RSC Advances

PAPER

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3. View Article Online

View Journal | View Issue

aDpto. de Quımica Organica y Quımic

Campus Universitario, E-28871 Alcala

javier.delamata@uah.es; rafael.gomez@uahbNetworking Research Center on Bioengin

(CIBER-BBN), SpaincLaboratorio InmunoBiologıa Molecular, H

Mara~non and Instituto de Investigacion

Spain. E-mail: mmunoz.hgugm@salud.maddCentro de Investigaciones Biologicas, Conse

(CSIC), Madrid, Spain

† These authors contributed equally to th

Cite this: RSC Adv., 2013, 3, 23445

Received 1st July 2013Accepted 27th September 2013

DOI: 10.1039/c3ra43338b

www.rsc.org/advances

This journal is ª The Royal Society of

Study of cationic carbosilane dendrimers as potentialactivating stimuli in macrophages

Javier Sanchez-Nieves,†ab A. Judith Perise-Barrios,†bc Paula Ortega,ab Angel L. Corbı,d

Angeles Domınguez-Soto,d M. Angeles Mu~noz-Fernandez,*bc Rafael Gomez*ab

and F. Javier de la Mata*ab

Cationic carbosilane dendrimers with two ammonium groups per branch [GnO3(SiONN)m]2m+ (n¼ 1,m¼ 6;

n ¼ 2, m ¼ 12; n ¼ 3, m ¼ 24), derived from the 1,3,5-trihydroxybenzene (1,3,5-C6H3(OH)3) core, have

been synthesized from reactions of dendrimers functionalized with Si–Cl bonds and the alcohol amine

2-{[2-(dimethylamino)ethyl]methylamino}ethanol (HO(CH2)2N(Me)(CH2)2NMe2) and further addition of

MeI. The cationic dendrimers obtained were soluble in water, although they decomposed slowly in this

medium. The toxicity and inflammatory activity of these dendrimers were evaluated in M1 macrophages

and the results compared with the known carbosilane dendrimer 2G-NN16, derived from a Si atom core.

The data showed good toxicity profiles for dendrimers [GnO3(SiONN)m]2m+ of first and second

generation and 2G-NN16 and also that these dendrimers are not sensed as activating stimuli for

macrophages and, as consequence, they do not cause an inflammatory response.

1. Introduction

Dendrimers are macromolecules with well-dened size andstructure, exibility, monodispersity and multivalent molecularsurface1–6 that have attracted attention of a diversity ofelds,3,6–12 as it has been observed that the multivalency raisesthe activity of the functions attached to the dendrimer withrespect to individual molecules (dendritic effect). For biomed-icine, this dendritic effect would lead to a stronger interactionof the dendrimer with its surrounding and this property hasfound a variety of applications.13–20 It is generally accepted thatthe use of dendrimers as therapeutic agents requires, besideslow cytotoxicity, adequate solubility in aqueous media.21

However, previous studies also warned about the importanceof evaluating the inammatory effect of molecules with a futurebiological application,22 as this factor will favour infectionprocesses. For that reason, it has been suggested the evaluationof cytokines release to establish the pattern of cytokines, as itcan be modied by these kinds of molecules,23 and will serve as

a Inorganica, Universidad de Alcala,

de Henares (Madrid), Spain. E-mail:

.es; Fax: +34 91 885 4683

eering, Biomaterials and Nanomedicine

ospital General Universitario Gregorio

Sanitaria Gregorio Mara~non, Madrid,

rid.org

jo Superior de Investigaciones Cientıcas

is work.

Chemistry 2013

predictor of safety during the rst steps of the evaluationprocess of any molecule.24,25

Macrophages are widely distributed cells that play a criticalrole in defence, tissue repair and homeostasis.26 They act as arst line of defence to detect and eliminate foreign particles(microorganisms, toxic macromolecules, damaged or deadcells) by phagocytoses or secretion of enzymes, production ofcytokines or reactive oxygen and nitrogen species. There is onemain group of macrophages called M1 (classically activatedmacrophages). During the initial stages of an inammatoryresponse, polarization takes place and these cells exhibit potentmicrobicidal properties, and for this reason they are consideredto be pro-inammatory cells.27 It is well known that macro-phages secrete cytokines when they are confronted with apathogen, and in the particular case of M1 macrophages, theyhave a typical pathogen-stimulated cytokine prole.28

Our group is interested in the applications of carbosilanedendrimers in biomedicine. The framework of these den-drimers is built with low polar bonds such as C–C and C–Sibonds, and as consequence they present high hydropho-bicity.29,30 However, functionalization of the periphery with verypolar moieties has turned them hydrophilic and thus suitablefor biomedical applications.31–35 We have shown that cationiccarbosilane dendrimers are water soluble and could be used asinternalizing agents for gene delivery,31,36–40 bactericides32,33,41

and also showed interactions with drugs,42 whereas watersoluble anionic carbosilane dendrimers present promisingantiviral properties.43,44 Others, have decorated the surface ofcarbosilane dendrimers with carbohydrates and studied theirapplications in biomedicine.35,45–48

RSC Adv., 2013, 3, 23445–23453 | 23445

Fig. 1 Drawing of 2nd generation cationic dendrimer derived from Si atom core2G-ONN16 previously synthesized in our group.31

RSC Advances Paper

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

We have previously reported the synthesis of cationic car-bosilane dendrimers of generations 1–3 functionalized withdiammonium groups at the periphery bonded to the dendrimerthrough Si–O bonds.31 The idea to introduce this system was, onone hand, to give water solubility and, on the other hand, toease the release of nucleic acid due to the instability of the Si–Obond in aqueous media. However, of the three generations, therst generation turned to be too unstable to carry out any kindof experiments in vitro, and the third generation showed solu-bility problems. Fortunately, we could study second generationdendrimer (named as 2G-ONN16, Fig. 1). These studies showedthat the presence of two cationic ammonium groups per branchtogether with the hydrolysable Si–O bond conferred interestingproperties to this dendrimer.31,36–38,49–56

