Editing Synthesis of pNP-PEG-PE-R8

pNP-PEG-PE.<br>

1. Dissolve 100 mg of DPPE&nbsp; in chloroform to obtain a 50 mg/ml of solution. <br>

2. Add 2 ml of TEA.<br>3. Add 2 g of (pNP)2-PEG dissolved in 20 ml of chloroform.<br>

4. Incubate overnight at room temperature with stirring under argon.<br>

5. Remove the organic solvents&nbsp; using a rotary evaporator.<br>6. Add 0.01 M HCl.<br>

7. Sonicate using a water bath to form pNP-PEG-PE micelles.<br>

8. Separate unbound PEG pNP on a column with Sepharose CL-4B using the 0.01 M HCl as an eluent. <br>

9. Pooled fractions containing pNP-PEG-PE.<br>

10. Freeze-dried.<br>

11.Further purify pNP-PEG-PE by&nbsp; RP-HPLC preparative column using methanol/ 0.01 M HCl (70/30, v/v) as a mobile phase. <br>

12. Collect the fractions containing pNP-PEG-PE.<br>

13. Remove mobile phase using a vacuum evaporator. <br>

14. Store the powder at − 20 °C.<br>

Synthesis of <br>

15. Dry 20 mg of pNP-PEG-PE&nbsp; from&nbsp; chloroform solution with argon and then by overnight incubation under high<br>vacuum. <br>

16. Hydrate the resulting film and disperse by vortex for 5 min in the 5-mg/ml R8 solution in 0.01 M HCl, at the pNP-PEG-PE-to-R8 molar ratio of 1-to-1. <br>

17. Add 4 ml of Tris buffer, pH 9.0 to the suspension, mix and incubate overnight at 4 °C under an argon atmosphere. <br>

18. The modified R8 becomes amphiphilic and forms micelles, separate these micelles from the non-reacted free R8 by the overnight dialysis against distilled water at 4 °C using a Spectrum dialysis bag (MWCO of 5 kDa).<br>

19. Freeze-dried. Stoe the R8-PEG-PE powder at − 20 °C.<br>

Ref: siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene <br><br>

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