Preparation of atorvastatin calcium-loaded liposomes using thin-film hydration and coaxial micromixing methods: A comparative study

Abstract

Development of techniques to produce nanoformulations in a controlled and reproducible manner is of great importance for research, clinical trials, and industrial scale-up. This research aimed to introduce a cost-effective micromixing approach for the nanoassembly of liposomes and compared with thin-film hydration (TFH) method. Numerical simulations and design of experiments (DOE) by response surface methodology (RSM) were used to evaluate the effects of input parameters on liposome properties, aiming to identify optimal conditions. Anionic liposomes without or with atorvastatin calcium (ATC) produced using TFH and the micromixing methods showed similar characteristics in size (150-190 nm), PDI (<0.2), and zeta potential (-50 to -60 mV). Both methods achieved about 70 % encapsulation efficiency with similar drug release profile for ATC-containing liposomes. Analysis of stability and DSC thermograms revealed comparable outcomes for liposomes prepared using both techniques. Nanoliposomes produced via both approaches indicated similar in vitro biological performance regarding cellular uptake and cell viability. The micromixing approach presented an alternative method to produce nanoliposomes in a one-step manner with high controllability and reproducibility without requiring specialized equipment. Compatibility of the micromixer with various solvents, including those detrimental to conventional microfluidic materials like PDMS and thermoplastics, enables exploration of a wide range of formulations.

Keywords: Coaxial micromixer; Comparison; Controllability; Microfluidics; Nanoliposome; Reproducibility; Thin-film hydration.

Graphical abstract

Fig. 1

Coaxial micromixer. (A) Schematic illustration for the components of the coaxial micromixer, (B) schematic of the assembled micromixer, (C) SEM image of the needle cross-section to show their diameters, (D) image of the experimental setup.

Fig. 2

(A) Schematic illustration of the overall computational domain; cylindrical symmetry was applied to the computational domain, (B) the comparison of mixing efficiency of T-shape micromixer (Aspect ratio = 0.5) in Re = 250 with the results of Cortes-Quiroz study.

Fig. 3

Numerical simulation of the mixer. (A) effect of TFR on the normalized concentration distribution of aqueous solution through the coaxial micromixer at FRR of 2, (B) effect of FRR on the normalized concentration distribution of aqueous solution through the coaxial micromixer at TFR = 150 μL/min. To conduct the numerical simulations, the concentration of the inlet solution for the inner needle (central needle) was set at zero, while the concentration of the inlet solution for the outer needle was set at one.

Fig. 4

Impact of input parameters of TFR, FRR, and lipid concentration on size, PDI, and zeta potential of the liposomes: A) TFR and FRR at LC = 48 mM, B) LC and TFR at FRR = 2, C) LC and FRR at TFR = 150 μL/min.

Fig. 5

Diameter of nanoliposomes. Size of nanoparticles reported as intensity prepared by TFH method and coaxial micromixer (A), TEM images of empty liposomes prepared by both TFH (B and C) and micromixing (TFR = 150 μL/min and FRR = 2) (D and E) methods.

Fig. 6

DSC curves of nanoliposomes. Thermograms of empty (L(Empty)) and ATC-containing (L(ATC)) liposomes prepared by both TFH (F) and micromixing ((M), TFR = 150 μL/min and FRR = 2) methods.

Fig. 7

Stability study. The stability of empty liposomal formulations produced by TFH and micromixing (TFR = 150 μL/min and FRR = 2) methods during one month at 4 °C, 25 °C and 37 °C in terms of particle size (A), PDI (B) and zeta potential (C). Data are denoted as mean ± S·D, (n = 3). The difference in the stability of liposomes comparing three investigated temperatures is not significant for any of the methods (P > 0.05).

Fig. 8

Comparison of release profiles. The in vitro release of ATC from liposomes produced by TFH and micromixing (TFR = 150 μL/min and FRR = 2) methods in Histidine/dextrose buffer pH (6.5) at 37 °C. Data are demonstrated as mean ± S·D, (n = 3). The difference in the release profile between the two methods is not significant (P > 0.05).

Fig. 9

The cytocompatibility comparison of nanoliposomes. Viabilities of J774 macrophages cell line treated with nanoliposomes without drug (L(Empty)) or with drug (L(ATC)) at 37 °C for 48 h. Cell viability of empty and ATC-loaded liposomes prepared by both TFH (F) and micromixing ((M), TFR = 150 μL/min and FRR = 2) methods showed no significant differences in various concentrations (P > 0.05). Data are presented as mean ± S·D, (n = 3).

Fig. 10

The in vitro cellular uptake of liposomes. A) Uptake of DiR-labeled nanoliposomes without drug (L(Empty)) or with drug (L(ATC)) by J774 macrophage cell line incubated at 37 °C for 4 h. The groups treated with liposomes prepared by TFH (F) and micromixing ((M), TFR = 150 μL/min and FRR = 2) methods compared to the control group (untreated macrophages) showed a significant level of cellular uptake. B) The mean fluorescence intensity (MFI) of different liposome uptake by the J774 cell line. Data are presented as mean ± S·D, (n = 3). Statistical differences in terms of significance are shown as (*: P < 0.05, **: P < 0.01).