Thus, in this work we have used M1 macrophages as sensorcells to determine whether dendrimers can be detected by theorganism as a foreign particle. Here the advantage is that, due tothe characteristic of these cells, they respond to most exogenousmolecules, causing the release of pro-inammatory cytokinesand nally triggering an inammatory response. The results havebeen compared with those obtained using a bacterial endotoxin(lipopolysaccharide, LPS). LPS is a complex glycolipid composedof a hydrophilic polysaccharide middle and a hydrophobicdomain known as lipid A. LPS is a major component of the outermembrane of Gram-negative bacteria and one of the most potentmicrobial initiators of inammation.57

Scheme 1 Synthesis of neutral and cationic dendrimers. (i) mHO(CH2)2N(Me)(CH2

23446 | RSC Adv., 2013, 3, 23445–23453

To carry out this research we have synthesized a new familyof carbosilane dendrimers with Si–O bonds at the peripheryderived from 1,3,5-trihydroxybenzene (1,3,5-(HO)3C6H5), thatintroduces rigidity in the core and forces the branches to extentoutwards.43,58 The aim to obtain a new group of dendrimers is tocompare and improve the results yet obtained with the den-drimer derived from a Si atom core with respect to stability andsolubility in aqueous media.

2. Results and discussion2.1 Synthesis of dendrimers

Reaction of dendrimers decorated with terminal Si–Cl bondsGnO3(SiCl)m (n ¼ 1, m ¼ 6; n ¼ 2, m ¼ 12; n ¼ 3, m ¼ 24)58 with2-{[2-(dimethylamino)ethyl]methylamino}ethanol led to theamine dendrimers GnO3(SiONN)m (n ¼ 1, m ¼ 6 (1); n ¼ 2, m ¼12 (2); n ¼ 3,m ¼ 24 (3)) (Scheme 1) in high yield (around 85%).The nomenclature employed indicates generation (Gn), corederived from 1,3,5-trihydroxybenzene (O3) and a peripheryfunctionalized with mSiO(CH2)2N(Me)(CH2)2NMe2 groups(SiONN). They were obtained as colorless oils soluble in organicsolvents, not aliphatic, but unstable toward protic solvents andmoisture.

The NMR spectroscopy conrmed the formation of den-drimers 1–3. The 1H NMR spectra showed a shiing of the Megroups of terminal SiMe2 moieties from ca. d 0.35 in the startingcompounds GnO3(SiCl)m to d ca. 0.05 in compounds 1–3. Theintegration of the aromatic protons with respect to the newOCH2 and NMe groups supports the complete derivatization ofthe starting materials. The carbon atoms of these Me groupswere observed at about d �2.0 in compounds 1–3, whereas inthe precursors were observed about d 1.9. In the 29Si NMR, theresonance corresponding to the outermost Si–O groups wasobserved about d 18, clearly shied to lower frequency than thesignal belonging to the initial SiCl groups (d ca. 31). Finally, 15NNMR showed two resonances for the two different N atoms at dca. �351.5, for the inner one, and at d ca. �356.5 for the outerone. Analyses by GPC or MS failed because of the instability ofthe Si–O bonds.

Neutral dendrimers 1–3 were transformed into cationic onesby addition of excess MeI in THF, obtaining the compounds[GnO3(SiONN)m]

2m+ (n ¼ 1, m ¼ 6 (4); n ¼ 2, m ¼ 12 (5); n ¼ 3,m¼ 24 (6)) (Scheme 1) as white solids in high yield about (85%).Compounds 4–6 (Fig. 2) were now soluble in high polar solventsand also in water, although in this medium they decomposedslowly. The quaternization of the N atoms was clearly shownby NMR spectroscopy (DMSO-d6) by the shiing to higher

)2NMe2, excess NEt3, Et2O, r. t., 16 h. (ii) Excess MeI, THF, r. t. 48 h.

This journal is ª The Royal Society of Chemistry 2013

Paper RSC Advances

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

frequency of the resonances involved in this change withrespect to the neutral compounds. The methyl groups bound tothe nitrogen atoms were observed in the 1H NMR spectra at d ca.

Fig. 2 Drawing of cationic dendrimers synthesized in this work 4–6.

This journal is ª The Royal Society of Chemistry 2013

3.20 and in the 13C NMR spectra at d ca. 52.0. Similarly, thechemical shis of the N atoms were detected at d ca. �326.5, forthe inner one, and at d ca. �331.5 for the outer one.

RSC Adv., 2013, 3, 23445–23453 | 23447

Table 1 Hydrodynamic radii of dendrimers 1–3 and G2Si(SiONN)8 in CHCl3 at 25 �C

Compound G1O3(SiONN)6 G2O3(SiONN)12 G3O3(SiONN)24 G2Si(SiONN)8rH (10�9 m) 1.57 1.42 2.95 0.64

RSC Advances Paper

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

2.2 Size of dendrimers by DOSY

To compare the size of the different dendrimers reported in thiswork among them and with the related dendrimer derived froma Si atom core, we have carried out DOSY NMR experiments(Table 1) to obtain the hydrodynamic radii (rH) of neutral den-drimers 1–3 and G2Si(SiONN)8, precursor of cationic 2G-ONN16.The cationic dendrimers present aggregation in solution andany comparison would fail. For these experiments we used asinternal reference the residual CHCl3 of the deuterated solventusing the approach of Wilkins et al.:59

rHðdendrimerÞ ¼ DðsolventÞDðdendrimerÞ

� rHðsolventÞ

where D is the diffusion coefficient obtained from the NMRexperiment and rH(solvent) is 2.65 A.60

The results obtained showed that rst and second genera-tion dendrimers of polyphenoxo core, with six and twelvefunctional groups, present similar sizes but are smaller thancorresponding third generation dendrimer, with twenty fourfunctional groups. This situation has been found in otherdendrimers.61,62 However they are clearly bigger than secondgeneration dendrimer derived from Si atom core, with eightfunctional groups. This difference reects the higher size andrigidity of the polyphenoxo core, which force the branches ofdendrimers to spread away from the core. These results thusoffer the possibility to work with different dendrimers thatcontain the same type of peripheral functionalities but differ inthe number of them and also in their size.

