Advances in manufacturing processes provide the ability for the high throughput production of liposomes containing a range of moieties including small molecules and biologicals (including proteins and nucleic acids for prophylactic and therapeutic applications). Whilst rapid quantification methods for small molecules are generally well established, the ability to rapidly quantify liposomal entrapment of proteins is limited. Indeed, most standard protein quantification techniques (including the BCA assay and Reverse phase-high performance liquid chromatography (RP-HPLC)) measure protein encapsulation indirectly by measuring the amount of non-incorporated drug, and subtracting from the initial amount of protein added. However, this can give inaccurate and misrepresentative results. To address this, we have developed a range of methods to directly quantify protein entrapment within liposomes. The encapsulation efficiency within neutral, anionic and cationic liposome formulations was determined by three techniques; BCA assay, RP-HPLC and HPLC coupled to an evaporative light scattering detector, (HPLC-ELSD). All three methods are reliable for the quantification of protein, with linear responses and correlation coefficients of 0.99, and LOQ for all three methods being less than 10 µg/mL. Here within, we provide three methods for the rapid and robust quantification of protein loading within liposomal (and other bilayer) vesicle systems.
Figure 1. Ovalbumin calibration curves for three quantification techniques. The calibration curves for microBCA (A-C), include Intraday curves (A), Interday curves (generated over 3 separate days) (B), alongside the average (C). Calbiration curves were also generated for RP-ELSD (D-F), including Intraday curves (D), Interday curves (generated over 3 separate days) (E), as well as the average (F). The same was also generated for the HPLC-ELSD (G-I); Intraday curves (G), Interday curves (H), and the average (I). Results represent the average of at least three replicate measurements ± standard deviation (SD).
Figure 2. Effect of increasing liposome concentration on micro BCA absorbance with no ovalbumin added. Three liposomal formulations were produced using microfluidics (FRR 3:1 and 1:1, TFR 10 mL/min) DSPC:Chol, DSPC:Chol:PS and DSPC:Chol:DOTAP and assessed for BCA absorbance interference. Results represent the average of at least three replicate measurements ± standard deviation (SD).
Figure 3. Ovalbumin calibration curves solubilised in water with liposomal formulation (DSPC:Chol 10:5 wt/wt) at a fixed final concentration of 1 mg/mL per well, to establish LOD and LOQ values using micro BCA, following empty liposome blank removal (A). Intraday curves were generated (B) within the same day, while Interday curves were generated over 5 separate days (C). Accuracy was determined at three concentrations, each in replicate (D), while intraday and interday precision was calculated across three different concentrations with %RSD shown (E-F).
Figure 4. Ovalbumin calibration curves solubilised in water with liposomes and solubilisation mixture (50/50 v/v IPA/water), to establish LOD and LOQ values using micro BCA (A). Intraday curves were generated (B) within the same day, while Interday curves were generated over 5 separate days (C). Accuracy was determined at three concentrations, each in replicate (D), while intraday and interday precision was calculated across three different concentrations with %RSD shown (E-F).
Figure 5. Comparative study between three protein quantification techniques, RP-HPLC, HPLC- ELSD and BCA assay. All three liposomal formulations were made using microfluidics. The DSPC:Chol and DSPC:Chol:PS formulations were made at a 3:1 FRR and 15 mL/ min TFR (4 mg/ mL initial lipid and 0.25 mg/ mL initial ovalbumin concentration). The DSPC:Chol:DOTAP formulation was produced at a 1:1 FRR, the ovalbumin was adsorbed onto the surface by passing pre-made DSPC:Chol:DOTAP formulation through the microfluidics nanoassemblr. All results were measured three times, with the average encapsulation and ovalbumin loading calculated.
Figure 6. Bland and Atlman plot analysis for the comparison of three analytical techniques. Plot of differences between three analytical techniques (RP-HPLC, HPLC-ELSD and BCA assay) on the y-axis, versus the mean of the three analytical techniques for the three formulations (DSPC:Chol, DSPC:Chol:PS and DSPC:Chol:DOTAP). The calculated mean is -8.8 (horizontal solid line), with the bias represented by the gap between the mean and the dashed lines. All formulations were measured three times for the encapsulation efficiency, with each measurement plotted.