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Development of a prelabeling approach for a targeted nanochelator

Supplemental Information

Prelabeling table summary

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Development of a prelabeling approach for a targeted nanochelator

Table 1. Summary of reaction conditions, percent ***M DOTA-NCS incorporated into PEI for the 33 reaction conditions.

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Development of a prelabeling approach for a targeted nanochelator

conditions / isotope / labeling / Conjugation conditions
Time / pH / Total Vol / Time / Temperature / Product / sd / n=
min. / (mL) / min. / (ºC) / %
1 / Ac / 5 / 8.5 / 0.500 / 60 / RT / 21.7, 13.9 / 2
2 / Ac / 10 / 8.5 / 0.500 / 60 / RT / 39.6, 19.4 / 2
3 / Ac / 20 / 8.5 / 0.500 / 60 / RT / 29.9, 30 / 2
4 / Ac / 30 / 8.5 / 0.500 / 60 / RT / 29.9. 28 / 2
5 / Ac / 45 / 8.5 / 0.500 / 60 / RT / 30.9, 30 / 2
6 / Ac / 60 / 8.5 / 0.500 / 60 / RT / 27.4, 12 / 2
7 / Ac / 45 / 8 / 0.500 / 105 / RT / 26.7 / 1
8 / Ac / 45 / 8.5 / 0.500 / 105 / RT / 30.8 / 1
9 / Ac / 45 / 9 / 0.500 / 105 / RT / 28.6 / 1
10 / Ac / 45 / 9.5 / 0.500 / 105 / RT / 22.7 / 1
11 / Ac / 45 / 10 / 0.500 / 105 / RT / 33 / 1
12 / Ac / 45 / 10.5 / 0.500 / 105 / RT / 10.9 / 1
13 / Ac / 45 / 8 / 0.500 / 180 / RT / 31.8 / 1
14 / Ac / 45 / 8.5 / 0.500 / 180 / RT / 26.8 / 1
15 / Ac / 45 / 9 / 0.500 / 180 / RT / 29.8 / 1
16 / Ac / 45 / 9.5 / 0.500 / 180 / RT / 31.6 / 1
17 / Ac / 45 / 10 / 0.500 / 180 / RT / 26 / 1
18 / Ac / 45 / 10.5 / 0.500 / 180 / RT / 27 / 1
19 / Cu / 45 / 8.0 / 0.130 / 60 / RT / 46.1 / 8.7 / 3
20 / Cu / 45 / 8.5 / 0.130 / 60 / RT / 44.1 / 4.4 / 3
21 / Cu / 45 / 9.0 / 0.130 / 60 / RT / 37.7 / 2.4 / 3
22 / Cu / 45 / 9.5 / 0.130 / 60 / RT / 43.1 / 5.0 / 3
23 / Pm / 45 / 8.5 / 0.160 / 15 / RT / 33.8 / 0.6 / 3
24 / Pm / 45 / 8.5 / 0.160 / 30 / RT / 35.6 / 1.8 / 3
25 / Pm / 45 / 8.5 / 0.160 / 60 / RT / 35.1 / 2.1 / 3
26 / Pm / 45 / 8.5 / 0.160 / 15 / 55ºC / 19.5 / 4.1 / 3
27 / Pm / 45 / 8.5 / 0.160 / 30 / 55ºC / 21.6 / 1.9 / 3
28 / Pm / 45 / 8.5 / 0.160 / 60 / 55ºC / 19.5 / 7.7 / 3
29 / Pm / 45 / 8.0 / 0.500 / 60 / RT / 29.0 / 1.7 / 3
30 / Pm / 45 / 8.5 / 0.500 / 60 / RT / 26.0 / 2.8 / 3
31 / Pm / 45 / 9.0 / 0.500 / 60 / RT / 25.0 / 0.2 / 3
32 / Pm / 45 / 9.5 / 0.500 / 60 / RT / 24.0 / 2 / 3
33 / Pm / 45 / 10.0 / 0.500 / 60 / RT / 23.0 / 2 / 3

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Development of a prelabeling approach for a targeted nanochelator

