Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)

The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems

Label-free imaging on waveguide platform with enhanced resolution and contrast

Chip-based Evanescent Light Scattering (cELS) utilizes the multiple modes of a high-index contrast optical waveguide for near-field illumination of unlabeled samples, thereby repositioning the highest spatial frequencies of the sample into the far-field. The multiple modes scattering off the sample with different phase differences is engineered to have random spatial distributions within the integration time of the camera, mitigating the coherent speckle noise. This enables label-free superior-contrast imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs) and liposomes, dynamics of living HeLa cells etc. We demonstrate a multi-moded straight waveguide as a partially coherent light source. For isotropic super-resolution, spatially incoherent light engineered via multiple-arms waveguide chip and intensity-fluctuation based algorithms are used. The proof-of-concept results are demonstrated on 100 nm polystyrene beads and resolution improvement of close to 2× is shown. cELS also realizes (2-10)× more contrast as opposed to conventional imaging techniques

Microscopy Meets Nanomedicine: The Challenge of Liposomes. Selecting, Understanding, and Adapting Imaging Techniques to Localize and Characterize Nanocarriers

Nanocarriers have brought several medical advances through better diagnostics and improved drug therapy. Yet, many promising preclinical findings were never translated into clinical success, consequently slowing drug development, and increasing its financial burden. By reliably predicting the nanoparticle fate at in vitro stages, the disappointment of suboptimal in vivo outcome could be avoided. To tackle the challenges of in vitro settings, this project aimed at gaining deeper insight on the interaction between nanocarriers and biological environment. Specifically, advance microscopy tools were used to visualize, characterize, and follow the biological fate of nanocarriers. As a back-to-basics approach, liposomes were chosen as model nanocarriers for their versatility, biocompatibility, and clinical relevance. When including a fluorescent molecule in the liposomal formulation, fluorescence dye and nanocarrier were found to affect each other’s properties in a manner dependent on environmental conditions (e.g., temperature, time, medium, and dye-specific chemistry). The fluctuations of fluorescence in the sample were further analyzed through image processing algorithms to obtain super resolution information from a diffraction-limited multi-frame acquisition. In parallel, to overcome some of the disadvantages often linked to the use of fluorescence, quantitative phase microscopy was optimized as a complementary label-free technique for the localization and characterization of liposomes in their hydrate state. Finally, fluorescence and label-free imaging were combined to determine the integrity of liposomes in nanofibers for topical administration. To understand the behavior of liposomes in cell culture, their internalization was followed using high throughput screenings, based on flow cytometry. These batch-mode results were validated in flow imaging and confocal microscopy. The behavior of liposomes, with and without PEG-coating, was compared in terms of intracellular localization and overall cellular response, resorting to the combination of fluorescence and label-free imaging (here, confocal and electron microscopy). Volumetric image correlation was then attempted, discussing benefits and limitations of the methods involved

Investigation of the influence of standard sequestering agents on the antimicrobial activity of a series of cytotoxic active pharmaceutical ingredients

L'interazione tra ciclodestrine (CD) e principi attivi (AP) in soluzione è stata testata con due metodi termodinamici (studi di solubilità di fase e studi di diffusione) per calcolare la stabilità del complessamento. Due antimicrobici sono quindi stati testati in combinazione con β-ciclodestrine modificate su colture di E. coli per verificare l'andamento della crescita batterica in funzione delle concentrazioni relative di AP e CD

Following the fate of dye-containing liposomes in vitro

The rather limited success of translation from basic research to clinical application has been highlighted as a major issue in the nanomedicine field. To identify the factors influencing the applicability of nanosystems as drug carriers and potential nanomedicine, we focused on following their fate through fluorescence-based assays, namely flow cytometry and imaging. These methods are often used to follow the nanocarrier internalization and targeting; however, the validity of the obtained results strictly depends on how much the nanosystem’s fate can be inferred from the fate of fluorescent dyes. To evaluate the parameters that affect the physicochemical and biological stability of the labeled nanosystems, we studied the versatility of two lipid dyes, TopFluor®-PC and Cy5-DSPE, in conventional liposomes utilizing well-defined in vitro assays. Our results suggest that the dye can affect the major characteristics of the system, such as vesicle size and zeta-potential. However, a nanocarrier can also affect the dye properties. Medium, temperature, time, fluorophore localization and its concentration, as well as their interplay, affect the outcome of tracing experiments. Therefore, an in-depth characterization of the labeled nanosystem should be fundamental to understand the conditions that validate the results within the screening process in optimization of nanocarrier

