mary poupot et al- design of phosphorylated dendritic architectures to promote human monocyte...

8/3/2019 Mary Poupot et al- Design of phosphorylated dendritic architectures to promote human monocyte activation

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The FASEB Journal Research Communication

Design of phosphorylated dendritic architectures topromote human monocyte activation

Mary Poupot,*,1 Laurent Griffe,,1 Patrice Marchand, Alexandrine Maraval,

Olivier Rolland,

Ludovic Martinet,* Fatima-Ezzahra LFaqihi-Olive,*Cedric-Olivier Turrin, Anne-Marie Caminade,,2 Jean-Jacques Fournie,*Jean-Pierre Majoral,,2 and Remy Poupot*,2

*INSERM, U.563, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, F-31300 France;Universit Paul-Sabatier, Toulouse, F-31400 France; and Laboratoire de Chimie de Coordinationdu CNRS, Toulouse cedex, France

ABSTRACT As first defensive line, monocytes are apivotal cell population of innate immunity. Monocyteactivation can be relevant to a range of immune condi-tions and responses. Here we present new insights intothe activation of monocytes by a series of phosphonic

acid-terminated, phosphorus-containing dendrimers.Various dendritic or subdendritic structures were syn-thesized and tested, revealing the basic structural re-quirements for monocyte activation. We showed thatmultivalent character and phosphonic acid capping ofdendrimers are crucial for monocyte targeting andactivation. Confocal videomicroscopy showed that afluorescein-tagged dendrimer binds to isolated mono-cytes and gets internalized within a few seconds. Wealso found that dendrimers follow the phagolysosomialroute during internalization by monocytes. Finally, we performed fluorescence resonance energy transfer(FRET) experiments between a specifically designed

fluorescent dendrimer and phycoerythrin-coupled anti-bodies. We showed that the typical innate Toll-likereceptor (TLR)-2 is clearly involved, but not alone, inthe sensing of dendrimers by monocytes. In conclusion,phosphorus-containing dendrimers appear as preciselytunable nanobiotools able to target and activate humaninnate immunity and thus prove to be good candidatesto develop new drugs for immunotherapies.Poupot,M., Griffe, L., Marchand, P., Maraval, A., Rolland, O.,Martinet, L., LFaqihi-Olive, F.-E., Turrin, C.-O., Cami-nade, A.-M., Fournie, J.-J., Majoral, J.-P., Poupot, R.Design of phosphorylated dendritic architectures to promote human monocyte activation. FASEB J. 20,

23392351 (2006)

Key Words: cellular immunotherapy targeting phosphorusdendrimers

Cooperative association energies, synergistic ef-fects, or receptor clustering, among others, are oftentargeted properties when dealing with dendrimers in-teracting with biological entities. These interactionshave been thoroughly studied during the last decade,bringing attention to the peculiar properties and bio-

medical potential of dendritic macromolecules (13)by virtue of well-defined structure, multivalency, orshape, and the possibility to precisely tune the degreeof branching and the flexibility of the dendriticskeleton or the hydrophilicity of the whole dendritic

object.Cell surface-mediated immunoregulation has beenshown to be highly relevant from the multivalencypoint of view. For instance, dendrimer glucosamineconjugates were safely used to prevent scar tissue for-mation via immunomodulatory and antiangiogenicproperties (4). Dendritic multiple antigenic peptideshave also proved to be promising pivotal compoundsfor immune response modification or immunodiagnos-tics (5, 6). The proof of the multivalent concept appliedto dendrimer science has also been illustrated by recentadvances in the development of novel dendritic ligandsfor lethal bacterial toxins or promising antiviral den-

dritic-based materials (7).Phagocytes of the innate immune system provide a

first line of defense against many common microor-ganisms and are essential for the control of commonbacterial infections. Among phagocytes, monocytesare white blood mononuclear cells that are precur-sors to macrophages (8). The cells of the innateimmune system play a crucial part in the initiationand subsequent direction of adaptive immune re-sponses. Moreover, because there is a delay of severaldays before the initial adaptive immune responsetakes effect, the innate immune response has a

critical role in controlling infections during thisperiod.

1 These authors contributed equally to this work.2 Correspondence: INSERM 563, Centre de Physiopatholo-

gie de Toulouse-Purpan, Hopital Purpan, BP3028, 31024Toulouse cedex 03, France. E-mail: [email protected]; Laboratoire de Chimie de Coordination duCNRS, 205 route de Narbonne, 31077 Toulouse cedex 04,France. E-mail: [email protected]; Laboratoire de Chimiede Coordination du CNRS, 205 route de Narbonne, 31077Toulouse cedex 04, France. E-mail: [email protected]

doi: 10.1096/fj.06-5742com

23390892-6638/06/0020-2339 FASEB


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Macrophages and neutrophils have surface receptorsthat have evolved to recognize and bind commonconstituents of many bacterial surfaces (9). Bacterialmolecules binding to these receptors trigger the cells toengulf the bacterium and also induce the secretion ofbiologically active molecules by these phagocytes.

In this study we looked for interactions betweenphosphorus-containing dendrimers (10, 11) and hema-topoietic cells of the human immune system. Synthesis

of a fluorescein isothiocyanate (FITC) -derived phos-phorylated dendrimer enabled us to monitor theseinteractions. By using this probe we showed that,among peripheral blood mononuclear cells (PBMC),monocytes were the main population targeted by den-drimers in vitro. Later on we showed that these mono-cytes were activated by phosphorylated dendrimers. Bysynthesizing a series of phosphonic acid or carboxylicacid surfaced dendrimers, we identified that phos-phonic acid groups as a major structural requirementfor phosphorus-containing dendrimer bioactivity. Theentire dendritic structure appeared as another struc-tural requirement for this bioactivity, as branches or

surface groups alone did not trigger monocyte activa-tion. Finally, we showed that sensing of dendrimer bymonocytes involved a typical receptor of innate immu-nity, namely the Toll-like receptor (TLR)-2.

