Supporting Information pH-degradable mannosylated nanogels for ...
Supporting Information
pH-degradable mannosylated nanogels for dendritic cell targeting Ruben De Coen,a Nane Vanparijs,a Martijn D. P. Risseeuw,a Lien Lybaert,a Benoit Louage, a Stefaan De Koker,b Vimal Kumar,b Johan Grooten,b Leeanne Taylor,c Neil Ayres,c Serge Van Calenbergh,a Lutz Nuhn,a* Bruno G. De Geesta* a
Department of Pharmaceutics, Ghent University, Ghent, Belgium. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. c Department of Chemistry, University of Cincinnati (OH), US. b
Figure S1. 19F-NMR spectrum of pentafluorophenyl acrylate.
Figure S2. 1H-NMR spectrum of pentafluorophenyl acrylate.
Figure S3. 1H-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S4. APT 13C-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S5. 2D Cosy spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S6. 2D HSQC spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S7. Synthesis of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide by reaction of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide with N-hydroxyethyl acrylamide in the presence of a silver trifluoromethanesulfunate catalyst.
Figure S8. 1H-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
Figure S9. APT 13C-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
Figure S10. 2D Cosy spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
RI [a.u.]
Figure S11. 2D HSQC spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
Figure S12. SEC traces of the p(TAManEAm72) macro CTA and the p(TAManEAm72-b-PFPA180) block copolymer.
Figure S13. 19F-NMR spectrum of a purified p(PFPAx) macro CTA, exemplified for P3 (sample code corresponds to the polymer composition listed in Table 1.
Figure S14.
1
H-NMR spectrum of polymerization reaction mixture Man3 (p(PFPA32-b-
TAManEAm14)) after 45 minutes of reaction.
Figure S15. 1H-NMR spectrum of all purified p(PFPAx-b-TAManEAmy) block copolymers. The sample codes (1), (2) and (3) correspond to the polymer compositions listed in Table 1: Man1, Man2 and Man3 respectively.
Figure S16. 1H-NMR spectrum of the purified p(PFPA65-b-TAGalEAm75) block copolymer.
Figure S17. (A) Chemical structure of biodegradable 2, 2’-bis(aminoethoxy)propane cross-linker, (B) chemical structure of non-degradable 2, 2’-(ethylenedioxy)bis(ethylamine) cross-linker.
200
Crosslinked Non-crosslinked
100
0 1
10
100
1000
size [nm] Figure S18. DLS size distributions of samples taken during nanogel synthesis of Man1D in DMSO. The solid line illustrates the self-assembled block copolymers before cross-linking, the dotted line depicts their disassembly after addition of chloroform.
Figure S19. 1H-NMR spectrum of Man1 after reaction of the PFP esters with 2-aminoethanol and de-acetylation of the mannosyl moieties with sodium methoxide/ methanol (note: No acetyl peaks around 2.2 – 1.8 ppm are present indicating full de-acetylation of the mannosyl moieties).
Figure S20. (A) UV spectrum before and after aminolysis of Man2 in DMSO. (B) UV-spectrum of Ellman’s assay Man2 and Man2D in 0.1 M TRIS HCl buffering solution.
Figure S21. Photographs of lectin-binding assay: the left side ((A) and (B)) depicts a ConA aggregated solution containing 20 µL of a 5 mg/mL Man1D solution in DPBS (+ CaCl2 and MgCl2). As depicted above, 20 µL of a 0.500 g/mL D-Mannose solution in DPBS was added to cuvet A and 20 µL of a 0.500 g/mL D-Galactose solution in DPBS was added to cuvet B. Upon addition of a huge excess of D-Mannose, the aggregates in cuvet A redissolved, while the addition of a similar amount of D-Galactose did not alter the precipitation at all.
fluorescence intensity [a.u.]
10 7 N-BL1 N-BL2 N-BL3 Control
10 6
10 5 1.00
0.33
0.10
nanogel concentration [mg/mL]
Figure S22. Fluorescent intensity in function of concentration of the different nanogels (n = 3, concentration is depicted in mg/mL).
