Chloroform-Injection (CI) and Spontaneous-Phase-Transition (SPT) Are Novel Methods, Simplifying the Fabrication of Liposomes with Versatile Solution to Cholesterol Content and Size Distribution

Abstract

Intricate formulation methods and/or the use of sophisticated equipment limit the prevalence of liposomal dosage-forms. Simple techniques are developed to assemble amphiphiles into globular lamellae while transiting from the immiscible organic to the aqueous phase. Various parameters are optimized by injecting chloroform solution of amphiphiles into the aqueous phase and subsequent removal of the organic phase. Further simplification is achieved by reorienting amphiphiles through a spontaneous phase transition in a swirling biphasic system during evaporation of the organic phase under vacuum. Although the chloroform injection yields smaller Z-average and poly-dispersity-index the spontaneous phase transition method overrides simplicity and productivity. The increasing solid/solvent ratios results in higher Z-average and broader poly-dispersity-index of liposomes under a given set of experimental conditions, and vice versa. Surface charge dependent large unilamellar vesicles with a narrow distribution have poly-dispersity-index < 0.4 in 10 μM saline. As small and monodisperse liposomes are prerequisites in targeted drug delivery strategies, hence the desired Z-average < 200 d.nm and poly-dispersity-index < 0.15 is obtained through the serial membrane-filtration method. Phosphatidylcholine/water 4 μmol/mL is achieved at a temperature of 10°C below the phase-transition temperature of phospholipids, ensuring suitability for thermolabile entities and high entrapment efficiency. Both methods furnish the de-novo rearrangement of amphiphiles into globular lamellae, aiding in the larger entrapped volume. The immiscible organic phase benefits from its faster and complete removal from the final product. High cholesterol content (55.6 mol%) imparts stability in primary hydration medium at 5 ± 3 °C for 6 months in light-protected type-1 glass vials. Collectively, the reported methods are novel, scalable and time-efficient, yielding high productivity in simple equipment.

Keywords: CI method; PDI; SPT method; Z-average diameter; Zeta potential; entrapped volume; liposomes; simple methods; stability study; topography of liposomes.

Figure 1

Comparison of CI and SPT liposomes produced with optimized parameters and without sizing. (a): CI method, (b): SPT method, (c): Z-av and PDI of unsized CI-liposomes, (d): Z-av, and PDI of unsized SPT-liposomes, (e): SEM image of unsized CI-liposomes, (f): SEM image of unsized SPT-liposomes.

Figure 2

AFM topography of liposomes produced with optimized parameters showing comparison between sized and unsized liposomes. (a): 2D image of unsized liposomes, (b): 3D image of the same sample, (c): 3D image of selected single liposomes showing larger size, more height and surface roughness, (d): 2D image of sized liposomes, (e): 3D image of sized liposomes, (f): 3D image of selected single liposomes showing flattened and smaller size and size-dependent lower height but smooth surface and high rigidity.

Figure 3

Accelerated stability study of optimized liposomes after sizing by serial membrane filtration with 0.2 µm membrane filter and stored in the primary hydration medium. (a): DLS results at 0-day. (b): DLS results after 180 days at 4 °C. (c): DLS results after 180 days at 25 °C. (d): Z-av and PDI vs. the number of days at 4 °C and 25 °C. The results are presented as mean ± SD.