1- or TLR2/TLR6-transfected HEK293 cells (M. Poupot et al., personal communication). This indicated that TLR2 is not the monocyte/macrophage receptor for phosphorus dendrimers, but may participate to a recognition/signal transducing complex, as shown for another member of the TLR family, namely TLR4, in mouse monocytes (26) . Moreover, the involvement of TLR2 in a signaling complex after phosphorus-containing dendrimer recognition by monocytes may not necessarily imply that these cells undergo an inflammatory maturation (8) . It was recently shown that mouse dendritic cells (27) or human monocyte-derived dendritic cells may undergo either inflammatory or anti-inflammatory maturation processes upon TLR2 triggering (28 , 29) .

Thus, phosphorus-containing dendrimers are a paradigm of versatile chemical tools affecting the expansion of biology through engineered nano-biotools and new therapeutic agents. These phosphorylated dendrimers represent an extraordinary set of virtually unlimited number of chemical structures. They clearly appear as new tunable therapeutic candidates to target and modulate human innate immunity via monocyte activation.


   ACKNOWLEDGMENTS
 
This work was supported by grants from Région Midi-Pyrénées ("Biothérapies" program), ARC (ARECA 2004), MENSR ("Action de Soutien à l’Innovation"); institutional funding from "Fonds Structurels Européens" (M.P.), INSERM, Paul Sabatier University, CNRS (fellowship to A.M.); industrial funding from Rhodia Ldt. (fellowships to L.G., P.M., O.R.). We thank Pr. Jean-Jacques Bonnet for continuous support.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication March 22, 2006. Accepted for publication June 23, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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