PAMAM amination

ForTomalia s dendritic PAMAM amine, depicted in the line notation as ... 

CM-chitosan ferminal ester group-PAMAM Terminai cartoxyi group-PAMAM Amine group In CM-chItosan Tertiary amine in PAMAM Hydrogen bond Lysozyme Carboxyl group in CM-chItosan and PAMAM... 

Newly synthesized PAMAM dendrimers have a defined size and shape and can be used in gene transfer experiments, but the efficiency of transfection can be greatly increased by a process called activation of the dendrimer. In activation, some of the tertiary amines are removed, resulting in a molecule with a higher... 

PAMAM dendrimers are synthesized in a multistep process. Starting from a multifunctional amine (for example ammonia, ethylenediamine, or tris(2-amino-ethyl)amine) repeated Michael addition of methylacrylate and reaction of the product with ethylenediamine leads to dendrimers of different generation numbers [1,9]. Two methylacrylate monomers are added to each bifunctional ethylenediamine generating a branch at each cycle. Unreacted ethylenediamine has to be completely removed at each step to prevent the initiation of additional dendrimers of lower generation number. Excess methylacrylate has also to be removed. Bridging between two branches of the same or of two different dendrimers by ethylenediamine can also be a problem, and has to be avoided by choosing appropriate reaction conditions. 

The addition of ammonia to excess methyl acrylate (a linear monomer), followed by amidation with excess ethylenediamine afforded the resultant cascade molecule, and thus Tomalia [37] created the commercially available PAMAM starburst series of dendrimers (2, Fig. 2). Related core molecules such as ethylenediamine and aminoalcohols and other functionalizable groups such as thiol moieties were used to prepare similar dendrimers [38]. This methodology is applicable to most primary amines, resulting in a 1 —> 2 branching pattern. Recently, examples of related Si-, [39] P-, [40] and metallo systems [41], which follow this linear monomer protocol have been reported. 

Fig. 4. Early generation PAMAM dendrimers with amine and ester/acid termini...

Figure 7.1 The synthesis of a PAMAM-type dendrimer proceeds from a diamine core [e.g., ethylene diamine (EDA)] by initial addition of the amines to the double bonds of methacrylate. Subsequent reaction of the methyl ester groups with EDA produces a G-0 dendrimer with four pendent amine groups. Another round of methacrylate and EDA additions results in a G-l PAMAM dendrimer containing eight primary amines.

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. 

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

The amines on the surface of PAMAM-type and other amine-containing dendrimers may be used to couple to aldehyde groups in other molecules, including those formed after periodate... 

Acylation reactions to block amine groups on PAMAM dendrimers with anhydride compounds are done in a similar manner to glycidol modification. The following protocol is based on the method of Majoros et al., 2005. 

In a fume hood, dissolve 270 mg of a G-4 dendrimer (PAMAM or other amine containing) in 2 ml of dry DMSO. Maintain a nitrogen blanket over the reaction to prevent oxidation. The use of a 3-necked flask and a heating mantle is recommended to control mixing and the proper temperature. 

The following protocol is based on the method of Singh (1998). Other amine-containing dendrimers besides the PAMAM type, such as the PrioStar dendrimers from Dendritic Nanotechnologies may be used as well. 

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