Figure S23. Flow cytometry gating strategy which illustrates that the MRhi subset is exclusively expressed DCs (CD11c positive cells).
pH-degradable mannosylated nanogels for dendritic cell targeting Ruben De Coen,a Nane Vanparijs,a Martijn D. P. Risseeuw,a Lien Lybaert,a Benoit Louage, a Stefaan De Koker,b Vimal Kumar,b Johan Grooten,b Leeanne Taylor,c Neil Ayres,c Serge Van Calenbergh,a Lutz Nuhn,a* Bruno G. De Geesta* a
Department of Pharmaceutics, Ghent University, Ghent, Belgium. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. c Department of Chemistry, University of Cincinnati (OH), US. b
Figure S1. 19F-NMR spectrum of pentafluorophenyl acrylate.
Figure S2. 1H-NMR spectrum of pentafluorophenyl acrylate.
Figure S3. 1H-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S4. APT 13C-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S5. 2D Cosy spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S6. 2D HSQC spectrum of 2, 3, 4, 6-tetra-O-acetyl-α-D-mannosylethyl acrylamide.
Figure S7. Synthesis of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide by reaction of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide with N-hydroxyethyl acrylamide in the presence of a silver trifluoromethanesulfunate catalyst.
Figure S8. 1H-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
Figure S9. APT 13C-NMR spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
Figure S10. 2D Cosy spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
RI [a.u.]
Figure S11. 2D HSQC spectrum of 2, 3, 4, 6-tetra-O-acetyl-β-D-galactosylethyl acrylamide.
Figure S12. SEC traces of the p(TAManEAm72) macro CTA and the p(TAManEAm72-b-PFPA180) block copolymer.
Figure S13. 19F-NMR spectrum of a purified p(PFPAx) macro CTA, exemplified for P3 (sample code corresponds to the polymer composition listed in Table 1.
Figure S14.
1
H-NMR spectrum of polymerization reaction mixture Man3 (p(PFPA32-b-
TAManEAm14)) after 45 minutes of reaction.
Figure S15. 1H-NMR spectrum of all purified p(PFPAx-b-TAManEAmy) block copolymers. The sample codes (1), (2) and (3) correspond to the polymer compositions listed in Table 1: Man1, Man2 and Man3 respectively.
Figure S16. 1H-NMR spectrum of the purified p(PFPA65-b-TAGalEAm75) block copolymer.
Figure S17. (A) Chemical structure of biodegradable 2, 2’-bis(aminoethoxy)propane cross-linker, (B) chemical structure of non-degradable 2, 2’-(ethylenedioxy)bis(ethylamine) cross-linker.
200
Crosslinked Non-crosslinked
100
0 1
10
100
1000
size [nm] Figure S18. DLS size distributions of samples taken during nanogel synthesis of Man1D in DMSO. The solid line illustrates the self-assembled block copolymers before cross-linking, the dotted line depicts their disassembly after addition of chloroform.
Figure S19. 1H-NMR spectrum of Man1 after reaction of the PFP esters with 2-aminoethanol and de-acetylation of the mannosyl moieties with sodium methoxide/ methanol (note: No acetyl peaks around 2.2 – 1.8 ppm are present indicating full de-acetylation of the mannosyl moieties).
Figure S20. (A) UV spectrum before and after aminolysis of Man2 in DMSO. (B) UV-spectrum of Ellman’s assay Man2 and Man2D in 0.1 M TRIS HCl buffering solution.
Figure S21. Photographs of lectin-binding assay: the left side ((A) and (B)) depicts a ConA aggregated solution containing 20 µL of a 5 mg/mL Man1D solution in DPBS (+ CaCl2 and MgCl2). As depicted above, 20 µL of a 0.500 g/mL D-Mannose solution in DPBS was added to cuvet A and 20 µL of a 0.500 g/mL D-Galactose solution in DPBS was added to cuvet B. Upon addition of a huge excess of D-Mannose, the aggregates in cuvet A redissolved, while the addition of a similar amount of D-Galactose did not alter the precipitation at all.
fluorescence intensity [a.u.]
10 7 N-BL1 N-BL2 N-BL3 Control
10 6
10 5 1.00
0.33
0.10
nanogel concentration [mg/mL]
Figure S22. Fluorescent intensity in function of concentration of the different nanogels (n = 3, concentration is depicted in mg/mL).
Figure S23. Flow cytometry gating strategy which illustrates that the MRhi subset is exclusively expressed DCs (CD11c positive cells).
