Particle Metrology Approach to Understanding How Storage Conditions Affect Long-Term Liposome Stability.

Lipid nanoparticles are a generic type of nanomaterial with broad applicability in medicine as drug delivery vehicles. Liposomes are a subtype of lipid nanoparticles and, as a therapeutic platform, can be loaded with a genetic material or pharmaceutical agents for use as drug treatments. An open question for these types of lipid nanoparticles is what factor(s) affect the long-term stability of the particles. The stability of the particle is of great interest to understand and predict the effective shelf-life and storage requirements. In this report, we detail a one-year study of liposome stability as a function of lipid composition, buffer composition/pH, and storage temperature. This was done in aqueous solution without freezing. The effect of lipid composition is shown to be a critical factor when evaluating stability of the measured particle size and number concentration. Other factors (i.e., storage temperature and buffer pH/composition) were shown to be less critical but still have some effect. The stability of these particles informs formulation and optimal storage requirements and assists with future developmental planning of a NIST liposome-based reference material. This work also highlights the complex nature of long-term soft particle storage in biopharmaceutical applications.

Microfluidic preparation of liposomes to determine particle size influence on cellular uptake mechanisms.

PURPOSE: This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40.6 nm to 276.6 nm. Liposomes were prepared by microfluidic hydrodynamic flow focusing, producing nearly-monodisperse populations and enabling size-dependent uptake to be effectively evaluated. METHODS: Populations of PEG-conjugated liposomes of various distinct sizes were prepared in a disposable microfluidic device using a simple continuous-flow microfluidic technique. Liposome cellular uptake was investigated using flow cytometry and confocal microscopy. RESULTS: Liposome uptake by Caco-2 cells was observed to be strongly size-dependent for liposomes with mean diameters ranging from 40.6 nm to 276.6 nm. When testing these liposomes against endocytosis inhibitors, cellular uptake of the largest (97.8 nm and 162.1 nm in diameter) liposomes were predominantly subjected to clathrin-dependent uptake mechanisms, the medium-sized (72.3 nm in diameter) liposomes seemed to be influenced by all investigated pathways and the smallest liposomes (40.6 nm in diameter) primarily followed a dynamin-dependent pathway. In addition, the 40.6 nm, 72.3 nm, and 162.1 nm diameter liposomes showed slightly decreased accumulation within endosomes after 1 h compared to liposomes which were 97.8 nm in diameter. Conversely, liposome co-localization with lysosomes was consistent for liposomes ranging from 40.6 nm to 97.8 nm in diameter. CONCLUSIONS: The continuous-flow synthesis of nearly-monodisperse populations of liposomes of distinct size via a microfluidic hydrodynamic flow focusing technique enabled unique in vitro studies in which specific effects of particle size on cellular uptake were elucidated. The results of this study highlight the significant influence of liposome size on cellular uptake mechanisms and may be further exploited for increasing specificity, improving efficacy, and reducing toxicity of liposomal drug delivery systems.

Microfluidic synthesis of PEG- and folate-conjugated liposomes for one-step formation of targeted stealth nanocarriers.

PURPOSE: A microfluidic hydrodynamic flow focusing technique enabling the formation of small and nearly monodisperse liposomes is investigated for continuous-flow synthesis of poly(ethylene glycol) (PEG)-modified and PEG-folate-functionalized liposomes for targeted drug delivery. METHODS: Controlled laminar flow in thermoplastic microfluidic devices facilitated liposome self-assembly from initial lipid compositions including lipid/cholesterol mixtures containing PEG-lipid and folate-PEG-lipid conjugates. Relationships among flow conditions, lipid composition, and liposome size were evaluated; their impact on PEG and folate incorporation were determined through a combination of UV-vis absorbance measurements and characterization of liposome zeta potential. RESULTS: PEG and folate were successfully incorporated into microfluidic-synthesized liposomes over the full range of liposome sizes studied. Efficiency of PEG-lipid incorporation was inversely correlated with liposome diameter. Folate-lipid was effectively integrated into liposomes at various flow conditions. CONCLUSIONS: Liposomes incorporating relatively large PEG-modified and folate-PEG-modified lipids were successfully synthesized using the microfluidic flow focusing platform, providing a simple, low cost, rapid method for preparing functionalized liposomes. Relationships between preparation conditions and PEG or folate-PEG functionalization have been elucidated, providing insight into the process and defining paths for optimization of the microfluidic method toward the formation of functionalized liposomes for pharmaceutical applications.

