Dear all, would like you to help me? The case, in my research I maked curcuminoid loaded in solid lipid nanoparticle (SLN) and I got the problem to calculate entrapment efficiency (EE). This is my composisition a part to make SLN, 0.1 gram curcuminoid, 0.5 gram poloxamer, 1 gram palmitic acid, all mix in 100 mL reverse osmotic water (RO). I followed yadav V et al. 2008 method to calculate EE.
1st. 3 mL SLN solution is setrifugated to separate SLN and unentrapped curcuminoid.
2th, the pelet (assuming contain curcuminoid in SLN) is added 3 mL methanol to extract and suck out the curcuminoid. Next is sentrifugated this solution to separate lipid and curcuminoid solution.
3th, solution contain 2 part, pelet and supernatant
4th. check supernatant absorbance 5th. by using a standart curve, the concentration of curcuminoid is 0.019489 mg/mL. 6th.EE = 0.019489 /1 = 1.948%.
Is my calculation correct?
Hi Tirta, why not using the unentrapped curcuminoid instead? The whole formula would be:
EE = ((Total conc. - Supernatant conc.)/Total conc.) * 100%
Supernatant conc = Supernatant from 1st centrifugation.
Radif
The calculation can be done as following :
first % TDL should be calculated :
The theoretical percentage of drug loading (TDL) can be calculated using the following
equation.
TDL = (Weight of drug added (g)/Weight of polymers and drug added (g) x 100
For example, if you use 2 g of drug and 8 g of polymer, then the theoretical loading is
2/10 x 100 = 20, hence the theoretical loading is 20% w/w. (Note: dominator in the previous equation always should contains total amount of excipients and drug).
U should validate an analytical method to calculate the actual loading and this will depend on you what instruments do you have, and etc............ But
Assume that, during analysis each gram of SLN contains 100 mg of drug.
Then actual loading is 100/1000 x100 = 10% w/w.
Therefore, the percentage of drug entrapment efficiency will be calculated according to the following equation:
%DEE= ( experimental drug loading/Theoretical drug loading (TDL)) x 100
As per the above example the DEE= 10/20 x100 = 50% w/w.
Lets assume the yield of the microsphere is 9 g. Remaining one gram lost during the
manufacture.
Strictly speaking, entrappment refers to material enshrinement INTO a carrier, whether of SLN or any other type. This excludes material adsorption or binding ONTO a carrier (or in the case of hollow carriers, such as bilayer vesicles, INTO such carriers shell / bilayer).
Practically speaking, association (with a carrier) is the more relevant term, as it encompasses entrappment AND adsorption, or binding. The reason is that both entrapped and bound molcules share, for certain while (i.e., until release) their carrier fate. The carrier biodistribution / bioprocessing therefore affects the cargo molecules biodistribution / bioprocessing, whereby cargo release characteristics normally differ for different association sites (carrier core, proximity to carrier surface, carrier outer surface, etc.). To understand a formulation properly, one should therefore study drug loading / release in sufficient detail to differentiate between, and describe quantitatively, all relevant association sites.
Separation of the carrier associated and carrier free cargo molecules, as proposed in the first answer, will arguably overestimate entrappment. The first reason is that it will include - in an ideal experiment - the adsorbed / bound cargo molecules into the entrapped cargo pool. In a real experiment, it will moreover include the cargo molecules contained in fluid between the 'separated' carriers in the centrifugation sediment as if they were trully associated with carriers. To correct for this error, one should determine the net concentration of carriers in the sediment and subtract the cargo molecules amount in the inter-carriers solvent, which forms one part of the sediment, from the overall cargo molecules amount measured in the sediment. Any kind of carrier separation, e.g. with an affinity column (or even better: carrier associated cargo detection), that avoids such 'contamination' is therefore superior to centrifugation. If applied in such contexts, the normalisation formula advocated in the second answer can prove useful; otherwise its use is just a detour - but not wrong.
To understand the last remark one can write, formally:
Association = Entrapment + Binding (
OF COURSE YOU ARE RIGHT.
BY % EE = ((Total conc. - Supernatant conc.)/Total conc.) * 100%.YOU GET SAME ANSWER.YOU ARE DOING RIGHT WELL DONE.YOU CAN DO BYE BOTH METHODE.
1.CALCULATE CONCENTRATION IN SUPERNATANT
2.CALCULATE CONCENTRATION IN PELLETS.
Dear Mr.Tirta Setiawan
Initially you have prepared 0.1g/100ml solution = 100mg/100ml or 1mg/ml solution of curcuminoid along with the polymers
From this you have taken only 3ml for centrifugation during your first step.
Let us suppose this 3ml contain only 3mg of drug as entrapped and as unentrapped as well.(Because the curcuminoid concentration of the prepared solution was 1mg/ml)
This 3ml.solution you have centrifuged during your step 2 and separated Supernant solvent (Solvent-A ) and the unentraped drug (X mg)
To this you have added 3ml of Methanol and pelet and centrifuged again then separated supernant liquid methanol (Solvent-B) to remove any further unentrapped curcuminoid present (Y mg).
Did you analyzed the Solvent-A and Solvent-B separately for the estimation of uentrapped curcuminoid present ? or have you taken only the Solvent-B for analysis?
If you have analysed both the solvents then
the amount of unentrapped curcuminoid = X + Y = Z mg
Then amount entrapped will be 3-Z = C mg. (Because the supernant liquid will contain only the unentrapped drug)
Then,
The % Entrapment efficiency = (Drug loading /Theoretical drug loading) x 100.
I agree with Gregor Cerv's explaination on the actual meaning of entrapment. For a simple ESTIMATION of %EE, most people will follow the method given by Ahmad Mohamad Zaiter.
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