TITLE: Liposome Encapsulation of Hydrophilic and Hydrophobic Drugs 
AUTHORS: Aisling  Field, James F Curtin, Janith Wanigasekara, Julie Rose Mae Mondala

[DESCRIPTION]
Dried reconstituted vesicles (DRV) are liposomes that are produced under mild conditions and can encapsulate very high amounts of hydrophilic solutes. These characteristics make this liposome type perfect for entrapment of drugs ( Antimisiaris, 2016). To produce DRV’s, the lipid ingredients are firstly dissolved in an organic solvent (chloroform), dried on a rotary evaporator to obtain a thin film, followed by lipid hydration (in the aqueous medium at a temperature greater than the phase-transition temperature) and lastly to homogenize and reduce the vesicle size, where the extrusion technique is used. This process produces heterogeneous multilamellar vesicles.  Cholesterol is added into the bilayer structure to aid liposome formation, and to stabilize against aggregation and drug leakage (Ahmad and Dwivedi, 2017).

The thin-film method is one of the most widely used liposome encapsulation methods. It is based on the generation of a thin film of lipids, formed on the inner wall of the rotary evaporator flask. The film is then hydrated with water. Before the hydration, it is integral that the lipid film is preheated above the lipids transitional temperature to enable a smoother creation of the bilayer, along with the vigorous shaking. This allows the film to peel off the flask and form liposomes. The liposomes generated are multilamellar vesicles of different sizes. The encapsulating substance can be added with the lipids before the formation of the thin film (hydrophobic compounds) or with the water (hydrophilic compounds). The advantage of this method is its high reproducibility even when working with small quantities of compounds (Šturm andPoklarUlrih, 2021).

This protocol describes the liposome encapsulation of hydrophobic and hydrophilic drugs using thin-film dispersed hydration method. Extrusion method with a polycarbonate membrane is used to make liposomes of suitable sizes that can be easily internalized by mammalian cells. 

[STEPS]
SECTION: Liposome Encapsulation
2. Stir the mixture for 15 min at above Tc of the lipid (60 ⁰C).

  

SECTION: Liposome Encapsulation
1. 
Dissolve 7 mmol of the lipid (DSPC), 3 mmol of cholesterol and 1:20 (w/w) Ursolic acid (lipophilic compound) in 5 ml chloroform in a round bottom flask.

   

SECTION: Liposome Encapsulation
3. 
Dry the lipid using a rotary evaporator at 40 ⁰C.

  

SECTION: Liposome Encapsulation
4. 
Further dry overnight by incubating above the Tc of the lipid (60 ⁰C) in a vacuum oven.
SECTION: Liposome Encapsulation
5. 
Re-hydrate lipid nanoparticle in 5 ml ultrapure water.
SECTION: Liposome Encapsulation
6. 
Liposome encapsulation of hydrophilic compounds are added after dehydration. Dissolve lipophilic compounds 1:20 (w/w) in 5 ml of ultrapure water. Re-hydrate lipid nanoparticles by adding the dissolved lipophilic compound in ultrapure water.  
SECTION: Liposome Encapsulation
7. 
Stir the liposome encapsulated compound at a temperature above the Tc of the lipid (60 ⁰C) for 30 min.

  

SECTION: Liposome Encapsulation
8. 
Vortex for 2 min.

  

SECTION: Liposome Encapsulation
9. 
Extrude at a temperature above the Tc of the lipid for approximately 22 passes using Avanti Mini Extruder Kit (Product Code: 610000). Do 11 passes with 100 nm pore size PC membrane and then another 11 passes using the 50 nm pore size PC membrane. This is done to have liposomes of between 50-100 nm in size.

  
Avanti Mini Extruder Procedure

SECTION: Liposome Encapsulation
10. 
Read particle size and zeta potential using Malvern Nano-ZS zetasizer.
SECTION: Liposome Encapsulation
11. 


 
 
Liposome Concentrations UA  0.35mg UA, 5.53mg DSPC, 1.16mg Cholesterol FITC 0.5ml FITC, 5.53mg DSPC, 1.16mg Cholesterol PI 0.5ml PI, 5.53mg DSPC, 1.16mg Cholesterol Control 5.53mg DSPC, 1.16mg Cholesterol 

 
 
13. Open zetasizer software and set to the desired measurement.
SECTION: Liposome Characterisation
12.  Turn on and warm up the Malvern Nano-ZS zetasizer for 1 hour for the laser to stabilize. 

  

SECTION: Particle Size
14. Measure the particle size of liposome samples by setting a manual SOP.
SECTION: Particle Size
15. Set the sample material to DTS0012 cuvette - polystyrene latex.

  

SECTION: Particle Size
16. Set the dispersant of water to a temperature setting of 25 ºC.
SECTION: Particle Size
17. Set the viscosity to 0.8872 cP.
SECTION: Particle Size
18. Set the refracted index to 1.330.
SECTION: Particle Size
19. Set the dielectric constant to 78.5.
SECTION: Particle Size
20. Set the equilibration time to 120 seconds.
SECTION: Particle Size
21. Set the measurement to 173º Backscatter.
SECTION: Particle Size
22. Fill the cuvette with the sample to a depth of approximately 1 cm.
SECTION: Particle Size
23. Place the cuvette with the sample in the cell of the zetasizer and analyse.
SECTION: Zeta potential
24. Measure the zeta potential of liposome samples by setting a manual SOP. 
SECTION: Zeta potential
25. Set the sample material to DTS1070 cuvette - polystyrene latex. 

  

SECTION: Zeta potential
26. Set the dispersant  water to a temperature setting of 25 ºC

SECTION: Zeta potential
27. Set the viscosity to 0.8872 cP.

SECTION: Zeta potential
28. Set the refracted index to 1.330.

SECTION: Zeta potential
29. Set the dielectric constant to 78.5.

SECTION: Zeta potential
30. Set the equilibration time to 120 seconds. 

SECTION: Zeta potential
31. Set the measurement to 173º Backscatter and 100 zeta run.
SECTION: Zeta potential
32. Fill the cuvette with the sample to a depth of approximately 1 cm.
SECTION: Zeta potential
33. Place the cuvette with sample  in the cell of the zetasizer and analyse.