Editing Synthesis Peptide-PEI

== Peptides<br>  ==

{| width="711" cellspacing="1" cellpadding="1" border="1"
|-
| width="200" | Name<br> 
| Seq<br> 
| Form<br> 
| Transfection Efficiency<br>
|-
| width="200" | Tat<sup>48–60</sup> 
| GRKKRRQRRRPPQ 
| <br> 
| <br>
|-
| width="200" | Tat<sup>49-60</sup><br> 
| RKKRRQRRRPPQ<br> 
| <br> 
| <br>
|-
| width="200" | Tat<sup>47–57</sup><br> 
| RKKRRQRRR-GGG<br> 
| <br> 
| <br>
|-
| width="200" | Tat2<br> 
| C(YGRKKRRQRRRG)2<br> 
| <br> 
| <br>
|-
| width="200" | Penetratin (Antp 43–58 ) 
| RQIKIWFQNRRMKWKK 
| <br> 
| <br>
|-
| width="200" | Transportan 
| GWTLNSAGYLLGKINLKALAALAKKIL 
| <br> 
| <br>
|-
| width="200" | TP10 
| AGYLLGKINLKALAALAKKIL 
| <br> 
| <br>
|-
| width="200" | Oligoarginine (R8) 
| RRRRRRRR 
| <br> 
| <br>
|-
| width="200" | MAP 
| KLALKLALKALKAALKLA 
| <br> 
| <br>
|-
| width="200" | MPG 
| GALFLGFLGAAGSTMGAWSQPKKKRKV 
| <br> 
| <br>
|-
| width="200" | MPGa 
| GALFLAFLAAALSLMGLWSQPKKKRKV 
| <br> 
| <br>
|-
| width="200" | CADY<br> 
| GLWRALWRLLRSLWRLLWRA-cya<br> 
| <br> 
| <br>
|-
| width="200" | LK15<br> 
| RKKRRQRRRGGGKLLKLLLKLLLKLLK-CONH<sub>2</sub><br> 
| <br> 
| <br>
|-
| width="200" | KALA <br> 
| WEAKLAKALAKALAKHLAKALAKALKACEA<br> 
| <br> 
| <br>
|-
| width="200" | Pep1 (Chariot)<br> 
| KETWWETWWTEWSQPKKKRKV<br> 
| <br> 
| <br>
|-
| width="200" | Tat<br> 
| RKKRRQRRRPPQ<br> 
| <br> 
| 4X10<sup>6</sup><br>
|-
| width="200" | Tat-CTHD<br> 
| RKKRRQRRRPPQC<br> 
| C-Terminal disulfide homodimer<br> 
| 1.2X10<sup>7</sup><br>
|-
| width="200" | Tat-NTHD<br> 
| CRKKRRQRRRPPQ<br> 
| N-Terminal disulfide homodimer<br> 
| 1.0X10<sup>7</sup><br>
|-
| width="200" | Cyclic-Tat<br> 
| CRKKRRQRRRPPQC<br> 
| Disulfide bond between C-terminal and N-terminal<br> 
| 3.8X10<sup>7</sup><br>
|}

== Polycondensation of cyclic Tat (cTatc--&gt;cGRKKRRQRRRGc)<br>  ==

20.8 mg of cTATc was dissolved in 145 uL phosphate buffered saline (PBS) and 70 uL DMSO. The reaction was carried out at room temperature for 96 h<br> 

<br> 

== Synthesis of PEI-Peptide<br>  ==

'''SPDP''': Succinimidyl 3-(2-pyridyl-dithio) propionate, NHS-Pyridyldithiol Crosslinkers, Water-insoluble (dissolve first in DMF or DMSO), SPDP crosslinker is membrane permeable, Pierce $133/50mg<br> 

[[Image:PEI SPDP Peptide.png]]<br> 

<br> 

6.6 mg of PEI (2kD) in 0.5 mL phosphate buffer (pH 8.0)&nbsp;<br> 

Add&nbsp; 0.5 mL of DMSO solution containing 3.8 mg of SPDP<br> 

Stir overnight at room temperature.<br> 

Dialyse the reaction mixture against water at 4 C overnight using a Spectra/Por dialysis membrane (MWCO 1000).<br> 

Freeze-dry the solution--&gt; 2.2 mg of PEI-SPDP<br> 

Add 2.0mg peptide to&nbsp; 0.9 mg PEI-SPDP&nbsp; in 1.0 mL of PBS. <br> 

React overnight at room temperature.<br> 

Dialyse the reaction mixture overnight using a Spectra/Por membrane (MWCO 1000).<br> 

Freeze-dry yielding a product.<br> 

Before use, dissolve PEI-Peptide conjugatein pure water.<br> 

<br> 

'''SMCC:''' NHS-Maleimide Crosslinkers, Non-cleavable Water-insoluble (dissolve first in DMF or DMSO), $90/50 mg<br> [[Image:Smcc conjugation.jpeg]]<br> 

'''Sulfo-SMCC'''<br>

[[Image:Sulfo-SMCC-conjugation.jpg]]<br>

<br> 

== 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> 

== mTat-PEI  ==

mTat: C-HHHHH-RKKRRQRRRR-HHHHH-C 

prepare mTat at 1 mM. 

Add DNA to mTat in 5% glucose solution, pH 7 

votex for 5 sec 

incubate at RT for 30 min, with lid open (expose to air 

shake for 90 min, open cap intermittently to allow air replenishment. 

