Editing Synthesis Peptide-PEI (section)

== Synthesis of P123-PEI-R13  ==

R13:RGDC-Tat (49–57) 

pluronic P123: BASF Corporation (Mount Olive, NJ) 

=== Synthesis of P123-PEI  ===

<br>Dry P123 (0.6 mmol)&nbsp; by coevaporation with anhydrous toluene in a vacuum at 50°C<br>

Dissolve&nbsp;Dried P123 in toluene/dichloromethane (3:1, 40 mL) and treat with bis-(trichloromethyl)-carbonate (0.356 g, 1.2 mmol) overnight. 

Evaporate the solution to dryness under a vacuum.

Dissolve the residue in toluene/dechloromethane (2:1, 30 mL) and treat with solid N-hydroxysuccinimide (0.240 g, 2.0 mmol), followed by anhydrous triethylamine (0.28 mL, 2.0 mmol). 

After 4 hours of stirring, filter the solution&nbsp; and evaporate to dryness. 

Purify the product on a silica gel column using stepwise elution with trichloromethane methanol (10%–15%, v/v) mixtures.

Dissolve LMW PEI (0.20 g, 0.10 mmol) in 10 mL anhydrous dichloromethane as Solution A

Dissolve the predetermined amount of activated P123 (0.01 mmol)&nbsp; in 10 mL anhydrous ethanol as Solution B.&nbsp;

Add Solution A and Solution B&nbsp; slowly to a base solution of 10 mL anhydrous dichloromethane with constant stirring. 

Let the reaction continue for overnight at room temperature.&nbsp;

Dialyse the conjugate&nbsp; using Spectra/Por 1 dialysis membrane (Spectrum, CA) against distilled water at 4°C for 2 days<br>Lyophilize. 

=== Conjugation of P123-PEI with R13  ===

<br> 

Add SMCC solution (3.33 mg/mL in dimethyl sulfoxide) dropwise to P123-PEI solution (9 mg/mL in 0.1M PBS) at a molar ratio of 10:1, 5:1 and 2:1 

Incubate the reaction mixture&nbsp; for 30 minutes at room temperature with gentle shaking. 

<br>P123-PEI was conjugated with R13 using SMCC as crosslinker. After the appropriate amount of SMCC solution (3.33 mg/mL in dimethyl sulfoxide) was added dropwise to<br>P123-PEI solution (9 mg/mL in 0.1M PBS) at a molar ratio of 10:1, 5:1, and 2:1, the reaction mixture was incubated for 30 minutes at room temperature with gentle shaking. The excess nonreacted crosslinker was removed by gel chromatography (Sephadex G-25; Pharmacia, Milton Keynes, UK). Thus, N-hydroxysuccinimide (NHS) eaters of SMCC have reacted with primary amines of P123-PEI to form amide bonds and produce the maleimide-activated P123-PEI. Then, R13 (10 mg/mL in 0.1M PBS) was reacted with the maleimide-activated P123-PEI at a molar ratio of 10:1, 5:1, and 2:1. The reaction mixture was stirred in the dark overnight at 4°C and then lyophilized after the nonreacted&nbsp; R13 was removed by ultrafiltration with Amicon Ultracentrifugal filter devices (Millipore, Billerica, MA). Then, three types of cationic polymer were obtained and named P123-PEI-R13<br>