How To Measure The Efficiency Of Drug Packaging?
The encapsulation efficiency is an important parameter of the nanodrugs delivery system and can be calculated by the following formula:
Encapsulation efficiency (%) = Weight of the drug in nanoparticles / (Weight of the drug in nanoparticles +Weight of the drug in medium) X100%
There are generally two methods for determining the drug encapsulation efficiency in a nanometer drug delivery system: 1) the free drug is separated from the nanocarrier and measured; 2) the free drug and the nanocarrier are not separated and measured directly. The first method requires the encapsulated drug to be stable and it should has no leakage during separation. The separation methods include dialysis, gel column chromatography, high speed centrifugation, centrifugal ultrafiltration and microcolumn centrifugation. The second method mainly uses fluorescence quenching and electron spin resonance spectroscopy.
The First Type Determination Method
1). Dialysis
The dialysis method uses a semi-permeable membrane to separate free drugs and then measures the drug concentration in the dialysate. The method is simple, accurate, and repeatable; the disadvantage is that it takes a long time, and the added dialysate may cause the encapsulated drug to further diffuse out of the carrier, resulting in a lower measured encapsulation rate than the theoretical value.
2). Gel Column Chromatography
Gel chromatography columns can take advantage of the difference in volume between nanoparticles and free drugs. Nanoparticles do not enter the micropores of human gels, while free drugs remain in the micropores of gels due to their small size. Commonly used gels are dextran gel columns and agarose gels. The method has a small dilution effect on the nanoparticles, and the problem is that the elution time is long.
3). High-speed Centrifugation
The high-speed centrifugation method generally uses a rotational speed of tens of thousands or even hundreds of thousands of revolutions per minute to separate the free drug and the drug-carrying nanoparticles according to the gravity difference. The method is simple in operation, does not dilute the sample, and does not cause drug leakage; however, the cost is high, the centrifugation time is longer (more than 1h), and the reproducibility is not good.
4). Centrifugal Ultrafiltration
Ultrafiltration is similar to dialysis, using a microporous membrane to separate free drugs and carriers. The sample is added to the ultrafiltration tube and centrifuged, the free drug can be centrifuged down, and the carrier remains on the filter membrane. According to the molecular weight of the free drug, an ultrafiltration membrane with a suitable pore size is selected. The method is convenient and fast, has no dilution effect, and the drug does not leak, with high accuracy and good reproducibility; the disadvantages are that the ultrafiltration membrane may adsorb the drug, and the ultrafiltration tube is more expensive.
The Second Type of Determination Method
1). Fluorescence Quenching
The phenomenon of fluorescence quenching is that water-soluble fluorescent substances such as calcein and carboxyfluorescein self-quench at high concentrations; after recovering to a low concentration range, the fluorescence intensity is directly proportional to the concentration. When these fluorescent substances are encapsulated in the aqueous phase of the nanocarrier at a high concentration, and then highly diluted in the dispersion medium, the fluorescent substances in the aqueous phase of the nanocarrier are self-quenched and no fluorescence is generated, and the concentration of the free drug is reduced to reproduce the fluorescence. When the carrier is destroyed and the fluorescent substance is released, the mass of the entire fluorescent substance can be measured, and the fluorescence intensity can be compared to determine the encapsulation efficiency. This method does not need to separate the nanocarriers, and directly measures the encapsulation efficiency, which is fast and accurate, and avoids errors caused by separation.
2). Electron Spin Resonance Spectroscopy
When a paramagnetic reagent such as iron hydride is added to the nanocarrier, the electron spin band of the external label will be significantly broadened. The difference in the diffusivity between the label and free label encapsulated inside the liposome is used to determine the encapsulation efficiency. For example, the electron spin resonance marker dsAT (which can penetrate freely into the liposome) and the line broadening agent chromium oxalate (which cannot penetrate into the liposome) are added to the liposome. When chromium oxalate is added, the dsAT spin resonance line enclosed in the liposome has not changed, and the free dsAT line broadens significantly. Therefore, the encapsulation rate can be calculated by measuring the encapsulated and unencapsulated dsAT.