MATERIALS AND METHODS

Synthesis procedures

All reactions were carried out in the absence of air usingstandard Schlenk techniques and vacuum line manipulations.Commercial samples were used as received. All solvents weredried before use. Thin-layer chromatography was carried outon Merck Kieselgel 60F254 precoated silicagel plates. Prepar-ative flash chromatography was performed on Merck Kiesel-

gel. Instrumentation: Bruker AC 200, AM 250, ARX 250, DPX300, AMX 400, Avance 500 (1H, 13C, and 31P NMR). Thenumber of atoms shown as a subscript refers to the genera-tion; the superscript number and letter refer to the O-C6H4(C1 linked to O) and C6H5 groups, respectively. Elementalanalyses were performed by the Service dAnalyse du Labora-toire de Chimie de Coordination (Toulouse, France). Thesynthesis and characterization of the FITC derivative 1b-G1(Fig. 1) and of compounds 2, 5, 7, 9, Xab-G1 (X2, 5, 7, 10,12) (Fig. 2) will be described elsewhere. The synthesis ofcompounds 11ab-Gn (n0, 1, 2) was carried out as reportedpreviously (12) from a cyclotriphosphazene core (13). Thesynthesis and characterization of two representative com-pounds are given below.

To a solution of (d-l) tyrosine (2 g, 11.05 mmol) in THF (4ml) was added a solution of formaldehyde (37% in water, 3ml, and 30.8 mmol) and dimethylphosphite (3 ml, 32.7mmol). The resulting mixture was stirred overnight at roomtemperature. The crude material was concentrated under

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

2340 Vol. 20 November 2006 POUPOT ET AL.The FASEB Journal


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reduced pressure and washed successively with 2 15 ml of

ethyl acetate and 2 15 ml of dichloromethane. The solid was dried under reduced pressure to afford 3 as a whitepowder (yield: 85%). 1H NMR (CDCl3 (CD)3OD): 2.81(dd, 2H, 2JHH14.2 Hz,

3JHH7.3 Hz, CHHCH); 3.04 (dd,2H, 2JHH14.2 Hz,

3JHH7.3 Hz, CHHCH); 3.223.51 (m,4H, PCH2); 3.70 (d, 6H,

3JHP8.1 Hz, POMe); 3.76 (d, 6H,3JHP8.1 Hz, POMe); 4.14 (t,

3JHP7.3 Hz, 1H, CH); 6.70 (d,2H, 3JHH8.5 Hz, C

2-H); 7.13 (d, 2H, 3JHP8.5 Hz, C3-H);

31P-{1H} NMR (CDCl3CD3OD): 31.04 (s, PO3Me2);13C-{1H} NMR (cluster of differentiation3OD): 35.05 (s,CH-CH2); 47.3 (dd,

1JCP167.3 Hz,3JCP9.3 Hz, PCH2);

52.89 (d, 2JCP7.0 Hz, POMe); 53.30 (d,2JCP7.0 Hz,

POMe); 66.60 (t, 3JCP6.9 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.mol1): 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 paratoluenesulfonicacid was refluxed for 36 h. The mixture was then cooled downto room temperature and concentrated under reduced pres-sure. The resulting crude oil was washed with a mixture ofpentane and diethylether. A sticky solid was obtained, dis-solved in THF, and precipitated with a mixture of pentaneand diethylether to afford 4 as a white powder that waspurified by column chromatography (eluent: CHCl3/MeOH,95/5) (yield: 90%). 1H NMR (CDCl3): 2.83 and 2.96 (ABpart of ABX syst, 2JHAHB14.1 Hz,

3JHAHX6.6 Hz,3JH-

BHX8.1 Hz, 2H, ArCHAHBCHX); 3.20 and 3.39 (AB part of ABX syst, 2JHAHB

2JHBHX16.5 Hz,2JHAHX4.5 Hz, 4H,

Figure 2. Dendrimer synthesis. Scheme 1)Synthesis of azabis(dimethyl)phosphonate armed phenols. a: (for tyramine) aqueousH2CO, HPO3Me2, THF, RT; (for tyrosine) aqueous H2CO, HPO3Me2, RT followed by MeOH, cat. PTSA, reflux. b: H2NR1, THF,MgSO4, RT; c: HPO3Me2; 50C; d: aqueous H2CO, HPO3Me2, THF, RT; Scheme 2) Synthesis of phosphonic acid-cappeddendrimers: G1 to Xa-G1 series (X2, 4, 5, 7, 8): 12 eq. of phenol X and 24 eq. of Cs2CO3 for 1 eq. of dendrimer G1 in THFat RT; G1 to 9a-G1: 4-hydroxybenzaldehyde, Cs2CO3, THF, RT, then HPO3Me2, THF, RT; Xa-G1 series to Xb-G1 series (X2,4, 5, 7, 8, 9): BrSiMe3, CH3CN, RT; MeOH RT, 60 min; HONa.

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CHAHBPX); 3.63 (s, 3H, COOMe); 3.72 (d, 6H,3JHP10.5

Hz, POMe); 3.74 (d, 6H, 3JHP10.8 Hz, POMe); 4.40 (t, 1H,3JHH7.2 Hz, CH); 6.79 (d, 2H,

3JHH7.9 Hz, C2-H); 7.08 (d,

2H, 3JHH7.9 Hz, C3-H); 7.28 (br s, OH); 31P-{1H} NMR

(CDCl3): 29.61 (s, PO3Me2);13C-{1H} NMR (CDCl3):

35.6 (s, CH2 Ar); 47.6 (dd,1JCP166.8 Hz,

3JCP9.8 Hz,PCH2); 51.8 (s, CO2Me); 53.9 (d,

3JCP6.8 Hz, POMe); 53.8(d, 3JCP6.8 Hz, POMe); 66.5 (t,

3JCP6.8 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 mol)in THF (5 l) was added cesium carbonate (383 mg, 1.175mmol) and phenol 4 (420 mg, 0.956 mmol). The reactionmixture was stirred for 36 h at room temperature, thencentrifuged and filtered through a pad of celite. The resultingclear solution was concentrated under reduced pressure (1ml), then precipitated with a large excess of pentane. Unre-acted phenol 4 was removed by dissolving the powder in 2 to5 ml of THF and subsequent precipitation with an excess ofether to afford dendrimer 4a-G1 as a white powder (yield:73%). 1H NMR (CDCl3): 2.793.10 (m, 24H, CH2Ar);3.103.50 (m, 66H, PCH2 and NCH3); 3.62 (d, 72H,3JHP10.6 Hz, POMe); 3.68 (d, 72H,

