Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

Abstract

Ferritin self-assembly has been widely exploited for the synthesis of a variety of nanoparticles for drugdelivery and diagnostic applications. However, despite the crucial role of ferritin self-assembly mechanism for probes encapsulation, little is known about the principles behind the oligomerization mechanism. In the present work, the novel "humanized" chimeric Archaeal ferritin HumAfFt, displaying the transferrin receptor-1 (TfR1) recognition motif typical of human H homopolymer and the unique salttriggered oligomerization properties of Archaeoglobus fulgidus ferritin (AfFt), was site-selectively labeled with N-(1-pyrenyl)maleimide on a topologically selected cysteine residue inside the protein cavity, next to the dimer interface. Pyrene characteristic fluorescence features were exploited to investigate the transition from a dimeric to a cage-like 24-meric state and to visualize the protein in vitro by two photon fluorescence microscopy. Indeed, pyrene fluorescence changes upon ferritin self-assembly allowed to establish, for the first time, the kinetic and thermodynamic details of the archaeal ferritins oligomerization mechanism. In particular, the magnesium induced oligomerization proved to be faster than the monovalent cation-triggered process, highly cooperative, complete at low MgCl 2 concentrations, and reversed by treatment with EDTA. Moreover, pyrene intense excimer fluorescence was successfully visualized in vitro by two photon fluorescence microscopy as pyrene-labeled HumAfFt was actively uptaken into HeLa cells by human transferrin receptor TfR1 recognition, thus representing a unique nano-device building block for two photon fluorescence cell imaging.

Design and Synthesis of Piperazine-Based Compounds Conjugated to Humanized Ferritin as Delivery System of siRNA in Cancer Cells

“…To circumvent these difficulties, a highly reactive cysteine per monomer was introduced by a point mutation as reported elsewhere. 20 , 37 This mutation (M54C) in the protein inner cavity offers the opportunity to covalently conjugate up to 24 thiol-reactive linkers, such as maleimide- or pentafluorobenzenesulfonamide-based compounds bearing positively charged side-chains ( PAs ) at pH 7.4 as predicted by ab initio calculation ( Figure S26 ). As illustrated in Scheme 3 , chemical conjugations were performed using the assembly disassembly mechanism of HumAfFt in very mild conditions, at pH 8.3, to promote the nucleophilic reaction between thiols and PAs linkers.…”

Nature of the N‐Substituted Alkyl Chain Influencing Emission Properties of Naphthalene Diimide (NDI) Via Excimer, and Exciplex Formation

“…It was found that at a lower salt concentration (0.01 M) emission quenching was less for NDI-OL, whereas at higher salt concentration (1 M) emission quenches by the following order; NaNO 2 > NaF > NaBr > Na 2 SO 3 � NaCl as seen for 464 and 556 nm emission respectively for NDI-OL and NDI-OD. [48] It is observed that at higher ionic strength does influence the excimer emission regardless of nature of salt and counter ion and the extensive quenching observed in the case of NO 2 À could be viewed as the effect of quenching by nitrite ions than the of ionic strength. Further, the effect of pH was studied in THF/water (1 : 99, v/v) mixture at varied pH range (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

Ferritin nanovehicle for targeted delivery of cytochrome C to cancer cells

“…Such procedure, however, is not amenable to the encapsulation of most proteins that display both acid or alkaline folding instability. In contrast, the ferritin from Archaeglobus fulgidus (AfFt) has emerged as an alternative to human ferritin homopolymers as it requires mild cargo material encapsulation conditions in view of the unique self-assembly properties that entail divalent cation driven assembly at neutral pH values 11 . AfFt assembles in a distinctive tetrahedral geometry as a result of a particular packing between four hexameric units into a unique 24-mer structure, which results in the formation of four wide triangular pores (45 Å) on the protein shell 12,13 .…”