<br> 

== Synthesis of PEG-g-PEI  ==

Dissolve 1 gram of PEI (MW 25,000) (40 μ mol)&nbsp;&nbsp;in 25 mL of a sodium phosphate buffer (pH 8.0).<br>Add 500 ug micrograms of PEG-SSA (100 μ mol, MW&nbsp;5,000) dissolved in 5 mL of dehydrated DMSO &nbsp;to the PEI solution in a drop-wise fashion. 

The<br>reaction was left at room temperature for 4 h. The<br>stoichiometry of the reaction was 1:2.5 (PEI:PEG-SSA).<br>The reaction was stopped by the addition of 60 mL of<br>deionized water followed by dialysis (MWCO 25,000).<br>The<br>conjugate was then freeze-dried and stored at − 20<br>o<br>C until use. The PEG-g-PEI was 1 analyzed by 1 H-NMR<br>using D 2 O as a solvent. The H-NMR spectra were<br>taken using a Brucker DRX 300 spectrometer operating<br>at 300 MHz. The chemical shift ( δ ) was measured in<br>ppm using 3-(trimethyl-silyl) propionic-2,2,3,3-d 4 acid<br>(TSP) sodium salt as an internal reference.<br>Formulation of KALA/PEG-g-PEI/DNA and KALA/PEI/<br>DNA Complexes<br>Ten milliliters of PEI (5 μ g/mL) and DNA (100 μ g/<br>mL) were separately prepared in an HBS (10 mM<br>HEPES, 30 mM NaCl, 1.5 mM sodium phosphate, pH<br>7.4) solution. One mL of the PEI solution was added to<br>1 mL of the DNA solution in a drop-by-drop manner.<br>The weight ratio of PEI and DNA in the solution mix-<br>ture was 1:20 (nitrogen/phosphate ratio = 1:5), and the<br>same amount of PEI was also formulated for making<br>PEG-g-PEI/DNA conjugates. Negatively charged com-<br>plexes were formed at this ratio. After incubation for 15<br>min, the PEI/DNA and PEG-g-PEI/DNA complex parti- 

<br> 

== TAT-PEG-PEI<br>  ==

Tat GRKKKRRQRC<br> 

α-vinyl sulfone-? -N-hydroxysuccinimide ester poly(ethylene<br>glycol) (NHS-PEG-VS) was purchased from Nektar Therapeutics (Huntsville, USA). The 25<br>kDa branched polyethylenimine (BPEI)<br> 

Activation of BPEI<br>19.2 mg (565 μmol) of bifunctional PEG (3.4 kDa), containing both an α-vinyl sulfone<br>and an ω-N-hydroxysuccinimide ester group, was weighed into a glass flask 4.293 μl of a<br>BPEI solution (corresponding to 12.15 mg/0.486 μmol BPEI; 282.4 μmol total amines) in<br>0.1M borate buffer at pH 5.5 were added and stirred. The activation reaction was carried out<br>for 4 hours at room temperature, followed by pH adjustment to 7 with 1N chloride acid and<br>additionally incubated for 2 hours at room temperature.<br>Coupling of the oligopeptide onto activated PEG-PEI<br>For coupling, 2.98 mg (2.26 μmol) of the oligopeptide TAT dissolved in 866 μl pure<br>water were added to the activated PEG-PEI yielding a theoretical degree of substitution of<br>approximately 1% based upon amine functions. The mixture was stirred for additional 2 hours<br>at room temperature.<br> 

<br> 

Coupling of the oligopeptide onto activated PEG-PEI<br>For coupling, 2.98 mg (2.26 μmol) of the oligopeptide TAT dissolved in 866 μl pure<br>water were added to the activated PEG-PEI yielding a theoretical degree of substitution of<br>approximately 1% based upon amine functions. The mixture was stirred for additional 2 hours<br>at room temperature.<br>Purification of TAT-PEG-PEI<br>Removal of unreacted PEG and low molecular weight residues was performed with an<br>ultrafiltration cell (Amicon, Bedford, USA) equipped with a 10 kDa molecular weight cut-off<br>membrane (Millipore, Bedford, USA) and 0.1 M borate buffer at pH 7.5 as eluent.<br>Amine concentration of the conjugate was determined by a recently described copper<br>assay [27].<br> 

<br> 

The (polyethylene glycol)-graft PEIs<br>(PEGPEI), were synthesized as previously described [28]. Briefly, PEGPEIs were obtained by<br>activating PEG-monomethyl ether with a hexamethylene diisocyanate. The coupling of PEG<br>to the amino groups of BPEI was carried out in anhydrous chloroform at 60 °C, which led to a<br>the PEI copolymers: (PEG5k) 2 PEI containing 2 PEG 5 kDa molecules per molecule of BPEI<br>and (PEG550) 35 PEI containing 35 550 Da molecules per molecule of BPEI.<br> 

<br> 

Briefly, PEGPEI was obtained by activating PEG-monomethyl<br>ether (5 kDa) with a hexamethylene diisocyanate. The coupling of PEG to the amino groups<br>of BPEI was carried out in anhydrous chloroform at 60 °C, which led to a PEI copolymer<br>containing 2 PEG 5 kDa molecules per molecule of BPEI.<br> 

[[Category:Paul]] [[Category:Transfection]]