3JHP10.6 Hz, POMe);3.75 (s, 36H, COOMe); 4.43 (t, 12H, 3JHH6.9 Hz, CH);7.017.09 (m, 36H, C0

2-H and C12-H); 7.237.27 (m, 24H,

C13-H); 7.597.63 (m, 18H, C13-H and CHN); 31P-{1H} NMR(CDCl3): 11.52 (s, N3P3); 29.42 (s, PO3Me2); 66.55 (s,PS); 13C-{1H} NMR (CDCl3): 32.78 (d,

2JCP11.9 Hz,NCH3); 35.24 (s, CHCH2Ar); 46.99 (dd,

1JCP165.5,3JCP9.2

Hz, PCH2); 51.48 (s, CO2Me); 52.42 (d,2JCP7.4 Hz, POMe);

53.16 (d, 2JCP6.3 Hz, POMe); 65.22 (s, CH); 121.05 (d,3JCP3.0 Hz, C0,1

2); 128.26 (s, C03); 130.55 (s, C1

3); 132.02 (s,C0

4); 134.61 (s, C14); 138.86 (d, 3JCP14.0 Hz, CHN);

149.14 (d, 2JCP6.5 Hz, C11); 151.24 (s, C0

1); 171.85 (s,CO2Me) ppm. Anal. Calc. for C240H360N27O114P33S6 (6662g.mol1): 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, 22.5mol) in acetonitrile (5 ml) maintained at 0C was added

dropwise trimethylsilylbromide (222 l, 1.68 mmol). Thereaction mixture was stirred at room temperature overnight,then evaporated to dryness under reduced pressure. Thecrude residue was dissolved with methanol (5 ml), vigorouslystirred for 2 h at room temperature, and evaporated todryness under reduced pressure. The resulting white solid was

washed with methanol (10 ml), then dried under reducedpressure. The acidic-terminated dendrimer was then trans-formed into its sodium salt as follows: the dendrimer wassuspended in water (1 ml/100 mg) and one equivalent ofsodium hydroxide per terminal phosphonic acid was added.The resulting solution was filtered (micropore, 0.1 ) andlyophilized to afford dendrimer 4b-G1 as a white powder(yield: 91%). 1H NMR (CD3CN/D2O): 3.274.06 (m,126H, Ph-CH2-CH, CH3-N-P1, N-CH2-P, OMe); 4.95 (s, 12H,

CH); 7.36 8.22 (m, 78H, CHN, Harom);31

P-{1

H} NMR(CD3CN/D2O): 12.04 (s, PO3HNa); 12.64 (s, N3P3);66.32 (s, PS); 13C-{1H} NMR (CD3CN/D2O): 31.85 (s,

Ar-CH2-CH); 33.16 (s, NCH3); 50.95 (d,1JCP138.4 Hz,

CH2-P); 51.42 (d,1JCP148.5 Hz, CH2-P); 52.63 (s, CO2Me);

68.57 (s, CH-CH2-N); 121.71 (s, C02, C1 (2)); 128.87 (s, C0

3);131.25 (s, C1

3); 132.29 (s, C0 (4)); 134.88 (s, C14); 140.44 (br

s, CHN); 149.74 (d, 2JCP6.5 Hz, C11); 151.13 (s, C0

1);171.91 (s, CO2Me) ppm.

8: Methylamine (25 mmol, 3 ml of a 33% solution inabsolute ethanol, 8 M) and 4-hydroxybenzaldehyde (20mmol, 2.5 g) were stirred in a flask for 1 day at RT. Thereaction mixture was concentrated under reduced pressureand dissolved in 5 ml of diethylether. A white solid was

obtained upon precipitation with pentane. This imine (17mmol, 2.3 g) was immediately reacted with dimethylphos-phite (18.7 mmol, 1.7 ml) with one drop of triethylamine.The reaction mixture was evaporated to dryness under re-duced pressure, and the resulting powder was flash chro-matographied on a pad of silica (acetone) to afford anaza-monophosphorylated phenol (12.2 mmol, 3.0 g, [31P]29.6 ppm), which was immediately reacted with aque-ous formaldehyde (33%, 24.4 mmol, 2 ml) and dimethylphos-phite (48.8l mmol, 5.5 ml) for 12 h at room temperature. Thecrude residue was evaporated to dryness, then purified by

column chromatography (silica, ethyl acetate, Rf0.35) toafford 8 in 55% overall yield.

1H NMR (CDCl3): 2.41 (s, 3H, NCH3); 2.61 (dd,2JHH15.3 Hz,

2JHP6.3 Hz, 1H, CHH); 3.12 (dd,2JHP15.6

Hz, 2JHH15.3 Hz, 1H, CHH); 3.32 (d, 3H,3JHP10.2 Hz,

POMe); 3.60 (d, 3H, 3JHP10.9 Hz, POMe); 3.67 (d, 3H,3JHP10.6 Hz, POMe); 3.73 (d, 3H,

3JHP10.9 Hz, POMe);4.05 (d, 2JHP23.9 Hz, 1H, CH); 6.74 (d,

3JHH7.8 Hz, 2H,Harom); 7.17 (d,

3JHH7.8 Hz, 2H, Harom); 9.08 (br s, 1H,OH); 31P-{1H} NMR (CDCl3): 28.1 (s, PO3Me2); 30.9 (s,PO3Me2);

