Paul.xu at 02:28, 16 January 2017

2017-01-16T02:28:38Z

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== 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 : Xi’an Hao-xuan Biological Technology Co., Ltd

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

== 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 mixture into ice (150 mL) and H2O2 (30%, 1 mL)

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

Vacuum drying for 12 h.

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

Exfoliate GO  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 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 Hummers’ method ==

from Flake graphite (99.95% purity, Aldrich).

== Preparation of USGO ==

Disperse GO (10 mg) in 10 ml MilliQ H2O

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

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

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

Stir at 70 ° C overnight.

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

Wash three times using MilliQ H2O

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

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

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

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

Stir the mixture overnight

Membrane-filtered (100 kDa, Millipore)

Wash three times with MilliQ H2O

Disperse in 10 mL of H2O.

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

== NGO-PEG-PEI  ==

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

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

Stir gently at room temperature for 10 min.

Add  PEI 2.5 mg/mL  for 5 min

Stir at room temperature for 6 h

Add EDC 1 mg/mL.

Wash mixture  3 ∼ 5 times with deionized water

== PEI-GO ==

Disper 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 EDC solution

Stir 30 min.

Add 15 ml of 10 mg/mL EDC

Stir overnight.

Add 1.6 g NaCl (1.6 g)

Spin 13 000 rpm for 2 h and collect  supernatant

Purify by ultrafiltration using a 100 K ultrafilter.

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

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

Ultrafiltration using water to  remove NaCl and urea

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

== Transfection ==

mixi  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]]

Synthesis of GO-PEI - Revision history

Paul.xu at 02:28, 16 January 2017