Amine-Dendrimers with Sulfo-NHS-LC-SPDP

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

Dissolve the amine-containing PAM AM dendrimer in methanol or a buffered aqueous medium at a pH of 7-9 (e.g., 50mM sodium phosphate, pH 7.5) and at a concentration of at least lOmg/ml. Note that Singh (1998) used a concentration of llOmg/ml in methanol, but other dendrimer concentrations should work equally well. For nonaqueous reactions, the addition of a proton acceptor may aid in driving the reaction to maximal yields (i.e., triethylamine or dimethylaminopyridine). 

Dissolve sulfo-NHS-LC-SPDP at a concentration of 20mM (5.2mg/ml) in DMSO or water. If water is used, the solution must be used immediately to prevent hydrolysis of the sulfo-NHS ester. 

With mixing, add an aliquot of the crosslinker to the dendrimer solution to provide the desired molar excess of reagent. For many applications, less than 10 pyridyl disulfide groups are needed per dendrimer molecule therefore, molar ratios in the range of 5-20 X excess of crosslinker over the amount of dendrimer present typically are used. 

React with mixing for at least 30 minutes at room temperature. Longer reactions may be used without problems. 

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