13C-{1H} NMR (CDCl3): 42.3 (t,3JCP6.3 Hz,

N-CH3); 49.2 (dd,1JCP164.1 Hz,

3JCP10.1 Hz, CH2); 52.58(d, 2JCP7.6 Hz, POMe); 52.97 (d,

2JCP6.9 Hz, POMe);53.08 (d, 2JCP6.9 Hz, POMe); 53.45 (d,

2JCP7.6 Hz,POMe); 65.2 (dd, 1JCP161.7 Hz,

3JCP13.5 Hz, CH); 115.4(s, C2); 120.9 (d, 2JCP3.5 Hz, C4); 131.8 (d, 3JCP9.1 Hz,C3); 157.8 (s, C1) ppm. Anal. Calc. for C13H23NO7P2 (367.3g.mol1): 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 mol) inTHF (2 ml) was added cesium carbonate (390 mg, 1.2 mmol)and phenol 8 (220 mg, 0.6 mmol). The reaction mixture wasstirred for 24 h at room temperature, then centrifuged andfiltered through a pad of celite. The resulting clear solution

was concentrated under reduced pressure (1 ml), thenprecipitated with a large excess of pentane. Unreacted phe-nol 4 was removed by dissolving the powder in 2 to 5 ml ofTHF and subsequent precipitation with an excess of ether toafford dendrimer 8a-G1 as a white powder (yield: 75%).

1H

NMR (CDCl3): 2.46 (s, 36H, CH-N-CH3); 2.65 (dd,2JHH15.3 Hz,2JHP7.4 Hz, 12H, CH2); 3.12 (dd,

2JHP15.5Hz, 2JHH15.3 Hz, 12H, CH2); 3.25 (d,

3JHP10.1 Hz, 18H,CH3-N-P); 3.303.90 (m, 144H, OMe); 4.2 (d,

2JHP23.4 Hz,12H, CH); 6.77.6 (m, 78H, Harom, CHN);

31P-{1H} NMR(CDCl3): 11.4 (s, N3P3); 27,5 (s, PO3Me2); 30.4 (s,PO3Me2); 65.4 (s, PS);

13C-{1H} NMR (CDCl3): 32.8 (d,2JCP12.3 Hz, CH3-N-P); 42.2 (t,

3JCP6.8 Hz, N-CH3); 49.3(dd, 1JCP164.0 Hz,

3JCP9.9 Hz, CH2); 52.3 (d,2JCP5.8 Hz,

OMe); 52.8 (d, 2JCP7.2 Hz, OMe); 53.4 (d,2JCP7.0 Hz,

OMe); 64.9 (dd, 1JCP138.1 Hz,3JCP11.9 Hz, CH); 121,1

(br s, C02, C1

2); 128.2 (s, C03); 128.4 (d, 2JCP3.1 Hz, C1

4);131.8 (s, C0

4); 131.8 (d, 3JCP8.2 Hz, C13); 139.0 (d,

3JCP14.5 Hz, CHN); 150.6 (d,2JCP6.9 Hz, C1

1); 151.2 (s,C0

1) ppm. Anal. Calc. for C204H312N27O90P33S6 (5797 g.mol1):

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 (3.97.102 mmol,

230 mg) at 0C in acetonitrile (5 ml) was added dropwisetrimethylsilylbromide (2.1 mmol, 280 l). The reaction mix-ture was stirred at room temperature overnight, then evapo-rated to dryness under reduced pressure. The crude residue

was dissolved with methanol (5 ml), vigorously stirred for 2 hat room temperature, and evaporated to dryness underreduced pressure. The resulting white solid was washed withmethanol (10 ml), then dried under reduced pressure. Theacidic-terminated dendrimer was then transformed into itssodium salt as follows: the dendrimer was suspended in water(1 ml/100 mg) and one equivalent of sodium hydroxide perterminal phosphonic acid was added. The resulting solution



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was filtered (micropore, 0.1 ) and lyophilized to afforddendrimer 8b-G1 as a white powder (yield: 88%).

1H NMR(CD3CN/D2O): 2.53.8 (m, 90H, CH3-N-P, CH-N-CH3,CH2, CH); 6.58.0 (m, 78H, Harom, CHN);

31P-{1H} NMR(CD3CN/D2O): 11.2 (s, PO3HNa); 14.1 (s, P0); 66.09 (s,PS); 13C-{1H} NMR (CD3CN/D2O): 35.5 (br s, CH3-N-P);44.8 (s, CH-N-CH3); 54.5 (d,

1JCP132.5 Hz, CH2); 70.5 (d,1JCP129.4 Hz, CH); 124.4 (br s, C0

2, C12); 129.4 (s, C0

3);131.4 (br s, C1

4); 135.7 (s, C04); 136.2 (br s, C1

3); 142.9 (m,CHN); 153.9 (s, C0

1); 154.1 (s, C11) ppm.

Blood samples, cells, and cell cultures

Fresh blood samples were collected from healthy adult do-nors, and PBMC were prepared on a Ficoll-Paque densitygradient (Amersham Biosciences AB, Uppsala, Sweden) bycentrifugation (800 g, 30 min at room temperature). Col-lected PBMC were washed twice and finally diluted at 1.5million cells/ml in complete RPMI 1640 medium, i.e., sup-plemented with penicillin and streptomycin, both at 100U/ml (Cambrex Bio Science, Verviers, Belgium), 1 mMsodium 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 flowcytometry) were positively selected from PBMC by magneticcell sorting on LS Separation Column (CD14 Microbeads,Miltenyi Biotec, Auburn, CA, USA) according to the manu-facturers instruction manual. Three million purified mono-cytes were cultured in 3 ml of complete RPMI 1640 mediumin 6-well plates. Sterile filtered solutions of the specifieddendrimers were added to cultures at a final concentration of20 M.

