Encapsulation Efficiency (EE) and Loading Efficiency (LE) Protocol

Encapsulation Efficiency (EE) and Loading Efficiency (LE) Protocol:

1. Prepare a standard curve for DOX (Doxorubicin):

   a. Dilute the DOX solution to eight different concentrations: 5, 10, 15, 20, 25, 30, 40, and 50 μg/mL, using ultrapure water as a blank control.

   b. Set the excitation wavelength of a microplate reader to 480 nm.

   c. Measure the absorbance values of the DOX solutions with different concentrations.

   d. Plot a standard curve of absorbance versus concentration.

   e. Determine the linear relationship between DOX concentration and absorbance value in the range of 0-50 μg/μL.

2. Encapsulation Efficiency (EE) measurement:

   a. After completing the doxorubicin encapsulation, take two samples of 200 μL DOX-loaded liposome solution.

   b. Adjust one sample to 500 μL with buffer and add 1 mL of acidic isopropanol for demulsification.

   c. React the sample on an oscillator for 10 minutes.

   d. Use 500 μL of HBS buffer as a blank control and add 1 mL of acidic isopropanol.

   e. Take 100 μL samples from each and add them to a 96-well plate.

   f. Set the wavelength of a microplate reader to 480 nm.

   g. Measure the absorbance values and calculate the doxorubicin content.

   h. Pass the other sample through a P-10 column and elute it with HBS buffer solution with pH 7.8.

   i. Collect the liposome part and use buffer solution to increase the volume to 500 μL.

   j. Perform demulsification and measure the absorbance value with a microplate reader.

   k. Calculate the DOX content, which represents the DOX content loaded by liposomes.

3. Calculation of Encapsulation Efficiency (EE):

   EE (%) = (Amount of drug loaded in liposomes / Amount of drug added) × 100%

4. Calculation of Loading Efficiency (LE):

   LE (%) = (Amount of drug in liposomes / Amount of liposomes) × 100%

5. Record the encapsulation efficiency (%EE) and loading efficiency (%LE) values for each type of liposome (DOPC-lip@DOX, DOPE-lip@DOX, DOPE-DVar7-lip@DOX). In this study, the encapsulation efficiency (%EE) and loading efficiency (%LE) were as follows:

   - DOPC-lip@DOX: %EE = 98%, %LE = 9.8%

   - DOPE-lip@DOX: %EE = 90%, %LE = 9.0%

   - DOPE-DVar7-lip@DOX: %EE = 93%, %LE = 9.3%