Isolation and Characterization of Precise Dye/Dendrimer Ratios

Abstract

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne `click' ligand, separation into sample containing precisely defined `click' ligand/particle ratios using reverse-phase high performance liquid chromatography (rp-HPLC), followed by reaction with excess azide-functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using rp-HPLC. These materials were characterized using 1H and 19F NMR, rp-HPLC, UV-Vis and fluorescence spectroscopy, lifetime measurements, and MALDI.

Generation 3 PAMAM dendrimer TAMRA conjugates containing precise dye/dendrimer ratios

“…In order to image the location of the material in biological environments, a fluorescent dye is often conjugated to the dendrimer scaffold; however, the Poisson distribution of dye/dendrimer ratios resulting from typical stochastic synthesis conditions [4–6] gives rise to a range of photophysical [7] and biodistribution properties that limit interpretation of these experiments.…”

“…Many groups have explored strategies to improve the control over fluorophore/dendrimer ratios since the relatively lower polydispersity index (PDI) of dendrimers and/or synthetic methodologies make this class of scaffold attractive for addressing the challenges [7–14]. Approaches include dye encapsulation [12–14], employing the core of the dendrimer as the dye [11], and convergent synthesis starting with the dye [8,9].…”

“…All of these approaches have particular advantages and limitations. Dye encapsulation is readily achieved by simple mixing but does not give homogeneous ratios [7] and rapidly leaches dye under biological conditions [13]. Convergent syntheses can give material structural control approaching molecular levels but is limited in terms of molecular weight (MW) that can be achieved and also gives dye/dendrimer ratios controlled by number of arms.…”

“…Use of the dye as the core avoids hydrophobic decoration of the dendrimer surface but can be synthetically arduous, increases dendrimer rigidity, and limits dye loading. In our approach, we have stochastically dye-modified the divergently synthesized, commercially available, poly(amidoamine) (PAMAM) dendrimer system and employed reverse-phase high performance liquid chromatography (rp-HPLC) to separate the resulting population into the desired precise ratio dye/dendrimer fractions [4,7,15]. In principle, this strategy has the advantages of being flexible across multiple generations of hydrophilic dendrimer and, in addition to providing the precise ratio materials for desired applications (i.e.…”

Distributions: The Importance of the Chemist’s Molecular View for Biological Materials

“…Our group has already published extensively on this work, as well as our research on characterizing, controlling, and eliminating heterogeneous distributions, in this journal 10,51 and others. 2−8,11,12 Here, we highlight a case in which we demonstrated in vitro the importance of explicit consideration of distributions in biological nanomaterials. 9 …”