Labeling experiment with the FITC-derived phosphorusdendrimer and competition experiments

Purified monocytes were incubated (30 min at 4C) witheither nonfluorescent dendrimer 2b-G1 (or dendrimer 12b-G1) in a range of concentrations between 9 nM and 50 M.Then a solution of FITC-dendrimer 1b-G1 (at 5 M) wasadded without prior rinsing. After 30 min of incubation at4C, monocytes were rinsed with PBS and analyzed by flowcytometry. The mean fluorescence intensity (mfi) correspond-ing to the FITC fluorescence was reported in relation to theconcentration 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 doneusing fluorochrome-conjugated monoclonal antibodies mAbfrom BD Biosciences (San Jose, CA, USA): clone UCHT1 foranti-CD3; clone G462.6 for anti-human leukocyte antigen-

A,B,C; clone 2331 (FUN-1) for anti-CD86; clone B159 foranti-CD56; clone 3G8 for anti-CD16; clone WM15 for anti-CD13; clone HI98 for anti-CD15; clone WM53 for anti-CD33;clone Tu36 for anti-human leukocyte antigen-DR; clone B-ly6for anti-CD11c; clone ICRF44 for anti-CD11b and clone M5E2for anti-CD14. Clone TL2.1 for anti-TLR2 was from BioLeg-end (San Diego, CA, USA). To compare the surface densitiesof 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 persample, and results were presented using the FACSDiva (BDBiosciences, San Jose, CA, USA) or WinMDI software.

For confocal microscopy, monocytes were stained withdendrimer 1b-G1 (20 M, 30 min at 37C), then samples wereprepared as already described (15) and examined using aLSM 510 confocal microscope (Carl Zeiss, Iena, Germany).

For colocation experiments, purified monocytes were firststained with red LysoTracker (DND-99, 1 M, 45 min at37C) and with TOTO-3 (TOTO-3 iodide 642/660, 1 M, 15min at 37C), both from Molecular Probes (Eugene, OR,USA). After rinsing, monocytes were incubated with den-drimer 1b-G1 (20 M) for 15 min at 37C. Then samples wereprepared as described (15) and analyzed with a LSM 510confocal microscope. Fluorescence curves for LysoTracker,TOTO-3, and dendrimer 1b-G1 were reported for a crosssection of one monocyte with AIM software.

Phagocytosis

Phagocytic activity of monocytes was measured by internaliza-

tion of Mycobacterium bovis BCG genetically modified to ex-press Green Fluorescent Protein (GFP) (from B. Gicquel,Institut Pasteur, Paris). Monocytes and bacteria were coincu-bated with a multiplicity of infection of 200, 1 h at 37C. Thencells were washed and analyzed by flow cytometry to detectGFP inside monocytes.

Nuclear Factor-B (NF-B) nuclear translocation

Nuclear extracts of purified monocytes treated 4 h at 37Cwith dendrimer 2b-G1 (20 M), peptidoglycan (5 g ml

1,Invitrogen, San Diego, CA, USA), or not treated were pre-pared (Nuclear Extract Kit, Active Motif, Carlsbad, CA,USA) according to the manufacturers instructions manual.Quantification of p50/p50 and p50/p65 NF-B in nuclearextracts was achieved using the TransAM NF-B p50 Chemikit (Active Motif) according to the manufacturers instructionmanual.

RESULTS

Synthesis of a FITC-derived phosphorus dendrimer tomonitor dendrimer-PBMC interactions

Phosphorus dendrimers used in this work (Table 1) were built from a cyclotriphosphazene core P3N3 viareiteration of a sequence of two reactions involving

nucleophilic substitution and condensation reactionsfor constituting OC6H4CHNN(Me)P(S) branches (10).Then the surface of these phosphorus dendrimerscould be decorated with a given number of phosphonicacid groups, either mono- (dendrimer 9b-G1),azamono- (dendrimer 5b-G1), symmetrical azabis-(dendrimers 2b-G1 and 4b-G1), or unsymmetricalazabis- (dendrimers 7b-G1 and 8b-G1) phosphonicgroups or carboxylate groups we described earlier(dendrimers 11b-G0, 11b-G1, and 11b-G2) (12). Toscreen by flow cytometry interactions between phos-phorus dendrimers and cells of the human immune



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TABLE 1. List of structures screened for monocyte activation

Monomer or dendritic structure Compound number

1b-G1

2b-G1

4

4b-G1

5

5b-G1

7b-G1

8

8b-G1

9b-G1

10b-G1

11b-G0

11b-G1

11b-G2

12b-G1



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system, we synthesized and characterized a FITC deriv-ative of this category of dendrimers. In this fluorescentdendrimer 1b-G1, a FITC group replaced statisticallyone of the 12 azabisphosphonic groups of the keycompound 2b-G1. This tagged compound 1b-G1 wasdesigned through an original synthesis involving or-thogonal reactivity (Fig. 1): the statistical reaction ofone equivalent of G1, a first-generation dendrimercapped with six PSCl2 functions (13), and one equiva-

lent of the sodium salt of 4-hydroxybenzaldehyde inTHF led to a dendrimer bearing one aromatic aldehydeand 11 reactive chlorine atoms. The nucleophilic sub-stitution of these chlorine atoms by an azabisphospho-nate derived from tyramine (compound 2) was realizedunder basic mild conditions in the presence of cesiumcarbonate in THF. The aldehydic proton could bedetected by means of 1H NMR, ensuring the orthogo-nality of this method even in the last step, whichinvolved the transformation of dimethylphosphonategroups to phosphonic acid termini in the presence oftrimethylsilylbromide and subsequent methanolysis. Fi-nally, the fluo-tagged fluorescein-5-thiosemicarbazide

was reacted with the remaining aldehyde function inwater to afford 1b-G1.

