Paul.xu at 15:45, 22 October 2014

2014-10-22T15:45:28Z

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== Peptides ==

{| width="711" cellspacing="1" cellpadding="1" border="1"
|-
| width="200" | Name 
| Seq 
| Form 
| Transfection Efficiency 
|-
| width="200" | Tat48–60
| GRKKRRQRRRPPQ
| 
| 
|-
| width="200" | Tat49-60 
| RKKRRQRRRPPQ 
| 
| 
|-
| width="200" | Tat47–57 
| RKKRRQRRR-GGG 
| 
| 
|-
| width="200" | Tat2 
| C(YGRKKRRQRRRG)2 
| 
| 
|-
| width="200" | Penetratin (Antp 43–58 )
| RQIKIWFQNRRMKWKK
| 
| 
|-
| width="200" | Transportan
| GWTLNSAGYLLGKINLKALAALAKKIL
| 
| 
|-
| width="200" | TP10
| AGYLLGKINLKALAALAKKIL
| 
| 
|-
| width="200" | Oligoarginine (R8)
| RRRRRRRR
| 
| 
|-
| width="200" | MAP
| KLALKLALKALKAALKLA
| 
| 
|-
| width="200" | MPG
| GALFLGFLGAAGSTMGAWSQPKKKRKV
| 
| 
|-
| width="200" | MPGa
| GALFLAFLAAALSLMGLWSQPKKKRKV
| 
| 
|-
| width="200" | CADY 
| GLWRALWRLLRSLWRLLWRA-cya 
| 
| 
|-
| width="200" | LK15 
| RKKRRQRRRGGGKLLKLLLKLLLKLLK-CONH2 
| 
| 
|-
| width="200" | KALA 
| WEAKLAKALAKALAKHLAKALAKALKACEA 
| 
| 
|-
| width="200" | Pep1 (Chariot) 
| KETWWETWWTEWSQPKKKRKV 
| 
| 
|-
| width="200" | Tat 
| RKKRRQRRRPPQ 
| 
| 4X106 
|-
| width="200" | Tat-CTHD 
| RKKRRQRRRPPQC 
| C-Terminal disulfide homodimer 
| 1.2X107 
|-
| width="200" | Tat-NTHD 
| CRKKRRQRRRPPQ 
| N-Terminal disulfide homodimer 
| 1.0X107 
|-
| width="200" | Cyclic-Tat 
| CRKKRRQRRRPPQC 
| Disulfide bond between C-terminal and N-terminal 
| 3.8X107 
|}

== Polycondensation of cyclic Tat (cTatc-->cGRKKRRQRRRGc) ==

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 

 

== Synthesis of PEI-Peptide ==

'''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 

[[Image:PEI SPDP Peptide.png]] 

 

6.6 mg of PEI (2kD) in 0.5 mL phosphate buffer (pH 8.0)  

Add  0.5 mL of DMSO solution containing 3.8 mg of SPDP 

Stir overnight at room temperature. 

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

Freeze-dry the solution--> 2.2 mg of PEI-SPDP 

Add 2.0mg peptide to  0.9 mg PEI-SPDP  in 1.0 mL of PBS. 

React overnight at room temperature. 

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

Freeze-dry yielding a product. 

Before use, dissolve PEI-Peptide conjugatein pure water. 

 

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

'''Sulfo-SMCC''' 

[[Image:Sulfo-SMCC-conjugation.jpg]] 

 

== Synthesis of P123-PEI-R13 ==

R13:RGDC-Tat (49–57)

pluronic P123: BASF Corporation (Mount Olive, NJ)

=== Synthesis of P123-PEI ===

 Dry P123 (0.6 mmol)  by coevaporation with anhydrous toluene in a vacuum at 50°C 

Dissolve 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  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)  in 10 mL anhydrous ethanol as Solution B. 

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

Let the reaction continue for overnight at room temperature. 

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

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

 

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  for 30 minutes at room temperature with gentle shaking.

 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 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  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 

== 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.

 

== Synthesis of PEG-g-PEI ==

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

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

 

== TAT-PEG-PEI ==

Tat GRKKKRRQRC 

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

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

 

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

 

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

 

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

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

Synthesis Peptide-PEI - Revision history

Paul.xu at 15:45, 22 October 2014