3. Biomedical assays3.1 Cytotoxicity of carbosilane dendrimers

As seen in previous reports, dendrimers can be cytotoxics for thecells in high concentrations. In order to establish the maximum

Fig. 3 (A) Dendrimers effect over macrophages viability analyzed by MTT assay. (BMacrophages were treated with dendrimers for 24 hours at different concentration

23448 | RSC Adv., 2013, 3, 23445–23453

concentration of a dendrimer that can be used without havingany toxic effects, the rst step was to evaluate the potentialtoxicity of cationic dendrimers [GnO3(SiONN)m]

2m+ (n¼ 1,m¼ 6(4); n ¼ 2, m ¼ 12 (5); n ¼ 3, m ¼ 24 (6)) and 2G-NN16 in M1macrophage cell cultures (Fig. 3). MTT assays revealed that,aer 24 hours, more than 80% of macrophages remained viablewhen exposed to a 5 mM concentration of cationic dendrimers 4and 5 (Fig. 3A), and up to 15 mM concentration of the 2G-NN16,whereas dendrimer 6 was clearly more toxic. The cell viabilitywas also studied by ow cytometry analysis, which showedsimilar values for dendrimers 4 and 5 (Fig. 3B) and slightlylower for 2G-NN16 (10 mM). Accordingly, 5 mM concentrationsfor dendrimers 4 and 5, and 10 mM for 2G-NN16 were used forall subsequent studies. Due to the high toxicity of the[G3O3(SiONN)24]

48+ dendrimer (6) on treated macrophages, thisdendrimer was discarded for subsequent macrophage assays.These data indicate higher level of toxicity for dendrimersderived from the polyphenoxo core 4 and 5, with twelve andtwenty four positive charges, with respect to 2G-NN16, withsixteen positive charges. This difference suggests an inuenceof the core, and hence external charge density, in the toxicity ofdendrimers with the same type of functional groups, probablydue to the higher exposure of the aromatic core because of itsrigidity.43

3.2 Study of macrophages activation

Pro-inammatory cytokines and chemokines are central to theprocess of development and maintenance of inammation.Cytokines are soluble peptides that are involved in many signaltransduction pathways regulating cell growth, differentiationand death, as well as recruitment of neutrophiles, macro-phages, and other cells to specic sites. It has been suggestedthat several cytokines are involved in particle-inducedinammation.63

) Dendrimers effect over macrophages viability analyzed by flow cytometry assay.s. This data represents the mean � SD from three independent donors.

This journal is ª The Royal Society of Chemistry 2013

Fig. 4 Cytokines release. Determination of TNF-a (A) and IL-12p40 (B) release by ELISA in culture supernatant of M1 macrophages. Shown are not treated cells (NT),cells stimulated by LPS (LPS), and treated with first [G1O3(SiONN)6]

12+ (4), second generation [G2O3(SiONN)12]24+ (5) and 2G-NN16 dendrimers for 24 h. The individual

and mean values of 3 independent experiments are shown (*p < 0.05, **p < 0.01).

Paper RSC Advances

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

The bacterial endotoxin LPS activates macrophages toproduce pro-inammatory cytokines such as tumour necrosisfactor-a (TNF-a) and IL-12.57 Production of these inammatorycytokines and mediators by macrophages contributes to theefficient control of growth and dissemination of invadingpathogens triggering an inammatory response.64

To establish whether dendrimers trigger M1 macrophageactivation, we confronted the macrophages cell cultures withdendrimers and their dendrimer-induced cytokine prole wasdetermined by ELISA assays. With regards to cytokine release,and in line with previous reports,28 LPS stimulation of M1macrophages, led to the production of TNF-a (Fig. 4A) and IL-12(Fig. 4B).

It was noted that macrophages do not release pro-inam-matory cytokines when they were confronted with dendrimers,thus indicating that, unlike LPS, these dendrimers do notactivate the macrophages (Fig. 4). First and second generationdendrimers 4 and 5 and 2G-NN16 did not induce the productionof TNF-a (Fig. 4A) and IL-12p40 (Fig. 4B) by M1macrophages. Inthis case, no effect of the core in macrophage response wasnoted for these dendrimers.

4. Conclusions

We have successfully synthesized a new family of carbosilanedendrimers derived from a 1,3,5-(HO)3C6H5 core with twoammonium groups per branch at the periphery and with Si–Obonds close to the surface. The three generations obtained wereperfectly water soluble in contrast with related dendrimerscontaining a Si atom core, which third generation derivative wasnot soluble. The polyphenoxo core generates clearly biggerdendrimers than the Si atom core, as has been showed by DOSYNMR experiments of neutral dendrimers. These new cationicdendrimers [GnO3(SiONN)m]

2m+ (n ¼ 1, m ¼ 6 (4); n ¼ 2, m ¼ 12(5); n ¼ 3, m ¼ 24 (6)) has allowed us to compare the effect ofgeneration and number of functional groups in biomedical

This journal is ª The Royal Society of Chemistry 2013

assays and also with our previous cationic dendrimer 2G-NN16,with the same type of periphery but derived from a Si atom core.

We have studied the behaviour of M1 macrophages in thepresence of the cationic dendrimers 4–6 and 2G-NN16, as thistype of cells is the rst line of defense to determine foreignparticles to the organism.We have observed that the toxicity of adendrimer is generation-dependent, in accordance withprevious results.31 Dendrimer 2G-NN16 was the less cytotoxic ofthese compounds, whereas the third generation dendrimer[G3O3(SiONN)24]

48+ presented the highest cytotoxicity level. Thedata indicates that the lower generation dendrimers can beused without having any cytotoxic effect, in contrast with thethird generation dendrimer 6.

This work was based on the use of the cytokines release as apredictor of safety during the rst steps of the evaluationprocess for a molecule with a future biological application. Ourdata demonstrates that 4 and 5 and 2G-NN16 are not promotingthe release of TNF-a and IL-12p40 pro-inammatory cytokinesby the macrophages, indicating that they are not recognized asforeign substances and consequently, do not trigger aninammatory response mediated by macrophages.

