Micelles were formed by film-hydration. To prepare
micelles with
10% functional groups on the surface, ben-PCL7-PEG2000 and ben-PCL7-PEG2000-DBCO were dissolved
in dichloromethane (DCM, Sigma-Aldrich, Saint Louis, MS, USA) and
mixed in a 9:1 weight ratio in a 5 mL glass vial (VWR, Radnor, PA,
USA). To this mixture, the hydrophobic photosensitizer mTHPC (meta-tetra(hydroxyphenyl) chlorine, Biolitec AG, Jena, Germany)
in tetrahydrofuran (THF, Sigma-Aldrich, Saint Louis, MS, USA) was
added at a 5% w/w mTHPC/polymer ratio. DCM and THF were evaporated
under vacuum. The obtained polymer film was hydrated to a 20 mg/mL
solution in PBS for 2 h at RT, gently heated to 60 °C, and filtered
through a 0.22 μm filter. To obtain micelles with 5% surface
coverage with VHH 7D12[Fluo], the DBCO-micelles were reacted
with 7D12[Fluo]-N3 in a 2:1 molar DBCO:N3 ratio. To ensure efficient click reaction kinetics, we kept
the N3 and DBCO concentration >100 μM. Reactions
were allowed to proceed overnight in the dark at 4 °C with continuous
shaking at 450 rpm. The final product was analyzed by SDS-PAGE under
reducing conditions (Fluorescein signal and CBB staining). Nontargeted
mTHPC-ben-PCL7-PEG2000 micelles were prepared
according to the same procedure, but without incubation with 7D12[Fluo]-N3.
micelles with
10% functional groups on the surface, ben-PCL7-PEG2000 and ben-PCL7-PEG2000-DBCO were dissolved
in dichloromethane (DCM, Sigma-Aldrich, Saint Louis, MS, USA) and
mixed in a 9:1 weight ratio in a 5 mL glass vial (VWR, Radnor, PA,
USA). To this mixture, the hydrophobic photosensitizer mTHPC (meta-tetra(hydroxyphenyl) chlorine, Biolitec AG, Jena, Germany)
in tetrahydrofuran (THF, Sigma-Aldrich, Saint Louis, MS, USA) was
added at a 5% w/w mTHPC/polymer ratio. DCM and THF were evaporated
under vacuum. The obtained polymer film was hydrated to a 20 mg/mL
solution in PBS for 2 h at RT, gently heated to 60 °C, and filtered
through a 0.22 μm filter. To obtain micelles with 5% surface
coverage with VHH 7D12[Fluo], the DBCO-micelles were reacted
with 7D12[Fluo]-N3 in a 2:1 molar DBCO:N3 ratio. To ensure efficient click reaction kinetics, we kept
the N3 and DBCO concentration >100 μM. Reactions
were allowed to proceed overnight in the dark at 4 °C with continuous
shaking at 450 rpm. The final product was analyzed by SDS-PAGE under
reducing conditions (Fluorescein signal and CBB staining). Nontargeted
mTHPC-ben-PCL7-PEG2000 micelles were prepared
according to the same procedure, but without incubation with 7D12[Fluo]-N3.