Targeting of FITC-derived dendrimer to monocytesin PBMC

We incubated the FITC-derived phosphorus dendrimer1b-G1 (20 M) for 30 min with human PBMC freshlyisolated from an healthy donor. Flow cytometry re- vealed that a CD3 CD56 CD14 population, thuscorresponding to monocytes was the only hematopoi-

etic population labeled with dendrimer 1b-G1 (Fig.3A). To further characterize the interaction betweenthe fluorescent dendrimer 1b-G1 and monocytes, wemagnetically purified human monocytes and filmed byconfocal video microscopy their interaction with den-drimer 1b-G1. Sequential images showed that den-drimer 1b-G1 rapidly bound within a few seconds tomonocyte surface (Fig. 3B) and was progressively inter-nalized within a few minutes and for hours (Fig. 3C).To distinguish the intracellular route of dendrimersafter their internalization by monocytes, we performedconfocal microscopy experiments using dendrimer1b-G1 and fluorescent specific molecular probes. Red

LysoTracker

is a specific fluorescent probe for intra-cellular acidic vesicles corresponding to phagolyso-somes in monocytes. Confocal microscopy showed co-location of LysoTracker and dendrimer 1b-G1 (Fig.3D), and depicted the phagolysosomial route as themain uptake mechanism of dendrimer by monocytes.Of interest in this colocation experiment was the strongdecrease of the FITC fluorescence in the acidic envi-ronment created by the fusion of phagosomes withlysosomes (16). However, using a specific fluorescentprobe for DNA (TOTO3), we could not detect anylocation of dendrimers in the nucleus.

Synthesis of a series of phosphorus-containingdendrimers

To decipher the structural requirements of phospho-rus-containing dendrimers to activate monocytes in

Figure 3. A) After a short incubation of PBMC with den-drimer 1b-G1, only CD3

CD56CD14 cells (i.e., monocytes)are labeled. B) Sequential images (first 64 seconds) fromconfocal videomicroscopy of purified monocytes (cytoplasmiclabeling with orange 5-(-6)-(4-chloromethyl(benzoyl)amino)tetramethylrhodamine [CMTMR]) incubated with den-drimer 1b-G1 emitting green fluorescence (white bar in thelower right image indicates 10 m). C) Membranous andinternal location at 15 min but only intracellular location at120 min of dendrimer 1b-G1 (white arrows) seen in confocal

microscopy (white bar in the 120 min image indicates 10m). D) Fluorescent labeling of purified monocytes withTOTO-3 (nuclear labeling in blue), LysoTracker (phagoly-sosomial labeling in red), and dendrimer 1b-G1 (in green).Left image is phase contrast microscopy; central image isconfocal microscopy, and the right graph reports fluores-cence curves along the white arrow in central image.



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culture, we synthesized a set of variously surfaceddendrimers with either phosphonic (mono-, azamono-,and azabisphosphonic acids) (Fig. 2) or carboxylicacids. We also synthesized a subdendritic structure: aphosphorus-containing branch (10b-G1) used for thedendritic outgrowth of dendrimers, ending withazabisphosphonic acids. These compounds are all listedin Table 1.

First, we synthesized nonfluorescent dendritic struc-tures bearing different phosphonic derivatives on theirsurface following a standardized procedure. This methodinvolved the nucleophilic substitution of the 12 chlorineatoms ofG1 by various phenol compounds equipped withvariously functionalized phosphonate derivatives, the lat-ter being transformed into their phosphonic acid deriva-tives after the grafting on dendrimers. Thus, our mainsynthetic efforts aimed at designing these phenols (Fig. 2)according to two different pathways. Symmetrical azabis-(dimethyl)phosphonate phenolic derivatives like 2 and 3were obtained from a catalyst free Kabachnik-Fields reac-tion involving aqueous formaldehyde, tyrosine, and dim-ethylphosphite. The acidic function of 3 was routinely

converted into its methyl ester. Other phenol derivatives with unsymmetrical azabis(dimethyl)phosphonate twee-zers like 7 and 8 were generated in three steps. A4-hydroxy-imine was generated and hydrophosphinylated with dimethylphosphite to afford azamono(dimeth-yl)phosphonate intermediates, the latter being convertedto azabisphosphonates 7 and 8 by a Kabachnik-Fieldsprocedure. Nucleophilic substitution of phenols 2, 4, 5, 7,and 8 on G1 was achieved in nearly quantitative yields inthe presence of cesium carbonate in THF, and thephosphonic acid-terminated dendrimers were finally ob-tained by treatment with trimethylsilylbromide in aceto-nitrile and subsequent methanolysis/salification process

to afford dendrimers 2b-G1, 4b-G1, 5b-G1, 7b-G1, and8b-G1, respectively. Dendrimer 9b-G1 was obtained by adirect Abramov-Pudovik reaction of dimethylphosphiteon a dendrimer capped with aromatic aldehyde functions(Fig. 2). Other dendrimers capped with carboxylategroups (series 11b-Gn generation n ranging from 0 to 2)were obtained from cinnamic acid-terminated dendrim-ers by addition of stoichiometric amounts of aqueoussodium hydroxide. A substructure of compounds 1b-G1and 2b-G1, namely 10b-G1, was obtained from parame-thoxybenzaldehyde following a routine dendritic out-growth and surface function derivatization, as describedfor its parent compound. Starting from a fluorescent core

bearing five aldehyde groups, a fluorescent dendritic toolwas also synthesized following the same procedure (Fig.4). This dendrimer 12b-G1 possessed fluorescence prop-erties that permit FRET experiments with phycoerythrin(PE) -tagged targets.

Azabisphosphonic phosphorus-containing dendrimers promote monocyte activation

At first we checked that dendrimer 2b-G1 bound tohuman monocytes. We assessed this point by displacingthe binding of the fluorescent dendrimer 1b-G1 to the

monocyte cell surface by competing with nonfluores-cent dendrimer 2b-G1 (Fig. 5A). Then we culturedpurified human monocytes with dendrimer 2b-G1 overa few weeks and compared them to control nonstimu-lated monocytes. Within 3 to 6 days of culture, mono-cytes in culture with dendrimer 2b-G1 underwent mor-phological changes (Fig. 5B); they also remained viableover longer periods than control monocytes (Fig. 5C).Among early events during culture of monocytes withdendrimer 2b-G1, cells underwent phenotypic changes.Expression of a series of monocyte receptors andmarkers was analyzed by flow cytometry. CD14 andhuman leukocyte antigen (HLA)-DR were down-regu-lated as well as other markers when monocytes werecultured with dendrimer 2b-G1 (Fig. 6A). As an addi-tional proof for monocyte activation, we measurednuclear relocation of the NF-B transcription factor.Despite their high level of basal NF-B activation,purified monocytes cultured from 4 to 8 h with den-drimer 2b-G1 had increased nuclear relocation ofNF-B (Fig. 6B). We also observed that within 3 to 6days of culture, monocytes in culture with dendrimer

2b-G1 exhibited increased phagocytic activity towardMycobacterium bovisBCG genetically modified to expressGFP-M.b.BCG (Fig. 6C). Moreover, neither morpholog-ical nor phenotypical criteria indicated that monocytescultured with dendrimer 2b-G1 matured toward den-dritic cells. Taken together, these results and observa-tions indicated that monocytes were activated by den-drimer 2b-G1.