Our data also suggest that these dendrimers could be used insubsequent studies to avoid the unwanted effects that havebeen had until now with similar compounds, since these newdendrimers could be used free of inammatory complicationsin future studies. In summary, these results drive us to a verypromising line of research, because dendrimers could be usedas a safety tool for biomedical applications.

5. Experimental section5.1 General considerations

All reactions were carried out under inert atmosphere andsolvents were puried from appropriate drying agents (THF).NMR spectra were recorded on a Varian Unity VXR-300 (300.13(1H), 75.47 (13C) MHz) or on a Bruker AV400 (400.13 (1H), 100.60(13C), 40.56 (15N), and 79.49 (29Si) MHz). Chemical shis (d) are

RSC Adv., 2013, 3, 23445–23453 | 23449

RSC Advances Paper

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

given in ppm. 1H and 13C resonances were measured relative tosolvent peaks considering TMS ¼ 0 ppm, meanwhile 15N and29Si resonances were measured relative to external liquid NH3

(but converted to the IUPAC CH3NO2 standard) and TMS,respectively. When necessary, assignment of resonances wasdone from HSQC, HMBC, COSY, TOCSY and NOESY NMRexperiments. Elemental analyses were performed on a Perkin-Elmer 240C. Compounds Karstedt's Pt catalyst, HO(CH2)2N-(Me)(CH2)2NMe2, NEt3, and MeI (Aldrich) were obtained fromcommercial sources. Compounds GnO3(SiCl)m (n¼ 1,m¼ 6; n¼2, m ¼ 12; n ¼ 3, m ¼ 24)58 and 2G-NN1631 were synthesized aspublished.

5.2 Synthesis of compounds

Synthesis of G1O3(SiONN)6 (1). HO(CH2)2N(Me)(CH2)2NMe2(0.373 g, 2.55 mmol) was added to a solution of G1O3(SiCl)6(0.500 g, 0.41 mmol) and NEt3 (0.70 mL g, 5.10 mmol) in Et2O(150 mL) at room temperature and the mixture was stirred for16 h. Then, volatiles were removed under vacuum, Et2O (100mL) was added, the solution was ltered and the solventremoved under vacuum, obtaining compound 1 as a colourlessoil (0.67 g, 88%). NMR data (CDCl3):

1H NMR: �0.08 (s, 9H,SiMe), 0.06 (s, 36H, SiMe2), 0.59 (m, 30H, CH2Si), 1.33 (m, 18H,CH2CH2Si), 1.74 (m, 6H, OCH2CH2), 2.21 (s, 36H, NMe2), 2.26,(s, 18H, NMe), 2.35 (m, 12H, CH2NMe2), 2.52 (m, 24H, CH2NMe),3.65 (t, J ¼ 7.7 Hz, 12H, SiOCH2), 3.82 (t, J ¼ 7.6 Hz, 6H,ArOCH2), 6.03 (s, 3H, C6H3);

13C NMR:�5.1 (SiMe),�2.0 (SiMe2),13.8 (SiCH2), 17.8, 18.8, 21.0 (SiCH2 and SiCH2CH2), 22.6(OCH2CH2CH2), 33.3 (OCH2CH2CH2), 43.3 (NMe), 45.9 (NMe2),56.2, 57.4 and 60.0 (CH2N), 60.8 (OCH2CH2N), 67.6 (ArOCH2),93.7 (CH, C6H3), 160.9 (Cipso, C6H3);

15N NMR: �356.3 (NMe2),�351.6 (NMe); 29Si NMR: 1.8 (SiMe), 17.5 (SiMe2). Anal. calc. forC93H210N12O9Si9 (1893.51): C, 58.99; H, 11.18; N, 8.88; obt.: C,57.67; H, 10.21; N, 8.33%.

Synthesis of G2O3(SiONN)12 (2). Following the proceduredescribed above for 1, compound 2 was obtained as a colourlessoil (0.64 g, 85%) from G2O3(SiCl)12 (0.500 g, 0.19 mmol),HO(CH2)2N(Me)(CH2)2NMe2 (0.350 g, 2.39 mmol) and NEt3(0.67 mL, 4.78 mmol). NMR data (CDCl3):

1H NMR: �0.08 (s,18H, SiMe), �0.03 (s, 9H, SiMe), 0.07 (s, 72H, SiMe2), 0.53 (m,48H, CH2Si), 0.64 (m, 24H, CH2Si), 1.33 (m, 42H, CH2CH2Si),1.74 (m, 6H, OCH2CH2), 2.21 (s, 72H, NMe2), 2.27 (s, 36H, NMe),2.34 (m, 24H, CH2NMe2), 2.52 (m, 48H, CH2NMe), 3.66 (t, J¼ 7.7Hz, 24H, SiOCH2), 3.87 (bs, 6H, ArOCH2), 6.03 (s, 3H, C6H3);

13CNMR: �4.9, �5.1 (SiMe), �2.2 (SiMe2), 13.8 (SiCH2), 17.3, 17.4,18.2, 18.5, 20.6 (SiCH2 and SiCH2CH2), 22.3 (OCH2CH2CH2),33.9 (OCH2CH2CH2), 43.0 (NMe), 45.8 (NMe2), 55.9, 57.2 and59.8 (CH2N), 60.6 (OCH2CH2N), 67.4 (ArOCH2), 93.5 (CH, C6H3),160.8 (Cipso, C6H3);

15N NMR: �356.2 (NMe2), �351.7 (NMe);29Si NMR: 1.9 (SiMe), 17.4 (SiMe2). Anal. calc. forC189H438N24O15Si21 (3877.45): C, 58.54; H, 11.39; N, 8.67; obt.: C,58.02; H, 11.11; N, 8.32%.

