Monosaccharide coating modulate the intracellular trafficking of gold nanoparticles in dendritic cells

Abstract

Dendritic cells (DCs) have emerged as a promising target for drug delivery and immune modulation due to their pivotal role in initiating the adaptive immune response. Gold nanoparticles (AuNPs) have garnered interest as a platform for targeted drug delivery due to their biocompatibility, low toxicity and precise control over size, morphology and surface functionalization. Our investigation aimed to elucidate the intracellular uptake and trafficking of AuNPs coated with different combinations of monosaccharides (mannose, galactose, and fucose) in DCs. We used 30 unique polymer-tethered monosaccharide combinations to coat 16 nm diameter spherical gold nanoparticles and investigated their effect on DCs phenotype, uptake, and intracellular trafficking. DCs internalized AuNPs coated with 100 % fucose, 100 % mannose, 90 % mannose +10 % galactose, and 80 % mannose +20 % galactose with highest efficiency. Flow cytometry analysis indicated that 100 % fucose-coated AuNPs showed increased lysosomal and endosomal contents compared to other conditions and uncoated AuNPs. Imaging flow cytometry further demonstrated that 100 % fucose-coated AuNPs had enhanced co-localization with lysosomes, while 100 % mannose-coated AuNPs exhibited higher co-localization with endosomes. Furthermore, our data showed that the uptake of carbohydrate-coated AuNPs predominantly occurred through receptor-mediated endocytosis, as evidenced by a marked reduction of uptake upon treatment of DCs with methyl-β-cyclodextrins, known to disrupt receptor-mediated endocytosis. These findings highlight the utility of carbohydrate coatings to enable more targeted delivery of nanoparticles and their payload to distinct intracellular compartments in immune cells with potential applications in drug delivery and immunotherapy.

Keywords: Carbohydrates; Dendritic cells; Gold nanoparticles; Immune modulation; Monosaccharides; Surface coating; T cells.

Graphical abstract

Fig. 1

Schematic of the functionalization of gold nanoparticles with glycan-functionalized fluorescent polymers. A) co-polymerization of N-hydroxyethyl acrylamine and hostasol methacrylate using pentafluoropenyl (PFP) RAFT agent and the subsequent substitution of the PFP end group with an amino monosaccharide (galactosamine, mannosamine and fucosamine) B) Anchoring of glycopolymers on pre-formed 16 nm gold nanoparticles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Flowcytometry phenotypic analysis and gating strategy of costimulatory markers CD86, MHC1 and MHC2 for sAuNP conditioned DCs. Data show no change in the costimulatory markers suggesting no stimulatory effects of the sAuNP on DCs. Data shown is representative of 3 donors.

Fig. 3

Flowcytometry data show uptake studies for sAuNP conditioned DCs. Different combinations have shown to promote uptake more than others where increase uptake is seen in uncoated AuNPs, 100%mannose, 90%mannose+10%galactose, 80%mannose+20%galactose, 70%mannose+30%galactose, 60%mannose+40%galactose, 100%fucose, 90%fucose+10%galactose. AuNP uptake by DCs is subtantially abroagted at 4 °C (incubated on ice). Data show are MFI + -SD of >3 separate donors. Data shown are a mean ± SD of three independent donors where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 4

Uptake data obtained after the depletion of cholesterol on DC membrane using Methyl-β-cyclodextrins. A) There is a significant decrease in uptake of both coated and uncoated particles. This suggests that an active endocytosis is involved in the uptake of these particles however passive uptake is still involved in particle uptake B) DCs viability using Live/dead flowcytometry stain suggest that the treatment with 10 mM of Methyl-β-cyclodextrins did not affect DC viability. Data shown are a mean ± SD of three independent donors where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 5

Flowcytometric analysis for uptake, lysosomal and endosomal contents for sAuNP conditioned DCs after 24 h incubation. High lysosomal content is seen in 100%fucose, 80%fucose+20%galactose and 100%mannose. These conditions also expressed high endosomal content except for 100%mannose. Data shown as mean ± SD for 3 donors.

Fig. 6

Images representing sAuNPs uptake Lysosomes (orange), endosomes (pink) and nucleus (purple) in DCs. Data confirms uptake by the conditions where most uptakes are seen in 100 % fucose. It is also where most lysosomal co-localization can be noted followed by 80%fuc+20%gal. These conditions show reduced endosomal co-localization suggesting rapid particle delivery to lysosomal contents. On the other hand. 100%Mannose show increased endosomal co-localization suggested delayed delivery of the particles to endosomes. Uncoated AuNPs show increased uptake but reduced co-localization for both endosomal and lysosomal contents. Data shown are representative images of 60x of 3 donors with 1000 images analyzed per donor. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Illustrating Gold particle co-localization studies in lysosomal and endosomal contents in conditioned DCs. Data show that 100 % fucose, 80%fucose+20%galactose and 70%gal+30%man have significant increase in sAuNPs/lysosomal co-localization compared to uncoated AuNPs. These conditions show no significant increase in the sAuNPs/endosomal co-localization. On the other hand, 100%mannose show a significant increase in sAuNPs/endosomal co-localization while no significant increase in the sAuNPs/lysosomal co-localization compared to uncoated AuNPs. Data shown are mean ± SD of three independent donors where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 8

Depletion of cholesterol on DC membrane using Methyl-β-cyclodextrins. This enables the inhibition of the formation of lipid rafts and there for disabling active endocytosis but not passive endocytosis. This allows the understating of the means in which particles are internalized differentiating between active and passive endocytosis.

Fig. 9

Illustrates the mechanism of sAuNP uptake by dendritic cells (DCs) through active endocytosis. Upon internalization, the particles are encapsulated by a protein coat, resulting in invagination of the plasma membrane and the formation of an early endosome. Subsequently, the early endosome matures into a late endosome, which ultimately fuses with a lysosome. Within the lysosome, the enzymatic activity degrades the contents of sAuNP, which then proceed for antigen presentation on dendritic cells.