Non-Traditional Intrinsic Luminescence (NTIL): Dynamic Quenching Demonstrates the Presence of Two Distinct Fluorophore Types Associated with NTIL Behavior in Pyrrolidone-Terminated PAMAM Dendrimers

Abstract

Historically, poly(amidoamine) (PAMAM) dendrimers were the first macromolecular structures reported to exhibit "non-traditional intrinsic luminescence" (NTIL) properties. Initially, this unique intrinsic luminescent property suggested the possibility of dendrimer-based biological imaging without the need for conjugating external labels. Unfortunately, low NTIL intensity levels exhibited by most simple surface-modified PAMAMs presented a serious barrier to progress in that area. Unexpectedly, a simple surface modification of amine-terminated PAMAM dendrimers with N-(4-carbomethoxy)pyrrolidone groups (4-CMP) was found to dramatically increase NTIL fluorescence intensity (i.e., >50-fold) while substantially enhancing biocompatibility and reducing cytotoxicity/complement activation properties. This current study focuses on the use of conventional and time-resolved spectroscopic measurements to characterize the NTIL behavior of 4-CMP PAMAM dendrimers over four generation levels (i.e., G2−G5). We describe and discuss the impact of polymer size and composition on NTIL intensity levels and quantum yields. Fluorescence lifetime measurements revealed two discrete major lifetime components, which were similar for all dendrimer generations and remained unaffected by changes in pH. Time-resolved fluorescence quenching studies involving a collisional quencher (methyl red) and a dynamic proximity quencher (nitrobenzoxadiazole dipeptide derivative) provided evidence for two spatially separated NTILtype emission sites within this 4-CMP PAMAM dendrimer series. In summary, these results provide important insights into the molecular-level NTIL mechanism and demonstrate the critical role of pyrrolidone surface modification as well as separate contributions made by interior dendrimer components to the observed enhancement of NTIL fluorescence intensity.

Synthesis, Internalization and Visualization of N-(4-Carbomethoxy) Pyrrolidone Terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in Mammalian Cells

“…Similarly, the values of three fluorescence lifetime components of the fluorescence decay curves measured for intrinsic fluorescence of PAMAM [G5:G3-(TREN)]- N -(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) (C) by the TCSCP method ( Table 1 ) were close to the values obtained for N -(4-CMP) PAMAM dendrimers ( ≈ 0.4 ns, ≈ 3 ns, ≈ 8 ns) [ 28 ]. However, intensity fractions of the respective components were somewhat different (ca.…”

“…More specifically, fluorescence lifetime analysis of core-shell tecto(dendrimer) structures confirmed that they manifested relevant NTIL fluorophore features that behaved analogously to previously characterized monomeric, N -(4-carbomethoxy pyrrolidone) (4-CMP) terminated PAMAM dendrimers [ 29 ]. For example, two relatively strong but comparable fluorophore centers, (i.e., corresponding to fluorescence decay curve components 2 and 3 in this study) are distinctly noted in this analysis [ 28 ]. It has previously been demonstrated that the brightest fluorophore (component 3, in our tecto(dendrimers) contributing half of total fluorescence output) is derived from interactions of tertiary amino groups and imidic acid moieties along the terminal PAMAM branches and that its brightness and quantum yield are enhanced by branch stabilization [ 54 , 55 , 56 ].…”

“…Excitation and emission fluorescence spectra of PAMAM [G5:G3-(TREN)]- N -(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) (C) were recorded at room temperature on a LS55 spectrofluorometer (Perkin-Elmer). Fluorescence lifetimes of nontraditional intrinsic luminescence (NTIL) emissions of PAMAM [G5:G3-(TREN)]- N -(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) (C) were measured in PBS (pH 7.4) as described previously [ 28 ]. Zeta potentials were measured in 10 mM HEPES buffer (pH 7.4) with Zetasizer Nano (Malvern Panalytical).…”

“…The objective of this cloaking process is to render drugs/drug carriers stealthy to in vivo protein opsonization, thereby reducing complement activation properties, while enhancing drug circulation times and reducing cytotoxicity. Furthermore, it should be noted that dendrimer surface “pyrrolidonylation” substantially enhances so-called “non-traditional intrinsic luminescence”(NTIL) properties [ 23 , 27 , 28 ]. This fluorescence enhancement allows direct in vivo bioimaging of the dendrimer nanoparticles without any need for conjugating traditional dyes or external staining.…”

“…This fluorescence enhancement allows direct in vivo bioimaging of the dendrimer nanoparticles without any need for conjugating traditional dyes or external staining. These studies demonstrated that pyrrolidone modified PAMAM-dendrimers manifest dramatically enhanced emission intensities (i.e., >50 fold) compared to unmodified amine terminated PAMAM dendrimers [ 28 , 29 ]. Recent proposed mechanisms to account for the NTIL phenomenon have concluded that these inexplicable blue emissions are largely due to the aggregation/clustering and/or physico-chemical confinement of normally non-emissive, electron-rich, hetero-atomic, functionalized moieties, (HASLs) (i.e., heteroatomic subluminophores) [ 30 ].…”