Glycodelivery Systems

The interaction of either simple or more complex carbohydrates with the receptors on the cell surface is appealing to introduce targeting specificity or favour the cellular uptake of general delivery systems.^{[102, 125]} Glycosylation is, however, most commonly used to improve the solubility of macrostructures,

Ca²⁺

NH₂ O

NH₂ O HN O

OH O O

O HO

HO O OH

O H

NH₂ O N

N H

different saccharides that have been used, trehalose, a disaccharide composed of two D-glucose units, has received much attention. Indeed, it was shown to inhibit the aggregation and folding of the polyglutamine-containing protein responsible for the formation of insoluble amyloid-like fibrils that lead to Huntington’s disease.^[129-131] Since then, it has been used to inhibit protein^[132-134] and nanoparticle aggregation,^[135] and has also been introduced on the sidechain of polymers, such as polymer 76 developed by Maynard and coworkers, to improve the solubility when conjugating it to proteins (Figure 35).^[136]

Figure 35. Trehalose used to a) inhibit protein and nanoparticle aggregation and b) functionalize the sidechain of polymers, such as in 76. Figure adapted from reference.^[135-136]

Glycosylation is also introduced to improve cellular uptake of CPPs. In a first example Lavielle et al. showed that the introduction of galactose units in CPP sequence 77 did not improve cellular uptake unless an alanine spacer was introduced giving 78 and suggesting the importance of spatial organization in cellular uptake (Figure 36).^[137-138] On the other hand, Deming and coworkers demonstrated that the alkylation of the sulfonium CPPs 7 with hydrophobic residue 79 gave lower activity and higher toxicity when compared to glycosylation with 80-81, that is galactose and glucose units, respectively.^[36] The

76

nS S S

O O

O

O O OH OH OH HO

OH OH

a) b)

improved cellular uptake obtained when monosaccharides are introduced in addition to the presence of units promoting counterion-mediated entry, suggests that binding to specific carbohydrate receptors can enhance cell translocation.

Figure 36. Glycosylated CPPs: a) introduction of galactose units without, 77, and with, 78 a spacer; b) alkylation of sulfonium polymer 7 leading to 79-81.

Figure adapted from reference.[137-138, 36]

Although there are not many examples of glycosylated CPPs, carbohydrates can be more frequently found as scaffolds to guanidinium-rich transporters. In this field, Chung and coworkers have reported delivery systems based on inositol (82),^[139] sorbitol (83),^[140] lactose (84)^[141] and trehalose (85),^[131] as shown in Figure 37. In all cases, the hydroxy groups of the carbohydrate are functionalized with guanidinium groups to obtain non-peptidic CPP-mimics which showed excellent uptake in different cell lines as well as in vivo. The role of the disaccharides as the CPP backbone enables to achieve a higher degree of functionalization and lack of cytotoxicity due to the biodegradability of the scaffold compared to common peptidic CPPs.[30, 131, 139-141]

: O

Figure 37. Carbohydrates used as scaffolds for non-peptidic CPPs 82-85. Figure adapted from reference.[139-141, 131]

Another example of carbohydrates functionalized with charges comes from the group of Tor. They were able to demonstrate that the conversion of the amine groups of the natural glycosidic antibiotics tobramycin 86 and neomycin B 87 into their guanidinium analogues, 88 and 89 respectively, enabled them to reach higher RNA affinity translated into higher anti-HIV activity (Figure 38).^{[30, 142]}

In addition, they reported a higher cellular uptake measured by flow cytometry of 89 compared to nona-arginine (R₉). This result indicated that the presence of the glycosidic backbone could help the translocation through the membrane with contributions coming from possible binding to carbohydrate receptors hypothesized in a later report.^[143]

O

Figure 38. a) Structure of antibiotics tobramycin 86 and neomycin B 87 and their guanidinium analogues 88 and 89, respectively. Count of HeLa cells after treatment with R₉ (filled) and 89 (empty). Figure adapted from reference.^[30]

As far as polymers are concerned, glycosylation is adopted to enable cellular uptake in addition to cell-specificity.[120, 125] By introducing different monosaccharides, selectivity towards one cell type over another can be achieved.

For example, when a polymeric-micelle was decorated with β-D-galactose units, melanoma cells could be targeted due to the binding to the overexpressed galectin-3 receptor.^[144] On the other hand, the functionalization of the side chain with α-D-mannose enables macrophage targeting.^[145] In a study conducted by Stenzel and coworkers in 2015,^[146] alkyne-containing polymer 90 was functionalized through CuAAC with azide-containing monosaccharides 91-92 to obtain glycopolymer 93 (Figure 39). By increasing the amount of β-D-fucose 92,

O

Figure 39. CuAAC between alkyne polymer 90 and azides 91-92, to obtain glycopolymer 93, and cellular uptake in cancer cell lines (AsPC-1, A451, OVCAR-3) versus healthy CHO cells revealing no uptake with 100% 91 and cancer cell-selectivity with 100% 92. Figure adapted from reference.^[146]

Moreover, Schubert et al. synthesized a small library of four different glycopolymers, 94-97, bearing α-D-glucose, α-D-mannose, α-D-galactose and β-D-fructose units, respectively (Figure 40).^[147] Glycosylation resulted in better solubility for all systems compared to the unsubstituted polymer, as well as lack of cytotoxicity in healthy cells. Moreover, enhanced uptake for 97, suggested binding to GLUT-5, a fructose-receptor overexpressed in human breast cancer cells and used in this study.^[147] Overall, these examples indirectly suggest that polymers decorated with carbohydrates can bind to specific-receptors on the cell membrane, triggering their cell entry, most likely by endocytosis, as proved in many cases.^[144-147]

O O

Figure 40. Structure of glycosylated polymers 94-97 and their cell viability indicating lack of toxicity in healthy L₉₂₉ cells. Figure adapted from reference.^[147]

Finally, there are also examples of cationic glycopolymers that are used for gene delivery applications to, once again, improve the solubility of the polymer-DNA/RNA conjugates and enable cell-selectivity.^[148-153] Narain and coworkers demonstrated the effectiveness of cationic glycopolymers in several examples, in terms of biocompatibility, lack of aggregation in physiological conditions and improved transfection efficiency of the DNA they were carrying.^[150-152]

The most common strategy to obtain cationic glycopolymers is to synthesize a block copolymer containing the carbohydrate in one block and an ammonium cation in the other.^[148-152] In the case of polymer 98, reported in 2016 by the Cao group, the positive charge is introduced using lysine residues, while the presence of the D-galactose units decreased the cytotoxicity of the cationic macrostructure

HN O HO

O O NH

R HO O

HO OH OH

S

HO O

HO S

HO OH

O HO OH S

OH OH

HO O

OH

OH OH

S

n

94 : R =

95 : R =

96 : R =

97 : R =

standard polyethylenimine, suggesting that a carbohydrate-mediated entry could enhance cellular uptake. Moreover, the polyplexes were found to enter through caveolae-mediated endocytosis with significant lysosomal escape over time due to the presence of the positive charge.^[148]

Figure 41. Structure of cationic glycopolymer 98 and its cellular uptake in H1299 cells followed by fluorescence microscopy showing lysosomal escape over time. Figure adapted from reference.^[148]

In conclusion, glycosylation is a common strategy to improve solubility, impart targeting functions and enhance or promote cellular entry. Indeed the presence of carbohydrate-specific receptors, such as GLUT transporters and lectins, on the cell surface can enable the recognition and binding of different glycoconjugates. The introduction, therefore, of saccharides to general delivery systems is to be considered as a strategy to allow or improve cellular uptake.