(201ah) A Novel Technique for Rapidly Synthesizing Small Unilamellar Liposomes with High Encapsulation Efficiencies

Since their initial discovery in 1964, liposomes have been recognized for their potential as drug delivery systems. These colloidal, vesicle-like particles are amphipathic and can accommodate both hydrophobic and hydrophilic molecules. However, despite being one of the most well studied nano-delivery systems, only a handful of formulations have received FDA approval. The foremost impediment in the development of liposomal delivery systems is that existing protocols used for synthesizing and encapsulating therapeutic laden small unilamellar liposomes are complex and require specialized facilities to produce populations of monodisperse particles. Furthermore, the encapsulation efficiencies of these techniques are excruciatingly low, with only ~1% of the total drug being sequestered within the core of the liposomes. This poses a major roadblock in the design and implementation of liposome delivery systems, both as point of care and mass produced therapeutics.

Results

Here, we demonstrate a unique approach for rapidly and efficiently producing, purifying, and concentrating small drug loaded liposomes with high encapsulation efficiencies using common benchtop equipment. We have thoroughly refined and customized an injection method to synthesize small unilamellar liposomes, with mean diameters of 80 nm and polydispersities of 0.13. This approach does not require the tedious secondary post processing steps (e.g. extrusion and ultracentrifugation) typically used in liposome synthesis. Following this, we efficiently encapsulate a wide range of dextrans (300 to 20,000 Da) representing small and large molecular drug formulations. This method does not affect the liposome characteristics, nor does it alter the chemistry of the therapeutic like active loading methods such as precipitation method do. We then demonstrate removal of 99.9% of the non-encapsulated molecules using a sequential filtration centrifugation technique, largely eliminating the need for repetitive ultracentrifugation protocols currently employed in nano-research facilities. Finally, the functional efficacy of loaded liposomes as drug delivery vehicles was validated by encapsulating the fluorescent chemotherapeutic doxorubicin and observing the liposomal release and subsequent uptake of the drug by metastatic breast cancer cells (MDA-MB-231) in vitro.

Conclusion

Our simplified technique addresses the existing challenges associated with liposome preparation in resource limited settings and offers significant potential for advances in translational pharmaceutical development, both in basic research and point-of-care formulations. It is our hope that with this efficient and accessible approach, the discovery of novel liposomal formulations may be better researched and more widely implemented in clinical settings.