Category: Formulation and Quality
Purpose: Liposomes are the most successful nanocarrier-based drug delivery platforms that have a history of both therapeutic enhancement and commercial triumph. Their ability to incorporate both hydrophilic and hydrophobic molecules makes them versatile and allows for combination of multiple therapeutic agents within the same formula. However, common methods for preparing liposomes involve thermal and mechanical shear stress, and the use of class 2 solvents, which can have adverse impact on sensitive chemical and/or biological moieties and raise concerns about residual solvent and formulation safety. The ethanol injection method can circumvent these complications as heat is not required, nanoscale particle size is achieved without homogenization/sonication, and the solvent used is safe (class 3). Moreover, the method is adaptable to continuous processing, which makes it suitable for large scale production.
Methods: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a lipid with low glass transition temperature (Tg), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (DSPE-PEG(2000) folate) were used in various proportions to design the liposomes. The lipids and docetaxel (DXL), a widely used anticancer drug, were co-dissolved in dehydrated ethanol and injected into continuously stirred water at various flow rates using a syringe pump (New Era Pump Systems, model NE-1000). Experiments were conducted at both room temperature and 4°C and alcohol was removed via evaporation. Particle size and zeta potential were measured via dynamic light scattering (NICOMP 380 ZLS), and DXL loading in liposomes was determined by HPLC analysis.
Results: Blank liposomes, DXL-liposomes and DXL-folate-liposomes were prepared using ethanol injection. The average particle size was 91 nm for blank liposomes, 96 nm for DXL-liposomes and 49 nm for DXL-folate-liposomes (Fig 1), which places them in a suitable size range for passive targeting of tumors via enhanced permeation and retention (EPR). Particle size was dependent on both the chemical composition and concentration of lipid. The average zeta potential was +30 mV for blank liposomes, +37 mV for DXL-liposomes, and +30 mV for DXL-folate-liposomes. The net positive charge on the liposomes is expected to aid their cellular uptake. Interestingly, the pumping flow rate of the ethanolic lipid solution (1.5, 2.5, 3.5, 4.5 ml/min) had minimal effect on particle size and uniformity. The key determining factors behind stable liposome formation with a narrow size range were the ability to introduce a continuous thin jet of the alcoholic solution of drug and lipids and the rate of non-turbulent stirring of the aqueous phase. We aimed for the lowest possible turbulence, determined by the stirring rate, the size of the mixing chamber, the size of the magnetic stirrer and the capillary size and shape of the injection needle. A stirring rate of 700 RPM and a flow rate of 2.5 ml/min through a 23G stainless steel needle produced the most stable monodisperse liposomes with narrow size range. Visually, this combination of parameters created a smooth fluid vortex that traveled from the point of injection to the bottom of the container. The drug loading efficiency was higher at 4°C (nearly 100%) compared to room temperature, and it was confirmed that some of the DXL precipitates in the aqueous phase at room temperature. DOPE liposomes incorporating cholesterol (16% w/w), DSPE-PEG(2000) folate (2% w/w) and DXL (30 µmol) were selected for further studies.
Conclusion: Ethanol injection was a simple and reproducible method to produce stable and uniform liposomes with a narrow size range and high drug loading capacity. This method eliminates the need for thermal and mechanical stress, and the class 2 solvents that are generally used for the preparation of liposomes. We identified suitable liposomes for future cytotoxicity studies in 2D and 3D culture models of MDA-MB-231, a triple negative breast cancer cell line.
Nandita Das– Professor of pharmaceutics & drug delivery, Butler University, Indianapolis, Indiana
Sudip Das– Professor of pharmaceutics & drug delivery and Director of graduate program, Butler University, Indianapolis, Indiana