Characterization of polyamidoamino (PAMAM) dendrimers using in-line reversed phase LC electrospray ionization mass spectrometry

Abstract

Generation 3 (G3) PAMAM dendrimers are symmetrical, highly branched polymers widely reported in the scientific literature as therapeutic agents themselves or as carrier scaffolds for various therapeutic agents. A large number of analytical techniques have been applied to study PAMAM dendrimers, but one that has been missing is in-line reversed phase LC electrospray ionization mass spectrometry (RP/LC/ESI/MS). To translate PAMAM dendrimers into therapeutic agents, a better understanding of their purity, stability and structure is required, and in-line RP/LC/ESI/MS is widely applied to all three of these analytical questions. In this study, we developed a robust in-line RP/LC/ESI/MS method for assessing stability, purity and structure of the G3 PAMAM dendrimers, and we also examined the reasons why previous attempts at method development failed. Using the RP/LC/ESI/MS method we uncovered several unique aspects of the chemistry of G3 PAMAM dendrimers. They are interconverted between two isomeric forms by dialysis, and under higher concentration levels there is an inter-molecular displacement reaction resulting, which degrades PAMAM dendrimers. Purification of G3 dendrimers by RP/LC was also previously unreported; so we slightly modified the LC/MS method for isolating individual components from a complex dendrimer mixture. Thus, we have developed a robust, comprehensive method for characterizing PAMAM dendrimers and their degradation.

‘Clickable’ cyclopentadienyl iron carbonyl complexes for bioorthogonal conjugation of mid‐infrared labels to a model protein and PAMAM dendrimer

“…The functionalized dendrimers described in this paper must be kept in solution at all times otherwise it is not possible to dissolve them again. Moreover, it has been shown that degradation occurs during storage, freeze–thaw and concentration of terminal amino or hydroxy PAMAM dendrimers, explaining why the commercial product is always sold as a 10% w/w solution . Consequently, compounds cannot be thoroughly dried before NMR recording.…”

“…Moreover, it has been shown that degradation occurs during storage, freezethaw and concentration of terminal amino or hydroxy PAMAM dendrimers, explaining why the commercial product is always sold as a 10% w/w solution. [43] Consequently, compounds cannot be thoroughly dried before NMR recording. Accordingly, to eliminate traces of MeOH, each sample is evaporated to dryness no more than 1 min, then dissolved twice with 0.8 mL MeOH-d 4 and evaporated.…”

Fluoride-Promoted Esterification (FPE) Chemistry: A Robust Route to Bis-MPA Dendrons and Their Postfunctionalization

“…These commercially available scaffolds are biodegradable and biocompatible [3], with long shelf-life and simple synthetic protocols and they fulfill all the necessary criteria for future technological breakthroughs in the field of biomedicine. They are definitely a viable alternative to the popular polyamidoamine (PAMAM) dendrimers [4,5], especially in light of recent reports detailing poor shelf-life [6] and high inherent toxicity [3,7,8]. The excellent features of monodisperse bis-MPA dendrimers and dendrons have led to their exploitation as precision polymers in an array of potential applications [9,10], for instance, as drug delivery carriers [11], self-assembled monolayers on gold nanoparticles [12], highly structured honeycomb films [13,14], functional hydrogels [15,16] and soft tissue-adhesives [17].…”

<p>PAMAM Dendrimer Nanomolecules Utilized as Drug Delivery Systems for Potential Treatment of Glioblastoma: A Systematic Review</p>

“…Commonly used assays for this are high-performance liquid chromatography (HPLC), mass spectrometry, capillary electrophoresis (CE), polyacrylamide gel electrophoresis (PAGE), and small-angle neutron scattering (SANS). [50][51][52][53] HPLC is a technique that helps identify and quantify the separate components of a mixture or solution. 50 The mixture is dissolved in a liquid solvent and pumped through a column containing a solid adsorbent material.…”

“…It can be used on both pure samples and mixtures. 51 Capillary gel electrophoresis (CE) is an electro-kinetic method for separating analytes by ionic mobility. It is typically used to characterize low generation dendrimers, yet becomes problematic when characterizing later generations, due to their similarities in charge density and interactions with the wall of the capillary.…”