Synthesis of pNP-PEG-PE-R8

pNP-PEG-PE.

1. Dissolve 100 mg of DPPE  in chloroform to obtain a 50 mg/ml of solution.

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

4. Incubate overnight at room temperature with stirring under argon.

5. Remove the organic solvents  using a rotary evaporator.
6. Add 0.01 M HCl.

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

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

9. Pooled fractions containing pNP-PEG-PE.

10. Freeze-dried.

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

12. Collect the fractions containing pNP-PEG-PE.

13. Remove mobile phase using a vacuum evaporator.

14. Store the powder at − 20 °C.

Synthesis of

15. Dry 20 mg of pNP-PEG-PE  from  chloroform solution with argon and then by overnight incubation under high
vacuum.

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.

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

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).

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

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