Synthesis of G3O3(SiONN)24 (3). Following the proceduredescribed above for 1, compound 3 was obtained as a colourlessoil (0.313 g, 83%) from G3O3(SiCl)24 (0.250 g, 0.05 mmol),HO(CH2)2N(Me)(CH2)2NMe2 (0.172 g, 1.17 mmol) and NEt3

23450 | RSC Adv., 2013, 3, 23445–23453

(0.32 mL, 2.35 mmol). NMR data (CDCl3):1H NMR: �0.11 (s,

45H, SiMe), �0.04 (s, 18H, SiMe), 0.05 (s, 144H, SiMe2), 0.58 (m,144H, CH2Si), 1.33 (m, 90H, CH2CH2Si), 1.74 (m, 6H, OCH2CH2),2.20 (s, 144H, NMe2), 2.26, (s, 72H, NMe), 2.36 (m, 48H,CH2NMe2), 2.51 (m, 96H, CH2NMe), 3.65 (t, J ¼ 7.7 Hz, 48H,SiOCH2), 3.84 (bs, 6H, ArOCH2), 6.03 (s, 3H, C6H3);

13C NMR:�2.9 (SiMe), �1.9 (SiMe2), 14.1 (SiCH2), 17.8, 18.6, 18.8, 20.6,21.0 (SiCH2 and SiCH2CH2), 22.8 (OCH2CH2CH2), 33.5(OCH2CH2CH2), 43.3 (NMe), 45.9 (NMe2), 56.2, 57.5 and 60.0(CH2N), 60.9 (OCH2CH2N), 67.8 (ArOCH2), 93.6 (CH, C6H3),160.9 (Cipso, C6H3);

15N NMR: �356.4 (NMe2), �351.6 (NMe);29Si NMR: 1.6 (SiMe), 18.2 (SiMe2). Anal. calc. forC381H894N48O27Si45 (7845.33): C, 58.33; H, 11.49; N, 8.57; obt.: C,57.45; H, 10.29; N, 8.06%.

Synthesis of [G1O3(SiONN)6(I)12] (4). MeI (0.25 mL, 3.80mmol) was added to a solution of 1 (0.300 g, 0.16 mmol) in THF(150 mL) and the mixture was stirred for 36 h, observingformation of a white precipitate. Aerward the volatiles wereremoved under vacuum and the white solid was washed withCH2Cl2, obtaining 4 (0.49 g, 86%). NMR data (DMSO-d6):

1HNMR: �0.05 (s, 9H, SiMe), 0.13 (s, 36H, SiMe2), 0.55 (m, 118H,CH2Si), 0.69 (m, 12H, CH2Si), 1.34 (m, 18H, CH2CH2Si), 1.68 (m,6H, OCH2CH2), 3.16 (s, 18H, NMe), 3.19 (s, 36H, NMe2), 3.56 (m,12H, CH2NMe2), 3.80–4.20 (m, 54H, CH2NMe2, SiOCH2 andArOCH2), 6.01 (s, 3H, C6H3);

13C NMR:�5.5 (SiMe),�2.6 (SiMe2),12.8 (SiCH2), 16.8, 17.3, 19.6 (SiCH2 and SiCH2CH2), 23.1(OCH2CH2CH2), 33.1 (OCH2CH2CH2), 50.1 (NMe), 52.6 (NMe2),55.7, 55.9 and 56.4 (CH2N), 64.9 (OCH2CH2N), 67.6 (ArOCH2),94.1 (CH, C6H3), 160.0 (Cipso, C6H3);

15N NMR: �331.7 (NMe2),�326.4 (NMe); 29Si NMR: 1.1 (SiMe), 20.6 (SiMe2). Anal. calc. forC105H246I12N12O9Si9 (3596.77): C, 35.06; H, 6.89; N, 4.67; obt.: C,35.36; H, 7.16; N, 5.04%.

Synthesis of [G2O3(SiONN)12(I)24] (5). Following the proce-dure described above for 4, compound 5 was obtained as whitesolid (0.45 g, 86%) from 2 (0.280 g, 0.07 mmol) and MeI(0.22 mL, 3.47 mmol). NMR data (DMSO-d6):

1H NMR: �0.09 (s,18H, SiMe), �0.03 (s, 9H, SiMe), 0.12 (s, 72H, SiMe2), 0.53 (m,48H, CH2Si), 0.68 (m, 24H, CH2Si), 1.32 (m, 42H, CH2CH2Si),1.66 (m, 6H, OCH2CH2), 3.22 (s, 180H, NMe2 and NMe), 3.60 (m,24H, CH2NMe2), 3.90–4.10 (m, 72H, CH2NMe and SiOCH2), 3.86(bs, 6H, ArOCH2), 5.99 (s, 3H, C6H3);

13C NMR:�5.4 (SiMe),�2.6(SiMe2), 13.8 (SiCH2), 16.8, 17.5, 17.7, 19.6 (SiCH2 andSiCH2CH2), 22.0 (OCH2CH2CH2), 32.3 (OCH2CH2CH2), 50.9(NMe), 52.5 (NMe2), 55.7, 57.9 and 56.3 (CH2N), 60.6(OCH2CH2N), 64.9 (ArOCH2), 93.5 (CH, C6H3), 160.0 (Cipso,C6H3);

15N NMR: �331.4 (NMe2), �326.5 (NMe); 29Si NMR: 1.3(SiMe), 20.4 (SiMe2). Anal. calc. for C213H510I24N24O15Si21(7283.98): C, 35.12; H, 7.06; N, 4.62; obt.: C, 35.69; H, 7.13;N, 4.23%.

