You can measure the non-encapsulated dye in the supernatant that you collected after nanoparticle preparation. Compare it to corresponding 100% dye concentration you used initially to prepare nanoparticles. Make sure you are using the fluorescent protocol for the plate reader. You can prepare different concentration of dye solution for standards.

I hope this helps.

Thank you Yasin,

I did that and my dye concentration is 3.3 ug/ml. How can I calculate the loading capacity and loading efficiency? 

I used 2mg/mL of the Rh123 encapsulated PLGA in the spectrophotometer.

Loading efficiency is amount (ug) of dye in per mg of recovered nanoparticles. Not sure about loading capacity.

Dr. Amira, That's really helpful! I appreciate your valuable time illustrating to me. I will work on that. 

For the determination of the sample loading,

Step 1.  centrifuge until you cannot observe absorbance in the supernatant. Normally water is enough.  alcohol can also be used to remove free dye or dye absorbed on the surface of particles.

Step 2. Freeze dry the particles as soon as you get clean particles.

Step 3. Dissolve, for example, 1 mg of dry particle into 10 mL DMF or other organic solvent.

Step 4. measure the UV abs and determine the conconcentration from calibration curve and the calculation the loading.

For the determination of the calibration curve,

Step 1. prepare 0.1mg/mL polymer solution. Use the same solvent as above. This is to be used as the solvent for the dye.

Step 2.  prepare dye solution with different concentrations using the solution in Step1.

Step 3: get the calibration curve and equation.

Note:

1. dilute the sample until the conc is in the calibration curve range. Use polymer organic solvent to dilute.

2. remember the dilution factors you have used.