No abstract

Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair.

The production of freeze-dried liposomes encapsulating drugs is considered a key challenge since the drugs are prone to leakage. The aim of this work was to study the effect of different saccharides on preserving the stability and drug retention capacity of a previously developed liposomal formulation, when subjected to a freeze-drying process. The protective role of trehalose, lactose, glucose, mannitol and sucrose, known for their cryo/lyoprotective effect, was tested by addition of different concentrations to liposomes. Sucrose, in a concentration dependent manner (8:1 sugar:lipids mass ratio) proved to be a suitable cryo/lyoprotectant of these liposomes. Effectively, this saccharide prevents the fusion or/and aggregation of the liposomal formulation, protecting the integrity of the freeze-dried empty liposomes. The liposomal formulation containing sucrose was studied in terms of morphology, concentration, and anticancer drugs retention ability. The study involved two drugs encapsulated in the aqueous core, methotrexate (MTX) and doxorubicin (DOX), and one drug located in the lipid bilayer, tamoxifen (TAM). After the freeze-drying process, liposomes with sucrose encapsulating drugs revealed high physical stability, maintaining their narrow and monodisperse character, however high leakage of the drugs encapsulated in the aqueous core was observed. Otherwise, no significant drug leakage was detected on liposomes containing the TAM, which maintained its biological activity after the freeze-drying process. These findings reveal that sucrose is a good candidate for the cryo/lyoprotection of liposomes with drugs located in the lipid bilayer.

Liposomes are one of the most important and extensively studied drug delivery system due to their ability to encapsulate different kinds of drugs. Exploiting the advantages of 1 H Nuclear Magnetic Resonance (NMR) spectrometry, we established a rapid and easy method for quantification of drugs encapsulated in liposomes. An internal standard, pyridine, was used for quantitative determination of drug concentration. Two different drugs were involved in this work, one hydrophilic, methotrexate disodium salt, and another hydrophobic, tamoxifen. The specificity and selectivity of the suggested method were evaluated by the absence of overlapping of at least one signal of each drug with pyridine in the NMR spectrum. The accuracy and precision of the method were assessed by adding a known amount of each drug to unloaded liposomes. Results obtained by quantitative NMR (qNMR) were validated and confirmed by comparing with two other traditional techniques, Ultraviolet-Visible (UV-vis) spectrophotometry and High-Performance Liquid Chromatography (HPLC). It was found that the results were consistent with the ones obtained from our proposed qNMR method. Considering all the experiments conducted in this study, we deliberate that qNMR can be a suitable tool for the determination of drugs encapsulated in liposomes.

Methotrexate (MTX) is a common drug used to treat rheumatoid arthritis. Due to the excessive side effects, encapsulation of MTX in liposomes is considered an effective delivery system, reducing drug toxicity, while maintaining its efficacy. The ethanol injection method is an interesting technique for liposome production, due to its simplicity, fast implementation, and reproducibility. However, this method occasionally requires the extrusion process, to obtain suitable size distribution, and achieve a low level of MTX encapsulation. Here, we develop a novel pre-concentration method, based on the principles of the ethanol injection, using an initial aqueous volume of 20% and 1:1 ratio of organic:aqueous phase (v/v). The liposomes obtained present small values of size and polydispersity index, without the extrusion process, and a higher MTX encapsulation (efficiency higher than 30%), suitable characteristics for in vivo application. The great potential of MTX to interact at the surface of the lipid bilayer was shown by nuclear magnetic resonance (NMR) studies, revealing mutual interactions between the drug and the main phospholipid via hydrogen bonding. In vivo experiments reveal that liposomes encapsulating MTX significantly increase the biological benefit in arthritic mice. This approach shows a significant advance in MTX therapeutic applications.