Synthesis of [G3O3(SiONN)24(I)48] (6). Following the proce-dure described above for 4, compound 6 was obtained as whitesolid (0.39 g, 84%) from 3 (0.250 g, 0.032 mmol) and MeI(0.19 mL, 3.06 mmol). NMR data (DMSO-d6):

1H NMR: �0.10 (s,45H, SiMe), �0.02 (s, 18H, SiMe), 0.11 (s, 144H, SiMe2), 0.52 (m,96H, CH2Si), 0.67 (m, 48H, CH2Si), 1.33 (m, 90H, CH2CH2Si),1.74 (m, 6H, OCH2CH2), 3.24 (s, 216H, NMe2 and NMe), 3.59 (m,48H, CH2NMe2), 4.00 (m, 150H, CH2NMe, SiOCH2 and ArOCH2),

This journal is ª The Royal Society of Chemistry 2013

Paper RSC Advances

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

6.03 (s, 3H, C6H3);13C NMR: �5.5 (SiMe), �2.9 (SiMe2), 16.8,

17.0, 17.5, 17.6, 17.8, 19.6 (SiCH2 and SiCH2CH2), 33.5(OCH2CH2CH2), 50.9 (NMe), 52.5 (NMe2), 55.5, 55.8 and 56.3(CH2N), 60.6 (OCH2CH2N), 60.9 (OCH2CH2N), 67.8 (ArOCH2),93.6 (CH, C6H3), 160.9 (Cipso, C6H3);

15N NMR: �331.5 (NMe2),�326.5 (NMe); 29Si NMR: 1.2 (SiMe), 20.3 (SiMe2). Anal. calc. forC429H1038I48N48O27Si45 (14658.40): C, 35.16; H, 7.12; N, 4.59;obt.: C, 35.66; H, 7.68; N, 4.13%.

Dosy experiments. These experiments were recorded onBruker AV400 at 298 K in CDCl3. The hydrodynamic radii werecalculated using solutions of about 10 mg of dendrimers in0.6 mL of CDCl3. The method employed avoids the knowledgeof solution viscosity and also interpretation of diffusion coeffi-cients, which are affected by possible inaccuracies in gradientpulse.

5.3 Macrophage differentiation and cell culture

Human peripheral blood mononuclear cells (PBMC) were iso-lated from buffy coats of healthy donors on a Ficoll-Hypaquedensity gradient (Rafer) according to standard procedures.Monocytes were puried from PBMC by magnetic cell sortingusing CD14 Microbeads (Miltenyi Biotec). Monocytes werecultured at 0.5 � 106 cells per mL for 7 days in RPMI mediumsupplemented with Foetal Bovine Serum at 37 �C in a humidi-ed atmosphere with 5% CO2 and containing RecombinantHuman Granulocyte Macrophage Colony Stimulating Factor(rh GM-CSF) (ImmunoTools) to generate M1 monocyte-derivedmacrophages. Cytokines were added every 2 days.

5.4 Cytoxicity assays

To determine the toxicity of the dendrimers on the macro-phages, dendrimers were added at day 7 at different doses from3 mM to 15 mM, and the toxicity was evaluated aer 24 hours oftreatment. The cell viability was determined by two methods.Firstly, by MTT (methylthiazolyldiphenyl-tetrazolium bromide)assay following manufacturer instruction (Sigma-Aldrich) formeasuring the activity of cellular enzymes that gives us infor-mation about cytotoxicity. Secondly, by 7AAD (7-amino-actinomycin D by Sigma-Aldrich) staining and were analyzed byow cytometry. Each experiment was performed in triplicate,and 3 different donors were evaluated.

5.5 Enzyme-linked immunosorbent assay (ELISA)

M1 macrophages were cultured for 7 days (as previouslydescribed), treated for 24 hours with dendrimers, and thesupernatants were tested for the presence of cytokines. Thesupernatants were evaluated aer 24 hours of treatment.The presence of cytokines were tested using commerciallyavailable ELISA for TNF-a (ImmunoTools), and IL-12p40 set(BD Pharmingen), following the protocols supplied by themanufacturers.

Acknowledgements

This work has been supported by grants from CTQ2011-23245(MINECO), Consortium NANODENDMED ref S2011/BMD-2351

This journal is ª The Royal Society of Chemistry 2013

(CM) and UAH2011/EXP-037 to R. G. and F. J. M. This work wassupported by grants from Fondos de Investigacion SanitariaISCIII (INTRASALUD PI09/02029, P509102669), FundacionEugenio Rodrıguez Pascual Red Tematica de InvestigacionCooperativa Sanitaria ISCIII (RETIC RD06/0006/0035 and RD12/0017/0037), Red Nacional de Biobancos (RD09/0076/00103),INDISNET S-2011-BMD2332, and FIPSE to M. A. M.-F. This workwas also supported by the Ministerio de Ciencia e Innovacion(grant SAF2011-23801) and Programa de actividades de I + D dela Comunidad de Madrid (RAPHYME) to A. L. C. This study wasalso supported by CIBER-BBN nanced by the Instituto de SaludCarlos III, with assistance from the European Regional Devel-opment Fund. We thank the Centers of Transfusion of Madridand Albacete for the buffy coats and Spanish HIV HGM BioBanksupported by ISC III (Grant no. RD09/0076/00103) and FIPSE.Also, we thank Dra. Maribel Clemente Mayoral for her technicalassistance and advice as cell culture technician (CA10/01274).

References

1 R. G. Denkewalter, J. F. Kolc and W. J. Lukasavage, US Pat.4289872, 1981.

2 F. Vogtle and C. A. Schalley, Top. Curr. Chem., 2003, 228.3 G. R. Newkome, C. N. Mooreeld and F. Vogtle, Dendrimersand Dendrons: Concepts, Syntheses, Applications, Wiley-VCH,Weinheim, Germany, 2001.

4 G. R. Newkome and C. D. Shreiner, Polymer, 2008, 49, 1–173.5 J. M. J. Frechet and D. A. Tomalia, Dendrimers and OtherDendritic Polymers, Wiley-VCH, Weinheim, Germany, 2002.

6 D. Astruc, E. Boisselier and C. Ornelas, Chem. Rev., 2010, 110,1857–1959.

7 T. Dutta, N. K. Jain, N. A. J. McMillan and H. S. Parekh,Nanomedicine: Nanotechnology, Biology and Medicine, 2010,6, 25–34.

