Cuarto de motores.

Labeling of A12-N3 and A12-biotin with Ru2+

A12-N3 and A12-biotin were prepared as described in Section 4.7. After removal of

residual peptide and thiol-reagents using size-exclusion chromatography in PBS, for both VHHs 2 mL was obtained with a concentration of 0.13 mg/mL as determined by BCA analysis. The pH of the VHH solution (15 nmol protein, 150 nmol NH2-groups) was

adjusted to 10.5 by the addition of 0.1 M aqueous NaOH. Hereupon Bis(2,2′-bipyridine)- (5-isothiocyanato-phenanthroline)ruthenium bis(hexafluorophosphate) (200 μL, 1 mg/mL in MQ containing 10% DMSO, 210 nmol) was added. The mixture was shaken for 3 hours, whereupon residual reagent was removed using a 10 kDa membrane Amicon Ultra centrifugal filter. After extensive washing against PBS, the Ru-labeled VHH was taken up in 1 mL of PBS.

Functionalization of BCN-polymersomes with A12-N3

Ru-labeled A12-N3 or A12-biotin (286 μL, 0.13 mg/mL, 2 nmol) was added to 250 μL of

polymersome solution (containing 2.1 nmol of BCN-functionalized polymer). The mixture was shaken at 4 ºC overnight and subsequently washed extensively with PBS using centrifugation (5 min, 3000 rpm) over a 100 nm pore size filter (Amicon UltraFree MC, polycarbonate membrane, diameter of 19 mm).

ICPMS analysis of A12-polymersome conjugate

Ru-labeled A12-N3, A12-biotin and the A12-N3 or A12-biotin functionalized polymersome

suspensions were lyophilized and subsequently destructed for 3 h in nitric acid (65%, 0.50 mL) at 80 ºC. After cooling the samples to room temperature, the samples were filtered over cotton, which was subsequently washed with MilliQ. An internal standard of InCl3

(1.50 mL of 0.97 mg/L) was added. The total volume of each sample was adjusted to 5.00 mL with MilliQ. The functionalization percentages were obtained by comparing the results obtained for A12-N3 to the results obtained for the A12-functionalized polymersomes. 5.8.5 ELISA assay for affinity testing

Binding assay of A12-polymersomes for Plexin D1 peptide

A 96-wells plate was coated with 100 μL of a 10 μg/mL neutravidin solution in 50 mM Na2CO3 buffer per well. The plate was coated overnight at 4 ºC, followed by washing with

PBS at room temperature (3 × 5 min incubation). Subsequently, a 50 μL solution of 0.6 μg/mL biotin-functionalized Plexin D1 peptide in PBS was added and allowed to bind to neutravidin for 1 hour at room temperature. Alternatively, only PBS was added to the wells. The unbound peptide was removed by washing with PBS at room temperature (3 × 5 min incubation). The plate was subsequently blocked using 5% marvel powder in PBS for 1 h at room temperature, after which the plate was washed with PBS at room

152 Dibenzoazacyclooctynes: Synthesis and Bioconjugation

temperature (3 × 5 min incubation). Three equivalents of A12-N3 (1, 72 μL, 1.5 nmol),

A12-polymersomes (7, 160 μL, 1.5 nmol), and BCN-polymersomes (6) that were reacted with A12-NH2 (2) (160 μL, 1.5 nmol) were then diluted five times with PBS and

subsequently added to the wells (100 μL/well). The wells were incubated for 2 hours at room temperature. After extensive washing, A12 was visualized using mouse anti-VSV in PBS (1:2000, 100 μL/well) which was incubated for 1 hour at room temperature. Unbound antibody was removed by washing with PBS at room temperature (3 × 5 min incubation) followed by HRP conjugated rabbit anti-mouse in PBS (1:5000, 1 mL/well) incubated for 1 hour at room temperature, after which the plate was again washed with PBS at room temperature (3 × 5 min incubation). The staining was performed by addition of 100 μL TMB solution (1 μL H2O2 and 10 μL of 10 mg/mL TMB in DMSO in 1 mL TMB buffer) per

well. When the solution in the wells turned yellow, the reaction was stopped by the addition of 100 μL of 2M H2SO4. The extinction was determined at λ = 450 nm. The

reaction was performed in duplicate and the average value was taken. 5.8.6 Biodistribution studies

In vivo biodistribution studies

The in vivo behavior of the A12-111In-polymersomes and Lys-111In-polymersomes was evaluated in 10 nude BALB/c mice (6-8 weeks, 20-25 grams) bearing s.c. OVCAR-3 tumors. The mice were divided in two groups: 5 animals were injected in their tail vein with 0.4 MBq (200 µL) A12-labeled polymersome solution, the other 5 animals received 0.4 MBq (200 µL) of Lys-labeled polymersomes. All animals were sacrificed by CO2/O2

asphyxation 24 h post-injection. Then, blood, muscle, tumor, lung, spleen, kidney, liver, stomach and intestine were collected, weighed and counted in a well-type γ-counter (Wizard, Perkin-Elmer, Waltham, MA). The percentage injected dose per gram (%ID/g) was calculated for each tissue. The animal experiments were approved by the local animal welfare committee and performed according to national regulations.

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Efficient enantioselective synthesis of organic compounds is of high importance for the fine chemical and pharmaceutical industries. One of the most efficient manners to induce enantioselectivity is by using the chiral environment of an enzyme. In this chapter, a non-selective metal catalyst complex was positioned in close proximity of the active site of the

lipase CalB via Strain-Promoted Azide Alkyne

Cycloaddition (SPAAC). Preliminary investigations were performed to assess if both catalytic systems (metal and enzyme) still showed activity.

6