142 Advances in Polymer Science Editorial Board: A. Abe. A.-C ...

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220 J. Roovers, B. Comanita Scheme 20 midomethyl (Pam) modified poly(styrene-co-divinylbenzene) beads, a third generation of poly (a,e-L-lysine) dendrimer with eight primary amines was obtained (see Scheme 20a). After capping each amine with a glycine spacer eight copies of a desired polypeptide are grown. A detailed critical description of the special conditions required in the synthesis of a dendrimer on a cross-linked polymer, including low loading of the resin beads, has been given [145]. The long hydrolyzable Pam spacer between the non-polar polymer and the more polar dendrimer is also important. The polypeptides chosen for the synthesis are specific antigens. The clusters of the antigens attached to the dendrimer elicit good antibody response without showing the problems associated with antigens bound to carrier proteins. In recent work Tam and Rao have attached unprotected peptides directly to a third generation poly(a,e-L-lysine) dendrimer [149]. As shown in Scheme 20b, the dendrimer is modified to contain eight aldehyde groups. These are then reacted with a cysteine terminated polypeptide with formation of a thiazolidine ring. The addition of the first five polypeptides occurs within 2 h at 18 ˚C but addition of subsequent peptide chains becomes progressively slower. After 67 h a
Dendrimers and Dendrimer-Polymer Hybrids 221 mixture of hepta-and octapeptide is obtained. The method which allows the use of unprotected polypeptides was expanded to other reactions producing oxime and hydrazone dendrimer-peptide linkages [150, 151]. These are, however, less stable than the thiazolidine ring at elevated pH. 5.2 Dendrimer-DNA Complexes Dendrimers with primary amines form complexes with nucleotides by electrostatic interaction between the cationic ammonium and anionic phosphate groups [152–157]. Such interactions are also known to occur with polylysine and other linear polycations. The first account of such interaction has been described for the case of PAMAM dendrimers with plasmid DNA encoding for either luciferase or bacterial b-galactosidase [156]. The complexation is monitored by DNA immobilization in electrophoresis [152, 155, 156] or by enhanced fluorescence anisotropy [153]. Complexes are completely immobile when the terminal amine and nucleotide base stoichiometry is around 1:1 to 1.5:1 [152, 155, 156]. Excess nucleotide shows no retardation of mobility, while excess dendrimer amine groups, i.e., 5:1 and higher, leads to positively charged complexes which migrate to the cathode [152, 155, 156]. It is observed that complexes are better prepared at low pH and low ionic strength [152]. Nevertheless, the dendrimer-DNA complexes are quite stable against variations in pH between 3 and 10 [152, 155] with some loss of complexation above pH 10 [153]. Ionic strengths between 50 mmol/l and 1.5 mol/l are tolerated. Disruption of the complex is only effected with strong ionic detergents [155, 157]. An important advantage of the complex is that it makes the DNA quite resistant to nuclease digestion [157]. The structure of the dendrimer-DNA complexes is not well characterized. It has been estimated that under conditions of maximum transfection, i.e., the process of bringing the DNA through the cell wall into the cell, the size of the luciferase plasmid is such that about 320 PAMAM dendrimers are present, although not all may be directly involved in the complexation [156]. Electron microscopy suggests that the dendrimer-DNA complexes form condensed aggregates [157]. Delong et al. have studied the complex formed between a third generation PAMAM dendrimer and a small 15 base oligonucleotide [152]. They found a broad distribution of MW by ultrafiltration with most material between 10 and 30 kD. Light scattering experiments, however, are obscured by the presence of a small amount of very high MW material. The PAMAM dendrimer-DNA complexes have been shown to promote transfection [152–156]. The earlier studies used reporter luciferase which allows for measurement of luciferase activity in host cells [155, 156]. Although there is some dependence on the cell type being invaded [155], experiments have shown that generations 6–8 dendrimers are most efficient in transfection [155, 156]. There is little transfection with generations 2–4 PAMAM dendrimers and no further improved efficiency with generations 9 and 10 dendrimers. The preferred dendrimer to DNA stoichiometry is 5:1 or higher [155, 156]. It has since
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142 Advances in Polymer Science Edi
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Branched Polymers I Volume Editor:
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Volume Editor Dr. Jacques Roovers I
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Preface While books have been writt
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Contents Synthesis of Branched Poly
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Synthesis of Branched Polymers by C
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72 N. Hadjichristidis, S. Pispas, M
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100 N. Hadjichristidis, S. Pispas,
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Poly(macromonomers): Homo- and Copo
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Poly(macromonomers), Homo- and Copo
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142 Advances in Polymer Science Editorial Board: A. Abe. A.-C ...

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