Number Concentration Measurements of Polystyrene Submicrometer Particles.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques. Diameter values were also measured where possible. The diameter values were found to agree within 20%, but the number concentration values differed by as much as a factor of two. Accuracy and reproducibility related with the different techniques are discussed with the goal of using number concentration standards for instrument calibration. Three of the techniques were used to determine SI-traceable number concentration values, and the three independent values were averaged to give consensus values. This consensus approach is proposed as a protocol for certifying SI-traceable number concentration standards.

Effect of Azide Preservative on Thermomechanical Aggregation of Purified Reference Protein Materials.

Protein aggregation can affect the quality of protein-based therapeutics. Attempting to unravel factors influencing protein aggregation involves systematic studies. These studies often include sodium azide or similar preservatives in the aggregation buffer. This work shows effects of azide on aggregation of two highly purified reference proteins, both a bovine serum albumin (BSA) as well as a monoclonal antibody (NISTmAb). The proteins were aggregated by thermomechanical stress, consisting of simultaneous heating of the solution with gentle agitation. Protein aggregates were characterized by asymmetric flow field flow fractionation (AF4) with light scattering measurements along with quantification by UV spectroscopy, revealing strong time-dependent generation of aggregated protein and an increase in aggregate molar mass. Gel electrophoresis was used to probe the reversibility of the aggregation and demonstrated complete reversibility for the NISTmAb, but not so for the BSA. Kinetic fitting to a commonly implemented nucleated polymerization model was also employed to provide mechanistic details into the kinetic process. The model suggests that the aggregation of the NISTmAb proceeds via nucleated growth and aggregate-aggregate condensation in a way that is dependent on the concentration (and presence) of the azide anion. This work overall implicates azide preservatives as having demonstrable effects on thermomechanical stress and aggregation of proteins undergoing systematic aggregation and stability studies.

Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Magnetic connectors for microfluidic applications.

We present a new type of microfluidic connector that employs a ring magnet on one side of the microfluidic chip and a disc magnet on the other side to produce a sealed connection between external tubing and inlets or outlets of microfluidic devices. The connectors are low-cost, simple to use and assemble, and reusable. We used numerical (finite element) simulations in order to optimize their geometry. Configurations that achieve interfacial forces in the range of 2 N to 15 N are discussed. Several types of gasket materials were explored. Finally, we demonstrate an application of these connectors in a microfluidic device used to generate liposomes.

Accurate optical analysis of single-molecule entrapment in nanoscale vesicles.

We present a nondestructive method to accurately characterize low analyte concentrations (0-10 molecules) in nanometer-scale lipid vesicles. Our approach is based on the application of fluorescence fluctuation analysis (FFA) and multiangle laser light scattering (MALLS) in conjunction with asymmetric field flow fractionation (AFFF) to measure the entrapment efficiency (the ratio of the concentration of encapsulated dye to the initial bulk concentration) of an ensemble of liposomes with an average diameter less than 100 nm. Water-soluble sulforhodamine B (SRB) was loaded into the aqueous interior of nanoscale liposomes synthesized in a microfluidic device. A confocal microscope was used to detect a laser-induced fluorescence signal resulting from both encapsulated and unencapsulated SRB molecules. The first two cumulants of this signal along with the autocorrelation function (ACF) were used to quantify liposome entrapment efficiency. Our analysis moves beyond typical, nonphysical assumptions of equal liposome size and brightness. These advances are essential for characterizing liposomes in the single-molecule encapsulation regime. Our work has further analytical impact because it could increase the interrogation time of free-solution molecular analysis by an order of magnitude and form the basis for the development of liposome standard reference materials.

Controlled self-assembly of monodisperse niosomes by microfluidic hydrodynamic focusing.

