Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006

2 Water-Soluble Carbosilane Dendrimers: Synthesis Biocompatibility and Complexation with ligonucleotides. An Evaluation for Medical Applications Jesus F. Bermejo, [a] Paula rtega, [b] Louis Chonco, [a] Ramon Eritja, [c] Rafael Samaniego, [d] Matthias Müllner, [a] Ernesto de Jesus, [b] F. Javier de la Mata,* [b] Juan Carlos Flores, [b] Rafael Gomez,* [b] and Mª Angeles Muñoz-Fernandez.* [a] [a] [b] [c] [d] Dr. J. F. Bermejo, L. Chonco, M. Müllner, Dr. M. A. Muñoz-Fernandez Laboratorio de Inmunobiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid (Spain) P. rtega, Dr. E. De Jesús, Dr. F. J. De la Mata, Dr. J. C. Flores, Dr. R. Gómez Departamento de Química Inorgánica, Universidad de Alcalá, Campus Universitario, E Alcalá de Henares (Spain) FAX: (+34) Dr. R. Eritja Instituto de Biología Molecular de Barcelona, CSIC, Jordi Girona, Barcelona (Spain) Dr. R. Samaniego Unidad de Microscopía Confocal, Hospital General Universitario Gregorio Marañón, Madrid (Spain) 1

3 2G-[{ -(C 6 H 3 )-3,5-( Me 2 ) 2 }] 8 (4) I II III Me 2 Me 2 Me 2 Me 2 4 1D 1 H MR, CDCl 3 Me 2 CDCl Me 2 C 6 H 3 - Ph -C H 2 Ph -C H2 -C H Me - -C 6 H Me 2 CDCl 3 C ipso ( - Me 2 ) C ipso (- ) C 6 H ppm Me 2 Me 1D 13 C MR, CDCl 3 1D 29 MR, CDCl 3 2

4 II III I 3

5 2G-[{( ) 2 (Me)( ) 2 Me 2 }] 8 (7) 2D{ 1 H 13 C} HSQC, CDCl 3, 300 MHz MR I a b c k d I e f l II g h m i j n l k b d,c,a f m g h + i j n 4

6 3G-[{( ) 2 (Me)( ) 2 Me 2 }] 16 (8) 4 1D 1 H MR, CDCl 3 5

7 Me 2 Me 2 Me CDCl 3 -C H 2 - -C H 2 - Me 1D 13 C MR, CDCl 3 Me Me 2 Me 1G-[{( ) 3 H 2 }] 4 (9) H 2 4 6

8 1D 1 H MR, CDCl 3 Me 2 - H 2 -C H 2 - H 2 - CDCl 3 1D 13 C MR, CDCl H 2 - CDCl 3 Me 2 7

9 1G-[{ -(C 6 H 3 )-3,5-( Me 3 + I ) 2 }] 4 (11) 2 I 4 Me 3 + DMS Me 2 1D 1 H MR, DMS-d 6 C 6 H 3 -C H 2 -C H 2 -C H C H D 13 C MR, DMS-d 6 8

10 C 6 H 3 - Me 3 + DMS-d 6 -C ipso ( - Me 3+ ) Me 2 - C ipso (- ) C 6 H D{ 1 H 13 C} HMQC, DMS-d 6, 300 MHz MR I a b k h Me c j e f 3 d i II g Me 3 2 I DMS j d h i k a, c g e 9

11 10

12 1G-[{( ) 2 (Me)( ) 2 Me 3 + (I )}] 4 (14) I 4 1D 1 H MR, DMS-d 6 Fragment of external units. Me 3 + DMS Me 1D 13 C MR, DMS-d 6 Me 3 + Me Me

13 12

14 2G-[{( ) 3 H 3 + Cl }] 4 (18) H 3 2 Cl H 3 4 1D 1 H MR, D 2 Me 2 - H 2 - H 2 - Me -C H 2 + 1D 13 C MR, D 2 13

15 H 2 Me 2 Me 14

16 DAPI LABELLIG DAPI labelling after 72 h of incubation with dendrimers: (1) dendrimer 1; (2) dendrimer 2; (3) dendrimer 12; (4) dendrimer 15; (5) dendrimer 18; (6) dendrimer Superfect TM ; arrow means apoptotic corpusculi Time-lapse confocal microscopy of cells treated for 72 h with dendrimer 1. Arrows mean cells moving over the time. 15

17 Table 1. Visual exam of PBMCs by phase-contrast light microscope treated with dendrimers 1, 2, 12, 15 and 18. Dendrimer 1 µm 5 µm 10 µm 20 µm 100 µm 1 Birrefringency diminution, increased cell mortality Diminution of alive cells mortality 2 aggregate formation + aggregates ++ aggregates Diminution of alive cells mortality 15 o high cell aggregate formation mortality 18 aggregate formation detritus mortality mortality SF Aggregates ++ Aggregates +++ ot tested ot tested ot tested 11

18 4G-PAMAM Aggregates + Aggregates ++ mortality ot tested 12

19 Table 2. Visual exam of erythrocytes by phase-contrast light microscope treated with dendrimers 1, 2, 12 and 15. Dendrimer 1 µm 5 µm 10 µm 20 µm 1 aggregate formation aggregate formation mortality aggregates ++ aggregates ++ mortality 12 aggregate formation + aggregates +++ aggregates +++ aggregates aggregates +/- aggregate +++ 4G-PAMAM Aggregates + Aggregates ++ Fucsiform cells Fucsiform cells 12

20 TRYPA BLUE STAIIG. A) Control; B) dendrimer 1 + D. 13

21 TRYPA BLUE UPTAKE USIG DEDRIPLEXES Percentage of cells positive for Trypan Blue after 72 h of incubation with dendriplexes and dendrimers. 14

22 control D + 1 D + 2 D + 12 D + 15 D + 18 D + SF D + 4G PAMAM D SF 4G-PAMAM 2 Dendrimer FLW CYTMETRY USIG DEDRIPLEXES Flow cytometry of PBMCs after 72 h of incubation with dendriplexes and dendrimers: evaluation of the percentage of cells with FW and SD corresponding to alive cells against the percentage of 15

23 cells corresponding to dead/apoptotic cells. Black areas denote dead cells and grey areas denote alive cells Apop/dead Alive Control D + 1 D + 2 D + 12 D + 15 D + 18 D + SF D + 4G- PAMAM D SF 4G-PAMAM 2 Dendriplexes and/or dendrimers 16