Alexa Fluor-Labeled Fluorescent Cellulose Nanocrystals for Bioimaging Solid Cellulose in Spatially Structured Microenvironments

Abstract

Methods to covalently conjugate Alexa Fluor dyes to cellulose nanocrystals, at limiting amounts that retain the overall structure of the nanocrystals as model cellulose materials, were developed using two approaches. In the first, aldehyde groups are created on the cellulose surfaces by reaction with limiting amounts of sodium periodate, a reaction well-known for oxidizing vicinal diols to create dialdehyde structures. Reductive amination reactions were then applied to bind Alexa Fluor dyes with terminal amino-groups on the linker section. In the absence of the reductive step, dye washes out of the nanocrystal suspension, whereas with the reductive step, a colored product is obtained with the characteristic spectral bands of the conjugated dye. In the second approach, Alexa Fluor dyes were modified to contain chloro-substituted triazine ring at the end of the linker section. These modified dyes then were reacted with cellulose nanocrystals in acetonitrile at elevated temperature, again isolating material with the characteristic spectral bands of the Alexa Fluor dye. Reactions with Alexa Fluor 546 are given as detailed examples, labeling on the order of 1% of the total glucopyranose rings of the cellulose nanocrystals at dye loadings of ca. 5 μg/mg cellulose. Fluorescent cellulose nanocrystals were deposited in pore network microfluidic structures (PDMS) and proof-of-principle bioimaging experiments showed that the spatial localization of the solid cellulose deposits could be determined, and their disappearance under the action of Celluclast enzymes or microbes could be observed over time. In addition, single molecule fluorescence microscopy was demonstrated as a method to follow the disappearance of solid cellulose deposits over time, following the decrease in the number of single blinking dye molecules with time instead of fluorescent intensity.

Internalization of (bis)phosphonate-modified cellulose nanocrystals by human osteoblast cells

“…The site-selective periodate oxidation on native CNCs allows cleavage of the C2-C3 bond of the glucopyranose ring, converting the respective vicinal hydroxyl into two aldehyde groups (Hlady and Buijs 1996;Kenawy et al 2015), while leaving the bulk material crystallinity unchanged (Grate et al 2015). The CNCs' modification with Aln and ApA molecules [presented in Scheme 1, according to the literature (Dash et al 2012)] was carried out using a Shiff-base coupling reaction between (bis)phosphonate amine (in ApA or Aln) and cellulose (CNC) aldehyde groups which are formed during the (periodate) oxidation process.…”

Design and Synthesis of Fluorescent Nanocelluloses for Sensing and Bioimaging Applications

“…The fact that the cell nucleus is dark suggests that CNCs indicator has little interaction with the nucleus. Good cell membrane penetration and non‐cytotoxicity make them useful in fluorescent bioimaging and drug delivery …”

“…Good cell membrane penetration and non-cytotoxicity make them useful in fluorescent bioimaging and drug delivery. [89,90,91,92,93,94] In addition to the cellulose nanocrystals used above, in 2018, Nei et al have developed an efficient labeling scaffold by using the cellulose-binding domain of Cellulomonas fimi CenA protein combined with pH-sensitive enhanced cyan fluorescent protein (CBD-ECFP) to achieve measurement of pH and Ca 2 + gradients by fluorescence intensity and lifetime imaging. The results demonstrated that CBD-ECPF has excellent pH sensitivity comparable to untagged ECFP (1.9-2.3 ns for pH [6][7][8].…”

Photoantimicrobial Biohybrids by Supramolecular Immobilization of Cationic Phthalocyanines onto Cellulose Nanocrystals

“…[32][33][34][35][36] Among them, only few examples have exploited CNC in combination with dyes, mainly porphyrinoids and rutheniumc omplexes,f or catalytic or biomedical applications. [24,[36][37][38][39][40][41][42] Our strategy to decorate CNC with Pc molecules relies on the octacationic zinc Pc (ZnPc)d erivatives 1 and 2,w hich can bind through electrostatic interactions to the negatively chargeds urfaceo fs ulfated CNC. Thes elected ZnPc compounds present eight pyridinium moieties that, in the case of 2,a re quaternized with am ethoxy(triethylenoxy) chain to further enhance hydrophilicity.Compound 2 also presents tert-butylphenyl substituents at the 5-positiono ft he pyridinium units, as sterically bulky groups supports that tune aggregation in aqueous solution.…”