Niosomes are synthetic membrane vesicles formed by self-assembly of nonionic surfactant, often in a mixture with cholesterol and dicetyl phosphate. Because of their inner aqueous core and bilayer membrane shell, niosomes are commonly used as carriers of treatment agents for pharmaceutical and cosmetic applications or contrast agents for clinical imaging applications. In those applications, niosomes are considered as a more economical and stable alternative to their biological counterpart (i.e., liposomes). However, conventional bulk method of niosome preparation requires bulk mixing of two liquid phases, which is time-consuming and not well-controlled. Such mixing conditions often lead to large niosomes with high polydispersity in size and thus affect the consistency of niosome dosage or imaging quality. In this study, we present a new method of niosome self-assembly by microfluidic hydrodynamic focusing to improve on the size and size distributions of niosomes. By taking advantage of the rapid and controlled mixing of two miscible fluids (i.e., alcohol and water) in microchannels, we were able to obtain in seconds nanoscaled niosomes with approximately 40% narrower size distributions compared to the bulk method. We further investigated different parameters that might affect on-chip assembly of niosomes, such as (1) conditions for the microfluidic mixing, (2) chemical structures of the surfactant used (i.e., sorbitan esters Span 20, Span 60, and Span 80), and (3) device materials for the microchannel fabrication. This work suggests that microfluidics may facilitate the development and optimization of biomimetic colloidal systems for nanomedicine applications.

Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This article provides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic. Current challenges limiting development of EVs for drug delivery are the lack of: standardized cell-based platforms for the production of EV-based therapeutics; EV reference materials that allow researchers/manufacturers to validate EV measurements and standardized measurement systems for determining the molecular composition of EVs.

Using Image Attributes to Assure Accurate Particle Size and Count Using Nanoparticle Tracking Analysis.

Nanoparticle tracking analysis (NTA) obtains particle size by analysis of particle diffusion through a time series of micrographs and particle count by a count of imaged particles. The number of observed particles imaged is controlled by the scattering cross-section of the particles and by camera settings such as sensitivity and shutter speed. Appropriate camera settings are defined as those that image, track, and analyze a sufficient number of particles for statistical repeatability. Here, we test if image attributes, features captured within the image itself, can provide measurable guidelines to assess the accuracy for particle size and count measurements using NTA. The results show that particle sizing is a robust process independent of image attributes for model systems. However, particle count is sensitive to camera settings. Using open-source software analysis, it was found that a median pixel area, 4 pixels2, results in a particle concentration within 20% of the expected value. The distribution of these illuminated pixel areas can also provide clues about the polydispersity of particle solutions prior to using a particle tracking analysis. Using the median pixel area serves as an operator-independent means to assess the quality of the NTA measurement for count.

Asymmetric flow field flow fractionation with light scattering detection - an orthogonal sensitivity analysis.

Asymmetric flow field flow fractionation (AF4) has several instrumental factors that may have a direct effect on separation performance. A sensitivity analysis was applied to ascertain the relative importance of AF4 primary instrument factor settings for the separation of a complex environmental sample. The analysis evaluated the impact of instrumental factors namely, cross flow, ramp time, focus flow, injection volume, and run buffer concentration on the multi-angle light scattering measurement of natural organic matter (NOM) molar mass (MM). A 2(5-1) orthogonal fractional factorial design was used to minimize analysis time while preserving the accuracy and robustness in the determination of the main effects and interactions between any two instrumental factors. By assuming that separations resulting in smaller MM measurements would be more accurate, the analysis produced a ranked list of effects estimates for factors and interactions of factors based on their relative importance in minimizing the MM. The most important and statistically significant AF4 instrumental factors were buffer concentration and cross flow. The least important was ramp time. A parallel 2(5-2) orthogonal fractional factorial design was also employed on five environmental factors for synthetic natural water samples containing silver nanoparticles (NPs), namely: NP concentration, NP size, NOM concentration, specific conductance, and pH. None of the water quality characteristic effects or interactions were found to be significant in minimizing the measured MM; however, the interaction between NP concentration and NP size was an important effect when considering NOM recovery. This work presents a structured approach for the rigorous assessment of AF4 instrument factors and optimal settings for the separation of complex samples utilizing efficient orthogonal factional factorial design and appropriate graphical analysis.