Dendritic structural requirements for humanmonocyte activation

Bioactivities of these dendritic and subdendritic struc-

tures and monomers were quantified by flow cytometryon morphological changes of monocytes (data notshown) and down-regulation of CD14 and HLA-DRsurface markers on these monocytes (Fig. 6D). Thisdown-regulation was quantified by the mfi-R (see Ma-terials and Methods): a low mfi-R indicated a strongactivation of monocyte, and vice versa. This studyshowed that monomers could not elicit monocyte acti-vation as dendrimer 2b-G1 did. The phosphorus-con-taining branch capped with an azabisphosphonic group(compound 10b-G1) promoted a better activation ofmonocyte in culture than monomers, but lower thanthat of dendrimer 2b-G1. Among phosphorus-contain-

ing dendrimers, molecules ending with carboxylic acidgroups (dendrimers 11b-G0, 11b-G1, and 11b-G2) en-abled lower activation of monocytes than moleculesending with phosphonic acid groups (dendrimers 2b-G1, 4b-G1, 5b-G1, 7b-G1, 8b-G1, and 9b-G1).

Molecular elements for monocyte activation byazabisphosphonic dendrimers

Finally, using a novel fluorescent dendritic nanobiotool(dendrimer 12b-G1), we searched for a potential mono-cyte receptor for 2b-G1-like dendrimers. In dendrimer



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12b-G1, one of the six dendritic branches was replacedby a fluorescent 3,4-diphenylmaleimide group (Table1). We tested the ability of dendrimer 12b-G1 to activatemonocytes in vitro. Despite lacking one branch over six,we showed that this dendrimer activated human mono-

cytes in the same way as dendrimer 2b-G1 (Fig. 7A).Dendrimer 12b-G1 was designed so that its fluorescencecharacteristics allowed FRET with PE, a commonlydescribed fluorochrome for flow cytometry (Fig. 7B). After monocyte labeling with dendrimer 12b-G1 andPE-coupled mAb against various typical monocytes re-ceptor, PE fluorescence emission was achieved by FRETif 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 againstCD14 (data not shown), could be stimulated to emitfluorescence by FRET from dendrimer 12b-G1 (Fig.

7C). This indicated that this typical innate receptor wassomehow involved in dendrimer sensing by monocytes.

DISCUSSION

Dendrimers are monodisperse polyfunctionalized hy-perbranched polymers whose nanometer size, topol-ogy, perfectly defined structures, multivalent charac-ter, and molecular weight can be rigorouslycontrolled during synthesis. The high density offunctionalities on their surface as well as their glob-ular shape confer to these special polymers versatileand unique properties that have been exploited inbiology and more intensively in material science(17). Among these nano-objects, phosphorus-con-taining dendrimers occupy a special space due to the

O

N

Ph

O

O

P

NP

N

PN

O

O

O

O

O

O

O

O

O

O

O

N

Ph

O

O

P

NP

N

PN

Cl Cl

Cl

ClCl

O

N

Ph

O

O

P

NP

N

PN

O

N

O

N

O

N

O

N

O

N

N

P N P

N

P

N

PNP

S

Cl

S

Cl

S

Cl

Cl

S

ClClCl

S

Cl

Cl

Cl

O

N

Ph

O

O

P

NP

N

PN

O

N N

P

SO

N

N

PS

O

N

NP

S

O

N

N P

S

O

N

N

P S

O

N

PO3RR'

PO3RR'

O

NPO3RR'

PO3RR'

O

N

PO3RR'

PO3RR'

O

N

PO3RR'

PO3RR'

O

NR'RO3P

R'RO3P

O

N

PO3RR'

PO3RR'

O

N

PO3RR'

R'RO3P

O

N

R'RO3P

R'RO3P

O

NPO3RR'

PO3RR'

O

N

PO3RR'

R'RO3P

c/

a/

b/

d/

e/

Figure 4. Dendritic FRET tool (12b-G1) synthe-sis. 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.



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reactivity of phosphorus, which allows preparation ofa large variety of dendritic and multidendritic mac-romolecules (18). The biocompatibility of somephosphorus dendrimers bearing anionic charges onthe end groups has been tested (19, 20). In this studywe took advantage of the versatility of dendrimers ingeneral 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 selectivelytargets monocytes among human PBMC. Bloodmonocytes and their tissue counterpart macrophagesare mononuclear phagocytes. On the one hand, theyare capable of engulfing endogenous matters, such ascellular apoptotic debris and injured or dead cells.On the other hand, they can also scavenge foreignsubstances such as whole infectious microorganismsor insoluble nano-objects; among these, phosphorusdendrimers as reported here. Moreover, we alsorealized that they are internalized via the phagolyso-

somial route.Normally, microorganisms internalized by macro-

phages are to be killed when lysosomal content isdelivered in phagosomes. In some cases, however,microorganisms can survive and even multiply withinthe macrophages: they are pathogens (e.g., Mycobac-teria, Salmonella, Leishmania, dengue virus. . . ). Thus,targeting drug delivery to macrophages to fightagainst parasitic infections is an attractive therapeu-tic strategy (21). With this aim, the two major pointsthat have to be addressed are selective targeting andinternal drug delivery to macrophages. Besides lipo-

somes, nanoparticles, and microspheres, phospho-rus-containing dendrimers are new nano-scale candi-dates. First, their versatility enables chemical bindingof different active drugs with specific antimicrobialactivities on one (or more) branch in place ofphosphonic acid groups of the dendritic structure.Second, they specifically target monocytes/macro-phages among PBMC. Third, they are internalized bymonocytes/macrophages. Fourth, drugs will be deliv-ered by the lytic content of lysosomes. To our knowl-edge, only poly(amidoamine) dendrimers have beenproposed so far for controlled site-specific drugdelivery (22).