8 C. M. Paleos, D. Tsiourvas, Z. Sideratou and L. Tziveleka,Curr. Top. Med. Chem., 2008, 8, 1204–1224.

9 W. D. Jang and K. Kataoka, J. Drug Delivery Sci. Technol.,2005, 15, 19–30.

10 H. Kobayashi and M. W. Brechbiel, Mol. Imaging, 2003, 2,1–10.

11 L. J. Twyman, A. S. H. King and I. K. Martin, Chem. Soc. Rev.,2002, 31, 69–82.

12 I. Cuadrado, M. Moran, J. Losada, C. M. Casado, C. Pascual,B. Alonso and F. Lobete, Advances in DendriticMacromolecules, ed. G. R. Newkome, Jai Press Inc.,Greenwich, 1999, vol. 3, pp. 151–191.

13 O. Rolland, C. O. Turrin, A. M. Caminade and J. P. Majoral,New J. Chem., 2009, 33, 1809–1824.

14 S. Svenson, Eur. J. Pharm. Biopharm., 2009, 71, 445–462.15 S. H. Medina and M. E. H. El-Sayed, Chem. Rev., 2009, 109,

3141–3157.16 M. A. Mintzer and E. E. Simanek, Chem. Rev., 2009, 109, 259–

302.17 D. G. Shcharbin, B. Klajnert and M. Bryszewska,

Biochemistry, 2009, 74, 1070–1079.18 N. Joshi and M. Grinstaff, Curr. Top. Med. Chem., 2008, 8,

1225–1236.

RSC Adv., 2013, 3, 23445–23453 | 23451

RSC Advances Paper

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

19 M. Longmire, P. L. Choyke and H. Kobayashi, Curr. Top. Med.Chem., 2008, 8, 1180–1186.

20 P. M. H. Heegaard, U. Boas and N. S. Sorensen, BioconjugateChem., 2010, 21, 405–418.

21 R. Duncan and L. Izzo, Adv. Drug Delivery Rev., 2005, 57,2215–2237.

22 G. F. Doncel, N. Chandra and R. N. Fichorova, J. AcquiredImmune Dec. Syndr., 2004, 37(suppl 3), S174–S180.

23 R. N. Fichorova, L. D. Tucker and D. J. Anderson, J. Infect.Dis., 2001, 184, 418–428.

24 G. F. Doncel andM. R. Clark, Antiviral Res., 2010, 88(suppl 1),S10–S18.

25 R. N. Fichorova, J. Acquired Immune Dec. Syndr., 2004,37(suppl 3), S184–S193.

26 A. Mantovani, A. Sica and M. Locati, Eur. J. Immunol., 2007,37, 14–16.

27 A. J. Fleetwood, T. Lawrence, J. A. Hamilton and A. D. Cook,J. Immunol., 2007, 178, 5245–5252.

28 F. A. Verreck, T. de Boer, D. M. Langenberg, L. van derZanden and T. H. Ottenhoff, J. Leukocyte Biol., 2006, 79,285–293.

29 A. W. van der Made and P. W. N. M. van Leeuwen, J. Chem.Soc., Chem. Commun., 1992, 1400–1401.

30 D. Seyferth, D. Y. Son, A. L. Rheingold and R. L. Ostrander,Organometallics, 1994, 13, 2682–2690.

31 J. F. Bermejo, P. Ortega, L. Chonco, R. Eritja, R. Samaniego,M. Mullner, E. de Jesus, F. J. de la Mata, J. C. Flores,R. Gomez and A. Mu~noz-Fernandez, Chem.–Eur. J., 2007,13, 483–495.

32 B. Rasines, J. M. Hernandez-Ros, N. de las Cuevas, J. L. Copa-Pati~no, J. Soliveri, M. A. Mu~noz-Fernandez, R. Gomez andF. J. de la Mata, Dalton Trans., 2009, 8704–8713.

33 P. Ortega, E. Fuentes-Paniagua, J. Sanchez-Nieves,J. M. Hernandez-Ros, J. L. Copa-Pati~no, J. Soliveri,M. A. Mu~noz-Fernandez, R. Gomez and F. J. de la Mata,Org. Biomol. Chem., 2011, 9, 5238–5248.

34 K. Matsuoka, M. Terabatake, Y. Esumi, D. Terunuma andH. Kuzuhara, Tetrahedron Lett., 1999, 40, 7839–7842.

35 K. Nishikawa, K. Matsuoka, M. Watanabe, K. Igai, K. Hino,K. Hatano, A. Yamada, N. Abe, D. Terunuma, H. Kuzuharaand Y. Natori, J. Infect. Dis., 2005, 191, 2097–2105.

36 P. Ortega, J. F. Bermejo, L. Chonco, E. de Jesus, F. J. de laMata, G. Fernandez, J. C. Flores, R. Gomez, M. J. Serramıaand M. A. Mu~noz-Fernandez, Eur. J. Inorg. Chem., 2006,1388–1396.

37 L. Chonco, J. F. Bermejo-Martın, P. Ortega, D. Shcharbin,E. Pedziwiatr, B. Klajnert, F. J. de la Mata, R. Eritja,R. Gomez, M. Bryszewska and M. A. Mu~noz-Fernandez,Org. Biomol. Chem., 2007, 5, 1886–1893.

38 N. Weber, P. Ortega, M. I. Clemente, D. Shcharbin,M. Bryszewska, F. J. de la Mata, R. Gomez andM. A. Mu~noz-Fernandez, J. Controlled Release, 2008, 132,55–64.

39 T. Gonzalo, M. I. Clemente, L. Chonco, N. D. Weber, L. Dıaz,M. J. Serramıa, R. Gras, P. Ortega, F. J. de la Mata, R. Gomez,L. A. Lopez-Fernandez, M. A. Mu~noz-Fernandez andJ. L. Jimenez, ChemMedChem, 2010, 5, 921–929.

23452 | RSC Adv., 2013, 3, 23445–23453

40 N. de las Cuevas, S. Garcıa-Gallego, B. Rasines, F. J. de laMata, L. G. Guijarro, M. A. Mu~noz-Fernandez andR. Gomez, Curr. Med. Chem., 2012, 19, 5052–5061.

41 P. Ortega, J. L. Copa-Pati~no, M. A. Mu~noz-Fernandez,J. Soliveri, R. Gomez and F. J. de la Mata, Org. Biomol.Chem., 2008, 6, 3264–3269.