Experimental Determination of Drug Diffusion Coefficients in Unstirred Aqueous Environments by Temporally Resolved Concentration Measurements

The diffusion coefficient (also known as diffusivity) of an active pharmaceutical ingredient (API) is a fundamental physicochemical parameter that affects passive diffusion through biological barriers and, as a consequence, bioavailability and biodistribution. However, this parameter is often neglected, and it is quite difficult to find diffusion coefficients of small molecules of pharmaceutical relevance in the literature. The available methods to measure diffusion coefficients of drugs all suffer from limitations that range from poor sensitivity to high selectivity of the measurements or the need for dedicated instrumentation. In this work, a simple but reliable method based on time-resolved concentration measurements by UV-visible spectroscopy in an unstirred aqueous environment was developed. This method is based on spectroscopic measurement of the variation of the local concentration of a substance during spontaneous migration of molecules, followed by standard mathematical treatment of the data in order to solve Fick's law of diffusion. This method is extremely sensitive and results in highly reproducible data. The technique was also employed to verify the influence of the environmental characteristics (i.e., ionic strength and presence of complexing agents) on the diffusivity. The method can be employed in any research laboratory equipped with a standard UV-visible spectrophotometer and could become a useful and straightforward tool in order to characterize diffusion coefficients in physiological conditions and help to better understand the drug permeability process

Multi-moded high-index contrast optical waveguide for super-contrast high-resolution label-free microscopy

The article elucidates the physical mechanism behind the generation of superior-contrast and highresolution label-free images using an optical waveguide. Imaging is realized by employing a high index contrast multi-moded waveguide as a partially coherent light source. The modes provide near-field illumination of unlabeled samples, thereby repositioning the higher spatial frequencies of the sample into the far-field. These modes coherently scatter off the sample with different phases and are engineered to have random spatial distributions within the integration time of the camera. This mitigates the coherent speckle noise and enhances the contrast (2–10) × as opposed to other imaging techniques. Besides, the coherent scattering of the different modes gives rise to fluctuations in intensity. The technique demonstrated here is named chip-based Evanescent Light Scattering (cELS). The concepts introduced through this work are described mathematically and the high-contrast image generation process using a multi-moded waveguide as the light source is explained. The article then explores the feasibility of utilizing fluctuations in the captured images along with fluorescence-based techniques, like intensity-fluctuation algorithms, to mitigate poor-contrast and diffraction-limited resolution in the coherent imaging regime. Furthermore, a straight waveguide is demonstrated to have limited angular diversity between its multiple modes and therefore, for isotropic sample illumination, a multiple-arms waveguide geometry is used. The concepts introduced are validated experimentally via high-contrast label-free imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs), liposomes, nanobeads and biological cells such as fixed and live HeLa cells

Experimental Determination of Drug Diffusion Coefficients in Unstirred Aqueous Environments by Temporally Resolved Concentration Measurements

The diffusion coefficient (also known as diffusivity) of an active pharmaceutical ingredient (API) is a fundamental physicochemical parameter that affects passive diffusion through biological barriers and, as a consequence, bioavailability and biodistribution. However, this parameter is often neglected, and it is quite difficult to find diffusion coefficients of small molecules of pharmaceutical relevance in the literature. The available methods to measure diffusion coefficients of drugs all suffer from limitations that range from poor sensitivity to high selectivity of the measurements or the need for dedicated instrumentation. In this work, a simple but reliable method based on time-resolved concentration measurements by UV–visible spectroscopy in an unstirred aqueous environment was developed. This method is based on spectroscopic measurement of the variation of the local concentration of a substance during spontaneous migration of molecules, followed by standard mathematical treatment of the data in order to solve Fick’s law of diffusion. This method is extremely sensitive and results in highly reproducible data. The technique was also employed to verify the influence of the environmental characteristics (i.e., ionic strength and presence of complexing agents) on the diffusivity. The method can be employed in any research laboratory equipped with a standard UV–visible spectrophotometer and could become a useful and straightforward tool in order to characterize diffusion coefficients in physiological conditions and help to better understand the drug permeability process