Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006
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) E-mail: mmunoz@cbm.uam.es 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-28871 Alcalá de Henares (Spain) FAX: (+34) 91 885 4683 E-mail: rafael.gomez@uah.es 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
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 3 - - - - Me 2 C 6 H 3 - Ph -C H 2 Ph -C H2 -C H 2 - - - Me - -C 6 H 3 - - Me 2 CDCl 3 C ipso ( - Me 2 ) C ipso (- ) C 6 H 3 - - - - 70 60 ppm Me 2 Me 1D 13 C MR, CDCl 3 1D 29 MR, CDCl 3 2
II III I 3
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
3G-[{( ) 2 (Me)( ) 2 Me 2 }] 16 (8) 4 1D 1 H MR, CDCl 3 5
Me 2 Me 2 Me - - - - CDCl 3 -C H 2 - -C H 2 - Me 1D 13 C MR, CDCl 3 Me 2 - - - - Me 2 Me 1G-[{( ) 3 H 2 }] 4 (9) H 2 4 6
1D 1 H MR, CDCl 3 Me 2 - H 2 -C H 2 - H 2 - CDCl 3 1D 13 C MR, CDCl 3 - - - - - - H 2 - CDCl 3 Me 2 7
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 2 + - C H 2 - - - 1D 13 C MR, DMS-d 6 8
- + - -C 6 H 3 - Me 3 + DMS-d 6 -C ipso ( - Me 3+ ) 65 60 Me 2 - C ipso (- ) C 6 H 3 - - 2D{ 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
10
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 - - - - 11
12
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
- + - - - H 2 Me 2 Me 14
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. 1 2 3 1 2 3 4 1 3 54 6 5 7 6 6 7 8 Time-lapse confocal microscopy of cells treated for 72 h with dendrimer 1. Arrows mean cells moving over the time. 15
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 +++ 12 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
4G-PAMAM Aggregates + Aggregates ++ mortality ot tested 12
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 + + 2 aggregates ++ aggregates ++ mortality 12 aggregate formation + aggregates +++ aggregates +++ aggregates +++ 15 aggregates +/- aggregate +++ 4G-PAMAM Aggregates + Aggregates ++ Fucsiform cells Fucsiform cells 12
TRYPA BLUE STAIIG. A) Control; B) dendrimer 1 + D. 13
TRYPA BLUE UPTAKE USIG DEDRIPLEXES Percentage of cells positive for Trypan Blue after 72 h of incubation with dendriplexes and dendrimers. 14
120 100 80 60 40 20 0 control D + 1 D + 2 D + 12 D + 15 D + 18 D + SF D + 4G PAMAM D 1 12 15 18 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
cells corresponding to dead/apoptotic cells. Black areas denote dead cells and grey areas denote alive cells. 120 100 80 60 Apop/dead Alive 40 20 0 Control D + 1 D + 2 D + 12 D + 15 D + 18 D + SF D + 4G- PAMAM D 1 12 15 18 SF 4G-PAMAM 2 Dendriplexes and/or dendrimers 16