In collaboration with the University of Chemistry and Technology Prague, who invited our team at the Institute of Physics (FZU), we ran in-situ experiments to check whether cholesterol crystallizes inside niosomes and, if it does, what crystal forms appear directly in liquid samples. Niosomes are tiny vesicles made from non-ionic surfactants and are often stabilized by adding cholesterol. But if too much cholesterol is used, the excess doesn’t fit into the membrane and can remain as separate microcrystals that is an unwanted outcome for drug delivery.
The article investigates how cholesterol is incorporated into niosomes, tiny vesicles made from non-ionic surfactants and commonly used as drug delivery carriers in medicine. Niosomes are similar to liposomes but are more stable and cost-effective, making them attractive for pharmaceutical applications. Cholesterol is usually added to niosomes to make their structure more stable and to improve their ability to trap drugs inside. However, if too much cholesterol is used, not all of it fits into the niosomal membrane, and some remains as separate cholesterol crystals in the solution. This is a problem because these crystals can cause unwanted effects in the body, such as acting as seeds for further crystal growth or contributing to blockages in blood vessels.
To address this, niosomes were prepared using two common methods and studied using advanced techniques like static and dynamic light scattering, X-ray diffraction, optical and confocal microscopy, and especially a novel approach called depolarized dynamic light scattering (DDLS). DDLS allows for detecting crystalline cholesterol in the liquid suspension without needing to dry the sample, which is vital because drying can change the structure and lead to misleading results. The study showed that DDLS, combined with cryo-transmission electron microscopy (cryo-TEM) and X-ray diffraction, is a fast and reliable way to check if any cholesterol crystals are present in niosomal samples.
The article proves there is a limit to how much cholesterol can be incorporated into the niosomal membrane. The cholesterol crystals formed when this limit is exceeded can be detected using the new DDLS-based method. The research provides a valuable quality control tool for the pharmaceutical industry, ensuring that niosomal drug carriers are safe and effective for medical use.
As part of this research, we performed in-situ screening of cholesterol forms and conducted powder diffraction experiments, which are necessary for detecting trace amounts of cholesterol crystals. The powder diffraction experiment was challenging due to the extremely low crystal content in the samples.
Fig. Results of the X-ray powder diffraction experiments show weak signals from individual cholesterol crystals. These measurements confirm that cholesterol is present in a crystalline state and that it can form multiple crystal structures. A - Comparison of XRPD patterns for niosomes made by organic-phase injection with slow addition and by thin-film hydration followed by ultrasound mixing. Even though the signals are weak, the distinct peak “fingerprints” show cholesterol is present in a crystalline state and that the two preparation routes give different crystal forms.