42 B. Rasines, J. Sanchez-Nieves, I. T. Molina, M. Guzman,M. A. Mu~noz-Fernandez, R. Gomez and F. J. de la Mata,New J. Chem., 2012, 36, 360–370.

43 B. Rasines, J. Sanchez-Nieves, M. Maiolo, M. Maly,L. Chonco, J. L. Jimenez, M. A. Mu~noz-Fernandez,F. J. de la Mata and R. Gomez, Dalton Trans., 2012, 41,12733–12748.

44 L. Chonco, M. Pion, E. Vacas, B. Rasines, M. Maly,M. J. Serramıa, L. Lopez-Fernandez, F. J. de la Mata,S. Alvarez, R. Gomez and M. A. Mu~noz-Fernandez, J.Controlled Release, 2012, 161, 949–958.

45 H. Oka, T. Onaga, T. Koyama, C.-T. Guo, Y. Suzuki, Y. Esumi,K. Hatano, D. Terunuma and K. Matsuoka, Bioorg. Med.Chem. Lett., 2008, 18, 4405–4408.

46 T. Mori, K. Hatano, K. Matsuoka, Y. Esumi, E. J. Toone andD. Terunuma, Tetrahedron, 2005, 61, 2751–2760.

47 A. Yamada, K. Hatano, K. Matsuoka, T. Koyama, Y. Esumi,H. Koshino, K. Hino, K. Nishikawa, Y. Natori andD. Terunuma, Tetrahedron, 2006, 62, 5074–5083.

48 J.-I. Sakamoto, T. Koyama, D. Miyamoto,S. Yingsakmongkon, K. I. P. J. Hidari, W. Jampangern,T. Suzuki, Y. Suzuki, Y. Esumi, T. Nakamura, K. Hatano,D. Terunuma and K. Matsuoka, Bioorg. Med. Chem., 2009,17, 5451–5464.

49 D. Shcharbin, E. Pedziwiatr, L. Chonco, J. F. Bermejo-Martın,P. Ortega, F. J. de la Mata, R. Eritja, R. Gomez, B. Klajnert,M. Bryszewska and M. A. Mu~noz-Fernandez,Biomacromolecules, 2007, 8, 2059–2062.

50 R. Gras, L. Almonacid, P. Ortega, M. J. Serramıa, R. Gomez,F. J. de la Mata, L. Lopez-Fernandez and M. A. Mu~noz-Fernandez, Pharm. Res., 2009, 26, 577–586.

51 T. Gonzalo, M. I. Clemente, L. Chonco, N. D. Weber, L. Dıaz,M. J. Serramıa, R. Gras, P. Ortega, F. J. de la Mata, R. Gomez,L. A. Lopez-Fernandez, M. A. Mu~noz-Fernandez andJ. L. Jimenez, ChemMedChem, 2010, 5, 921–929.

52 J. L. Jimenez, M. I. Clemente, N. D. Weber, J. Sanchez,P. Ortega, F. J. d. l. Mata, R. Gomez, D. Garcıa, L. A. Lopez-Fernandez and M. A. Mu~noz-Fernandez, BioDrugs, 2010,24, 331–343.

53 M. Pion, M. J. Serramıa, L. Dıaz, M. Bryszewska, T. Gallart,F. Garcıa, R. Gomez, F. J. de la Mata and M. A. Mu~noz-Fernandez, Biomaterials, 2010, 31, 8749–8758.

54 D. Shcharbin, E. Pedziwiatr, O. Nowacka, M. Kumar,M. Zaborski, P. Ortega, F. J. de la Mata, R. Gomez,M. A. Mu~noz-Fernandez and M. Bryszewska, Colloids Surf.,B, 2011, 83, 388–391.

55 R. Gras, M. I. Garcıa, R. Gomez, F. J. de la Mata, M. A. Mu~noz-Fernandez and L. A. Lopez-Fernandez, Mol. Pharmaceutics,2012, 9, 102–110.

56 E. Pedziwiatr-Werbicka, D. Shcharbin, J. Maly, M. Maly,M. Zaborski, B. Garbara, P. Ortega, F. J. de la Mata,

This journal is ª The Royal Society of Chemistry 2013

Paper RSC Advances

Publ

ishe

d on

01

Oct

ober

201

3. D

ownl

oade

d by

Ins

titut

o de

Inv

est B

iom

édic

as "

Alb

erto

Sol

s" o

n 24

/02/

2014

11:

59:4

3.

View Article Online

R. Gomez, M. A. Mu~noz-Fernandez, B. Klajnert andM. Bryszewska, J. Biomed. Nanotechnol., 2012, 8, 57–73.

57 J. Cohen, Nature, 2002, 420, 885–891.58 J. Sanchez-Nieves, P. Ortega, M. A. Mu~noz-Fernandez,

R. Gomez and F. J. de la Mata, Tetrahedron, 2010, 9203–9213.59 D. K. Wilkins, S. B. Grimshaw, V. Receveur, C. M. Dobson,

J. A. Jones and L. J. Smith, Biochemistry, 1999, 38, 16424.60 D. Zuccaccia and A. Macchioni, Organometallics, 2005, 24,

3476–3486.

This journal is ª The Royal Society of Chemistry 2013

61 F. E. Appoh, D. S. Thomas and H.-B. Kraatz,Macromolecules,2005, 38, 7562–7570.

62 I. Angurell, L.-I. Rodrıguez, O. Rossell and M. Seco,Organometallics, 2011, 30, 5771–5775.

63 T. Stoeger, C. Reinhard, S. Takenaka, A. Schroeppel, E. Karg,B. Ritter, J. Heyder and H. Schulz, Environ. Health Perspect.,2006, 114, 328–333.

64 M. Fujihara, M. Muroi, K. Tanamoto, T. Suzuki, H. Azumaand H. Ikeda, Pharmacol. Ther., 2003, 100, 171–194.

RSC Adv., 2013, 3, 23445–23453 | 23453