Detailed Synthesis of TrastuzumabPEI derivatives

Synthesis of PEI-Oregon Green® 488 (PEI-OG)

A 1.1 mL sample of (3.1x10-4M, 3.41x10-7 moles) PEI was added to a PD-10 column and the buffer was changed to 1 M sodium carbonate buffer (pH=8.9), then concentrated to 1 mL by centrifugation and added to a 2 mL reaction vial. In a separate vial 0.00153 g (3.008 x10-6 moles) of Oregon Green® 488 carboxylic acid succinimidyl ester was dissolved in 0.1 mL DMF and transferred into the reaction vial. The reaction was mixed with a labquake mixer overnight, purified with a PD-10 column with fractions 3-6 pooled and concentrated by centrifugation to 1 mL. A 0.05 mL portion was diluted to 4 mL and UV absorbance at 496 nm was used to determine a ratio of dye molecules to PEI. Sample concentration 2.84x10-4M.

SPDP PEI OG

A working solution of SPDP was prepared using 0.002 grams SPDP and the sample was dissolved in 0.4 mL of DMF. The PEI-OG sample was converted to the following buffer to PBS/0.005 M EDTA (pH=7.4). To a vial was added 0.002 mL portion of a (2.84x10-4 M) solution of PEI-OG (5.7 x 10-8 moles) followed by the addition of 0.0072 mL of the SPDP solution and the sample was incubated at rt for 30 min. A PD-10 desalting column was used to purify the reaction and fractions 3-5 were pooled and spun to ~1.2 mL with a 10 KDaCentricon® filter device. To quantify the amount of SPDP on the polymer a working solution of DTT was prepared with a concentration of 15 mg/mL in PBS/EDTA. Then ~ 0.1 mL of the SPDP modified PEI was to a vial and 0.01 mL of the DTT solution was added. The absorbance at 343 and 496 nm was measured before and 15 min after the addition of DTT. The molar ratio of SPDP to Oregon Green® 488 and PEI was determined.[[i]]

Trastuzumab PEIAC-OG

A 1 mL sample of a 4.75x10-5 M PEI-OG-SPDP (4.75x10-8 moles), 0.12523 g of bromoacetic acid (8.95x10-4 moles) and 2 mL of 1 M sodium bicarbonate were added to a vial and the reaction was incubated for 1 hour. The sample was concentrated to 0.5 mL with a 10 KDaCentricon® filter device and purified with a 10DG size exclusion column and concentrated to 1 mL. A DTT solution was prepared as described above with 0.5 mL of PBS EDTA. To the SPDP PEIAC- OG was added 0.1 mL of the DTT solution and the sample was incubated at rt for 1hour. The reaction was purified with a 10 DG size exclusion column followed by concentration to 1 mL with a size exclusion device. A 3.5 mL solution of Trastuzumab (4.4 x10-9moles) was spun to 0.7 mL with a 10 KDaCentricon® filter device. To the filter device was added 0.2 mL of the 4.75x10-5 M solution of the thiol containing PEIAC-OG (9.5 x10-9 moles), 0.3 mL of PBS EDTA and the sample was concentrated to 0.2 mL. The sample was purified with a Bio Gel p-100 with a bed volume of 1 mL. The ratio of PEI, Oregon Green® 488 and Trastuzumab was determined by UV-Vis.

Trastuzumab Pei ogspdp Ab

The previous procedure to synthesis Trastuzumab–PEI Oregon Green® 488 Acid was followed to synthesis Trastuzumab–PEI Oregon Green® 488 with the exception that the step to add bromoacetic acid was not used. For the reaction 7.13x10-9 moles of thiol modified PEI-Oregon Green® 488 and 2.2x10-9moles of Trastuzumab-Maleimide were used.

Synthetic scheme utilizing Sulfo-SMCC

Figure 7. The synthetic scheme used to make anTrastuzumab-PEI-(CH2COO-), based on a Sulfo-SMCC crosslinking method. Ratio PEI/OG/Ab A. 0.2/1.1/1, B. 0.4/2.1/1.

[i]