Encapsulation of Fluorescent Molecules by Functionalized Polymeric Nanocontainers:  Investigation by Confocal Fluorescence Imaging and Fluorescence Correlation Spectroscopy

Abstract

Nanocontainers (NCs) were prepared from amphiphilic triblock copolymers, having an average molecular weight of around 8000 g/mol, by using previously published preparation methods consisting of dispersing the polymer in an aqueous buffer solution containing molecules for encapsulation. A small molecular weight fluorophore, sulforhodamine B, as well as the fluorescent protein avidin labeled with Alexa 488 were encapsulated, and the resulting nanocontainers were characterized using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS). Nanocontainer size determination by FCS is very robust and compares well with results obtained from photon correlation spectroscopy: the measured diameters of the polymeric nanocontainers vary between 140 and 172 nm. Encapsulation of fluorescent molecules was determined by evaluating the molecular brightness of nanocontainers with an encapsulated fluorescently labeled protein (avidin-Alexa 488). Results indicate that the number of encapsulated avidin-Alexa 488 molecules corresponds well with the initial concentration of the fluorescently labeled protein and the encapsulated volume. A nanocontainer binding assay was developed using biotinylated fluorescently labeled nanocontainers. Binding of biotinylated nanocontainers to fluorescently labeled streptavidin was followed by fluorescence cross-correlation spectroscopy. The intrinsic dissociation constant, K(d), of labeled streptavidin to the ligand-modified nanocontainers is 1.7 +/- 0.4 x 10(-8) M, and about 1921 +/- 357 molecules of labeled streptavidin are bound to each nanocontainer.

Functionalization of Block Copolymer Vesicle Surfaces

“…The state of the art in terms of common conjugation chemistry that fulfills all or some of these requirements is summarized in Table 1. [26,29] Horseradish peroxidase [26] and an azidofunctionalized Candida antarctica Lipase B [29] Azide Alkyne 1,4-disubstituted 1,2,3-triazole PBD-b-PEG [30,31], PS-b-PAA [32] Mannose functional polyester dendron [30,31], dansyl probe [32], biotin [32], eGFP [32] Biotin Streptavidin Biotin-Streptavidin interaction PBD-b-PEG and PEG-b-PEE [33], PMOXA-b-PDMS-b-PMOXA [17,20,34,35] Superavidin-coated microspheres [33], antibody [36], polyguanylic acid [20,34], streptavidin-modified glass surface [17], fluorescently labeled avidin [33,35] …”

“…The first method that was used to attach ligands to the surface of preformed polymersomes was the biotin-streptavidin conjugation approach [33], which allows an arbitrary variation of ligands bearing biotin groups [17,20,34,35]. Ligands are bound indirectly by biotin-streptavidin-biotin interactions to the polymersome surface.…”

“…Ligands are bound indirectly by biotin-streptavidin-biotin interactions to the polymersome surface. The modification of block copolymers with biotin has been performed either by N,N'-dicyclohexylcarbodiimide/4-(dimethylamino)pyridine-activated esterification [17,20,34,35] or by preactivation of the terminal hydroxyl functionality by tresyl chloride [44] or 4-fluoro-3-nitrobenzoic acid [36,45] and subsequent reaction with biocytin [33,36]. Both the esterification and the tresylation method resulted in a good yield of biotin modified polymer, which is essential for streptavidin and thus ligand binding after self-assembly of the polymersome.…”

“…The binding of fluorescent dye-modified streptavidin to biotin modified polymersomes, with diameters between 140 and 172 nm, was demonstrated by fluorescence imaging [33], fluorescence correlation spectroscopy (FCS), as well as fluorescence cross-correlation spectroscopy (FCCS) [35]. A model system, investigated by FCS and FCCS and comprising rhodamine-green-biotin modified polymersomes and cyanin5-labeled streptavidin, was utilized in order to determine the number of binding sites and the dissociation constant, K D , of the receptor interaction between biotin-modified polymersomes and streptavidin [35]. An average number of 1921 ± 357 streptavidin-Cy5 per polymersome and an intrinsic K D of (1.7 ± 0.4)  10 −8 M were determined [35].…”

“…A model system, investigated by FCS and FCCS and comprising rhodamine-green-biotin modified polymersomes and cyanin5-labeled streptavidin, was utilized in order to determine the number of binding sites and the dissociation constant, K D , of the receptor interaction between biotin-modified polymersomes and streptavidin [35]. An average number of 1921 ± 357 streptavidin-Cy5 per polymersome and an intrinsic K D of (1.7 ± 0.4)  10 −8 M were determined [35]. The latter differs from the value of 10 −15 M given in the literature for the biotin-streptavidin interaction, which was explained by entropic and steric effects.…”