Editing Synthesis of GO-PEI

== Materials ==

Graphene oxide (GO): Tianjin Plannano Technology Co., Ltd.

Chloroacetic acid: Tianjin Fuchen Chemical Reagents Factory.

N-(3-dimethylamino propyl-N’-ethylcar-bodiimide) hydrochloride (EDC-HCl), N-hydroxysuc-cinimide (NHS), Succinic anhydride and pyridine: Chengdu Kelong Chemical Reagents Factory.

Paclitaxel and docetaxel&nbsp;: Xi’an Hao-xuan Biological Technology Co., Ltd

Aminated polyethylene glycol (NH2-PEG-NH2, Mw = 4000)&nbsp; Thermo Scientific&nbsp;







== Synthesis of Graphene Oxide ==

To a mixture of KMnO4 (6 g) and graphite (1 g), add Concentrated sulfuric acid and orthophosphoric acid (120:15 mL)

Heat to 50 ◦C and stir for 24 h.

Pour the resulting&nbsp;mixture into ice (150 mL) and H2O2 (30%, 1 mL)

Filter&nbsp;using a polycarbonate membrane. &nbsp;Wash the solid product with water, 30% HCl, and ethanol two times

Vacuum drying for 12 h.

== Synthesis of Reduced Graphene Oxide (RGO) ==

Exfoliate&nbsp;GO &nbsp;in water by ultrasonication to produce a homogeneous graphene oxide water based suspension (0.1 mg/mL).

Reduce the GO using glucose as the reducing agent at 95 ◦C for 2 h.

Filter the mixture by a polycarbonate membrane

Wash&nbsp;several times with water and ethanol to obtain reduced graphene oxide (RGO).

Compared with GO, RGO shows a much lower oxygen content with the majority of oxygen-containing groups such as carboxyl, hydroxyl and ketone moieties removed during the reduction process and thus usually has a poor water solubility.

== Carboxylate Graphene Oxide ==

To carboxylate Graphene oxide sheet, GO (10 mg) was added to water and sonicated to form an aqueous suspension. NaOH (0.12 g/mL) and chloroacetic acid (0.5 g) were then added, and the resultant mixture was sonicated for 3 h to form carboxyl group on the surface of GO. GO-COOH suspension was neutralized and purified by centrifugation at 10,000 rpm. The supernatant was discarded and the residue was washed with water twice.



== Modification of GO with aminated polyethylene glycol ==

Carboxylated GO was diluted by water (10 mL) and then bath sonicated with amino-terminated PEG4000 (100 mg) for 30 min, followed by addition of N-(3-dimethylaminopropyl-N’-ethylcarbodiiminde) hydrochloride (EDC-HCl) to reach a concentration of 40 mmol/L. Then the mixture was allowed to react overnight. The final reactants were dialyzed in dialysis bags (MWCO = 10,000) for 4 days to obtain the PEGylated GO.



== Conjugate PEI to Graphene Oxide (PEI-GO) ==

[[File:GO-PEI.png|RTENOTITLE]]

== Preparation of Graphene Oxide (GO) by modified&nbsp;Hummers’ method ==

from Flake graphite (99.95% purity, Aldrich).

== Preparation of USGO ==

Disperse GO (10 mg) in 10 ml MilliQ H2O

Add &nbsp;30 mg, 0.11 mmol of&nbsp;Nα,Nα-bis(carboxymethyl)-L-lysine hydrate (Aldrich)

Add &nbsp;50 mg, 0.89 mmol of KOH (Aldrich).

Bath-sonicate the mixture &nbsp;at 80 ° C for 2 h

Stir&nbsp;at 70 ° C overnight.

Membrane-filtered the mixture &nbsp;using 100 kDa membrane&nbsp;(Millipore)

Wash&nbsp;three times using MilliQ H2O



'''Preparation of PEI-g-USGO and PEI-g-GO'''

Disperse the above USGO or GO (10 mg) &nbsp;in MilliQ H2O (10 mL)

ADD 30 mg, 0.073 mmol of&nbsp;PEI (60 kDa, branched; &nbsp;Aldrich)

Add 50 mg (0.26 mmol)&nbsp;N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, Aldrich)

Stir the mixture overnight

Membrane-filtered (100 kDa, Millipore)

Wash&nbsp;three times with MilliQ H2O

Disperse&nbsp;in 10 mL of H2O.

Note: should not use&nbsp;N-hydroxysuccinimidyl ester (NHS) to replace&nbsp;EDC as it will cause&nbsp;precipitation after adding NHS.

== NGO-PEG-PEI&nbsp; ==

Mix GO solution ( ∼ 0.5 mg/mL &nbsp;) &nbsp;with 6-armed amine-terminated PEG (0.5 mg/mL&nbsp;) under sonication for 5 min.

Add&nbsp;EDC (0.5 mg/mL&nbsp;) then sonicate for&nbsp;5 min

Stir&nbsp;gently at room temperature for 10 min.

Add &nbsp;PEI 2.5 mg/mL &nbsp;for 5 min

Stir&nbsp;at room temperature for 6 h

Add EDC 1 mg/mL.

Wash mixture &nbsp;3 ∼ 5 times with deionized water





== PEI-GO ==

Disper&nbsp;10 mg GO dispered in 10 ml distilled water

Add 100 mg branched PEI 25 K in 10 mL distilled water

Add 5 ml of 10 mg/mL&nbsp;EDC solution

Stir&nbsp;30 min.

Add 15 ml of 10 mg/mL EDC

Stir&nbsp;overnight.

Add 1.6 g NaCl (1.6 g)

Spin 13 000 rpm for 2 h and collect &nbsp;supernatant

Purify by ultrafiltration using a 100 K ultrafilter.

Wash the retainer in the ultrafilter repeatedly &nbsp;with aqueous solution containing 10% NaCl and 10% urea to remove any unreacted PEI.

Free PEI in the ultrafiltrate can be&nbsp;detected by '''ninhydrin''', to confirm complete removal of unreacted PEI.

Ultrafiltration using&nbsp;water to &nbsp;remove NaCl and urea

Disperse&nbsp;in water to form a stable solution (∼1.57 mg/mL&nbsp;PEI-GO).

== Transfection ==

mixi &nbsp;PEI-GO (4.8 μg) and FITC siRNA (10 μg) in cell medium (500 μL) for 30 min

Agarose gel assay: ~4.4 μg of pEGFP can be bound by 1 μg of USGO-PEI ~1.4 μg of pEGFP can be bound by 1 μg of GO-PEI
[[Category:Transfection]] [[Category:Paul]]