Dear Farhad,

I have not done any curcumin experiments like these, but have seen experiments like this before.  Here are my thoughts and suggestions.

Yes, if the particles formed grow in size then you will have some co-addition of the light scattering of the particles and the absorption due to curcumin's presence in the particles.  As a first approximation, be certain to draw a baseline on each side of the curcumin's apparent absorption in the encapsulated particle spectrum so that you can to first order remove the scattering contribution.  

Do remember, just like the authors of your cited reference, to separate unincorporated curcumin away from the nanoparticles prior to assaying the nanoparticles.  In the ideal world, it would even be easier to analyze the unincorporated curcumin in the sera (thereby avoiding ligt scattering) that you remove from the nanoparticles, but with a diafiltration step that gets somewhat impractical.

Good hunting!

Mike Bender

Just dissolve the nanoparticles ("filtered, centrifuged or dialyzed") in low-molecular alcohol (ethanol, i-propanol) for making true (clear) solution, and measure the absorbance without scattering.

Best regards

1. dissolve curcumin in methanol with different concentrations. Preferable in micro gram concentrations (each ML of methanol you have to use new micropipette tip) 

2. record absorption value at 420 nm and find the leaner fit equation 

3.dry your predetermined volume of nanoparticle suspension, dissolve in methanol and sonicate few min in closed container (calculate your dilution factor)

4. record absorption at 420 nm and calculate loading efficiency using the standard plot. 

thanks all

can I use a protease to remove protein aggregate and then measure absorption in 420 for curcumin determination without scattering? 

Usually, this type of measurement is made by HPLC. In this case, you have a good separation of curcumin and no co-absorption if you choose the right experimental conditions.

If you only have a UV spectrophotomer, I recommend you to separate the nanoparticles by centrifugation at high speed (14000rpm) during a sufficient time to have a clear surpernactant. Then measure the unencapsulated curcumin in this surpernactant.

This is a common problem with lipid nanoparticles.

There are three method:

1. You can try with Sephadex G column. Pass your formulation through Sephadex G and collect the sample and determine the presence of drug with a suitable analytical method.

2. You can try with nanosep. Fill you formulation in the nanosep microcentrifuge tubes and centrifuge it. Then collect the filtrate and analyse the presence of drug in your sample with suitable analytical method.

3. You can try will ultracentrigue. If you have ultracentigue then use high speed like around 1 lakh of above rpm. Then collect the supernatant and determine the presence of free drug with a suitable analytical method.

Apart from the above, another method is there i.e. organic solvent method. But, you may get some interference in that method.

You can also try with second derivative method in UV.

HPLC method will overcome excipient interference and quantitation. Methanol: Acetone [70:30] mobile phase worked well in our experience. [You can use the same solvent composition to breakdown the matrix and release the curcumin from the dried NP composition, to make it available for estimation].