Studying human monocytes/macrophages in cul-ture with azabisphosphonic acid capped phosphorusdendrimer 2b-G1 over a few days, we documentedactivation features of these cells. The bioactivity ofseveral phosphorus-containing dendrimers, surfacedwith phosphonic acid or carboxylic acid groups, was

screened for their ability to activate monocytes. Wedemonstrated that surface phosphonic groups con-stitute an important determinant for the bioactivity,since phosphorus-containing dendrimers cappedwith carboxylic acid groups were much less active. Wealso prepared and tested a single phosphorus-con-taining branch (10b-G1) along with tyramine- ortyrosine-derived monomers used to cap dendrimersfor monocyte activation. These subdendritic com-pounds were poor activators. Taken together, theseresults indicate a strong requirement for all the3-dimensional scaffold of phosphonic acid surfacing

Figure 5. A) Labeling of monocytes by den-drimer 1b-G1 can be displaced by competing

with dendrimer 2b-G1

(IC5045 M). B)Mono-cyte activation is checked by microscopy: leftimage unstimulated monocytes, right im-age dendrimer 2b-G1 activated monocytes(cultures at day 3). These observations are con-firmed by flow cytometry by morphologicalchanges (increase in size and granularity). C)Inculture 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 mono-cytes without dendrimer (open circles).



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groups framed around the P3N3 core of phosphorus-containing dendrimers.

Monocyte/macrophage can take several aspects (23).Besides the early described classical activation pathway, analternative activation mechanism emerged in 1992 in themouse model (24). The classical activation of monocytes/macrophages is mediated by IFN- as primer, then tumornecrosis factor (TNF) or microbial trigger, whereas alter-native activation is mediated by interleukin (IL) -4, IL-13,and glucocorticoids (23, 25). The classical activation path-

way of monocytes evolves toward an inflammatory im-mune response (secretion of high levels of IL-1, IL-6,IL-12, and TNF-) while the alternative pathway evolvestoward an anti-inflammatory response (secretion of highamounts of IL-10 and IL-1 receptor antagonist) (25). Witha transcriptomic approach, we will further assess the typeof activation that monocytes/macrophages stimulatedwith phosphonic acid group-ended, phosphorus-contain-ing dendrimers undergo.

Using the fluorescent nano-biotool dendrimer 12b-G1especially designed to enable FRET with PE-conjugatedpartners, we showed that the typical innate receptor TLR2

is involved in the sensing of phosphorus-containing den-drimers. Nevertheless, these dendrimers were not recog-nized and sensed by TLR2-transfected HEK293 eukaryoticcells or by combined TLR2/TLR1- or TLR2/TLR6-trans-fected HEK293 cells (M. Poupot et al., personal commu-nication). This indicated that TLR2 is not the monocyte/macrophage receptor for phosphorus dendrimers, butmay participate to a recognition/signal transducing com-plex, 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 phos-phorus-containing dendrimer recognition by monocytesmay not necessarily imply that these cells undergo aninflammatory maturation (8). It was recently shown thatmouse dendritic cells (27) or human monocyte-deriveddendritic cells may undergo either inflammatory or anti-inflammatory maturation processes upon TLR2 trigger-ing (28, 29).

Thus, phosphorus-containing dendrimers are a para-digm of versatile chemical tools affecting the expansion ofbiology through engineered nano-biotools and new ther-apeutic agents. These phosphorylated dendrimers repre-

Figure 6. A)Phenotypical changes of monocyteactivated by dendrimer 2b-G1 (filled bars, un-treated monocytes: open bars). B) Monocytesare activated by dendrimer 2b-G1 as shown byNK-B p50 nuclear relocation in a chemilumi-nescent readout (RLU: relative luminescentunit) (basal level: open bars; with dendrimer2b-G1: filled bars; positive control: hatched bar,monocyte activation by peptidoglycan). Nu-clear extracts of stimulated Jurkat cells serve askit positive control (crossed bars). C)Monocyteactivation is also checked in flow cytometry byan increase of phagocytosis detected by inter-nalization of GFP-M.b.BCG. D)Screening of thebioactivity of various dendritic and subdendriticstructures on human monocytes (mfi-R forHLA-DR and CD14 markers). Open circles:carboxylic acid capped dendrimers (11b-Gn, nfrom 0 to 2), open squares: monophosphonicacid capped dendrimers (5b-G1, 9b-G1) andmonomer 5, open circles: bisphosphonic acid-capped dendrimers (2b-G1, 4b-G1, 7b-G1, 8b-G1), phosphorus-containing branch (10b-G1)and monomers 4, 8.



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sent an extraordinary set of virtually unlimited number ofchemical structures. They clearly appear as new tunabletherapeutic candidates to target and modulate humaninnate immunity viamonocyte activation.

This work was supported by grants from Region Midi-Pyrenees (Biotherapies program), ARC (ARECA 2004),MENSR (Action de Soutien a lInnovation); institutional

funding from Fonds Structurels Europeens (M.P.), IN-SERM, 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 continu-ous support.

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Figure 7. A) Bioactivity of dendrimer 12b-G1 measured bydown-regulation of HLA-DR and CD14 markers (filled bars)compared with the effect of dendrimer 2b-G1 (gray bars) andto untreated monocytes (open bars) after a 5 day culture. B)Spectral properties of dendrimer 12b-G1 compared withspectral properties of PE: dendrimer 12b-G1 achieves FRETon PE. Dotted lines: excitation spectra, full lines: emission

spectra. C) The involvement of TLR2 in the sensing ofphosphorus-containing dendrimers evidenced by FRET ex-periment (immunoglobulin control is a mouse immunglobu-lin isotype control).



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


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