Spectroscopic Studies as a Toolbox for Biophysical and Chemical Characterization of Lipid-Based Nanotherapeutics

Abstract

The goal of this study is to provide tools to minimize trial-and-error in the development of novel lipid-based nanotherapeutics, in favor of a rational design process. For this purpose, we present case-study examples of biophysical assays that help addressing issues of lipid-based nanotherapeutics' profiling and assist in the design of lipid nanocarriers for therapeutic usage. The assays presented are rooted in spectroscopic methods (steady-state and time-resolved fluorescence; UV-Vis derivative spectroscopy; fluorescence anisotropy and fluorescence lifetime image microscopy) and allow accessing physical-chemical interactions between drugs and lipid nanocarriers, as well as studying interactions between lipid-based nanotherapeutics and membranes and/or proteins, as this is a key factor in predicting their therapeutic and off target effects. Derivative spectroscopy revealed Naproxen's high distribution (LogD ≈ 3) in different lipid-based nanocarriers (micelles and unilamellar or multilamellar vesicles) confirming the adequacy of such systems for encapsulating this anti-inflammatory drug. Fluorescence quenching studies revealed that the anti-inflammatory drugs Acemetacin and Indomethacin can reach an inner location at the lipid nanocarrier while being anchored with its carboxylic moiety at the polar headgroup. The least observed quenching effect suggested that Tolmetin is probably located at the polar headgroup region of the lipid nanocarriers and this superficial location may translate in a fast drug release from the nanocarriers. Fluorescent anisotropy measurements indicated that the drugs deeply buried within the lipid nanocarrier where the ones that had a greater fluidizing effect which can also translate in a faster drug release. The drug binding strength to serum albumin was also compared for a free drug (Clonixin) or for the same drug after encapsulation in a lipid nanocarrier DSPC:DODAP (2:1). Under both conditions there is a strong binding to serum albumin, at one binding site, suggesting the need to produce a stealth nanosystem. Finally the cellular uptake of lipid nanocarriers loaded with Daunorubicin was investigated in cancer cells using fluorescence lifetime imaging microscopy. From the images obtained it was possible to conclude that even at short incubation times (15 min) there was a distribution of the drug in the cytoplasm, whereas for longer incubation periods (4 h) the drug has reached the nucleus.

Rational Development of Liposomal Hydrogels: A Strategy for Topical Vaginal Antiretroviral Drug Delivery in the Context of HIV Prevention

“…In addition, understanding the formulation targets as well as the barriers they impose is of major importance and may dictate new approaches in the formulation development process, such as drug–membrane interaction studies. These are also of use in providing better knowledge of the drugs and guiding the development of formulation [29,35].…”

“…The evaluation of possible changes in the microviscosity of the liposomal formulation upon drug encapsulation was also required, as it is a helpful parameter to be considered in the choice of the most adequate formulation. For example, if including the drug in the liposomal formulation alters considerably its microviscosity, it can result in the liposomal system becoming too fluid to carry the drug during the required time after administration, and the lipid composition should perhaps be revised to convey more rigid domains [29]. Microviscosity changes induced by TDF encapsulation in lipid formulations of different fluidity (DMPC, DPPC, DSPC) were further evaluated by determining T m (main phase transition temperature at which the liposomes undergo conformational changes from the gel phase to the fluid phase) and B (cooperativity of the phase transition, which provides evidences if the drug is homogeneously distributed).…”

“…The final lipid concentration in each sample was approximately 15–20% w / v . After the total dissolution of the compounds, the lipid suspensions were prepared as previously described [29] and later transferred into X-ray transparent glass capillaries with 1.5-mm diameter (Hilgenberg, Malsfeld, Germany), which were flame-sealed and stored at 4 °C until measurement.…”

Lipid Nanosystems and Serum Protein as Biomimetic Interfaces: Predicting the Biodistribution of a Caffeic Acid-Based Antioxidant

“…[19][20][21][22][23][24][25][26][27][28][29][30] In our previous works, as well as in other studies, it is reported the correlations between the biological action and cytotoxicity of drugs and their biophysical effects in biomimetic membranes, eg by altering lipid membrane packing, decreasing lipid membrane transition temperatures or disturbing cooperativity of lipid unit cells, with consequent membrane biophysical impairment. 21,[26][27][28]31,32 Additionally, several interesting reports 19,20,[33][34][35][36][37] highlighted the importance of biomimetic models based on lipid nanosystems (liposomes, monolayers and supported lipid bilayers) to acquire molecular and functional information. The aim is using this acquired information to support the development of new drugs with better selectivity and reduced side effects 34 .…”

“…21,[26][27][28]31,32 Additionally, several interesting reports 19,20,[33][34][35][36][37] highlighted the importance of biomimetic models based on lipid nanosystems (liposomes, monolayers and supported lipid bilayers) to acquire molecular and functional information. The aim is using this acquired information to support the development of new drugs with better selectivity and reduced side effects 34 . This information can also assist to the design of new chemical entities with improved efficacy and reduced toxicity, 19,34,37 or even to understand the interaction of drug delivery nanosystems with biomembranes.…”