Dendrimer conjugation methods

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 purified SPDP-modified dendrimer of step 5 in 50 mM sodium phosphate, 0.15M NaCl, pH 7.5, or in DMSO at a concentration of at least lOmg/ml. Add a 10-20 X molar excess of an amine-reactive fluorescent molecule (i.e., NHS-rhodamine or a hydrophilic NHS-Cy5 derivative see section on fluorescent probes). React with mixing for 1 hour at room temperature. Purify the fluorescently labeled SPDP-modified dendrimer using gel filtration or ultrafiltration. Follow the method of either step 7 or 8 to conjugate the dendrimer to another protein or molecule. 

The following method for carbohydrate conjugation to dendrimers may be used to couple a variety of reducing sugars to amine-dendrimers, including saccharides, longer-chain carbohydrates, and even complex glycans after release from a protein (see Chapter 1, Section 4.6). 

Figure 7.20 The multivalent surface of dendrimers can be used to couple biotin groups and labels for detection in immunoassays. One such conjugate was made by coupling NHS-biotin and a maleimido-iron chelate to an amine-dendrimer for use in an unique carbonyl metallo assay method.

Drugs are encapsulated in dendrimers primarily using two methods by covalent dendrimer-drug conjugation or non-covalent encapsulation of drugs. 

Another method to create hybrid nanoparticles is encapsulation of smaller nanoparticles, such as dendrimers, into larger nanoparticles, such as polymeric nanoparticle and liposomes using a double emulsion method.[36] Double emulsions may be formed with either the water-in-oil-in-water (W/OAV) or the oil-in-water-in-oil types (O/W/O). The W/O/W double emulsion method is more commonly used as it stabilizes the particles in the aqueous phase and encapsulates hydrophilic materials into a variety of polymers or copolymers with a controlled particle size. [37] This method allows for the preparation of hybrid nanoparticles that have two-size scale, i.e., polymeric nanoparticles of 50-200 nm and encapsulated dendrimers or other polymer conjugates of <10 nm).[36,38] The encapsulation process is affected by several parameters such as the chemical composition of the outer layers, size and surface charges of dendrimers, and ratios of the outer and inner nanoparticles. [36,25]... 

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