Functional materials for microscale genomic and proteomic analyses.

The design of functional materials for genomic and proteomic analyses in microscale systems has begun to mature, from materials designed for capillary-based electrophoresis systems to those tailored for microfluidic-based or 'chip-based' platforms. In particular, recent research has focused on evaluating different polymer chemistries for microchannel surface passivation and improved DNA separation matrix performance. Additionally, novel bioconjugate materials designed specifically for electrophoretic separations in microscale channels are facilitating new separation modalities.

Using light scattering to evaluate the separation of polydisperse nanoparticles.

The analysis of natural and otherwise complex samples is challenging and yields uncertainty about the accuracy and precision of measurements. Here we present a practical tool to assess relative accuracy among separation protocols for techniques using light scattering detection. Due to the highly non-linear relationship between particle size and the intensity of scattered light, a few large particles may obfuscate greater numbers of small particles. Therefore, insufficiently separated mixtures may result in an overestimate of the average measured particle size. Complete separation of complex samples is needed to mitigate this challenge. A separation protocol can be considered improved if the average measured size is smaller than a previous separation protocol. Further, the protocol resulting in the smallest average measured particle size yields the best separation among those explored. If the differential in average measured size between protocols is less than the measurement uncertainty, then the selected protocols are of equivalent precision. As a demonstration, this assessment metric is applied to optimization of cross flow (V(x)) protocols in asymmetric flow field flow fractionation (AF(4)) separation interfaced with online quasi-elastic light scattering (QELS) detection using mixtures of polystyrene beads spanning a large size range. Using this assessment metric, the V(x) parameter was modulated to improve separation until the average measured size of the mixture was in statistical agreement with the calculated average size of particles in the mixture. While we demonstrate this metric by improving AF(4) V(x) protocols, it can be applied to any given separation parameters for separation techniques that employ dynamic light scattering detectors.

A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array.

A novel microscale device has been developed to enable the one-step continuous flow assembly of monodisperse nanoscale liposomes using three-dimensional microfluidic hydrodynamic focusing (3D-MHF) in a concentric capillary array. The 3D-MHF flow technique displays patent advantages over conventional methods for nanoscale liposome manufacture (i.e., bulk-scale alcohol injection and/or sonication) through the on-demand synthesis of consistently uniform liposomes without the need for post-processing strategies. Liposomes produced by the 3D-MHF device are of tunable size, have a factor of two improvement in polydispersity, and a production rate that is four orders of magnitude higher than previous MHF methods, which can be attributed to entirely radially symmetric diffusion of alcohol-solvated lipid into an aqueous flow stream. Moreover, the 3D-MHF platform is simple to construct from low-cost, commercially-available components, which obviates the need for advanced microfabrication strategies necessitated by previous MHF nanoparticle synthesis platforms.

Microfluidic-enabled liposomes elucidate size-dependent transdermal transport.

Microfluidic synthesis of small and nearly-monodisperse liposomes is used to investigate the size-dependent passive transdermal transport of nanoscale lipid vesicles. While large liposomes with diameters above 105 nm are found to be excluded from deeper skin layers past the stratum corneum, the primary barrier to nanoparticle transport, liposomes with mean diameters between 31-41 nm exhibit significantly enhanced penetration. Furthermore, multicolor fluorescence imaging reveals that the smaller liposomes pass rapidly through the stratum corneum without vesicle rupture. These findings reveal that nanoscale liposomes with well-controlled size and minimal size variance are excellent vehicles for transdermal delivery of functional nanoparticle drugs.

Dietary CdSe/ZnS quantum dot exposure in estuarine fish: bioavailability, oxidative stress responses, reproduction, and maternal transfer.

Continued development, use, and disposal of quantum dots (QDs) ensure their entrance into aquatic environments where they could pose a risk to biological organisms as whole nanoparticles or as degraded metal constituents. Reproductive Fundulus heteroclitus were fed a control diet with lecithin, diets containing 1 or 10 µg of lecithin-encapsulated CdSe/ZnS QD/day, or a diet containing 5.9 µg CdCl2/day for 85 days. Cadmium concentrations in liver, intestine, and eggs were quantified with inductively coupled plasma mass spectrometry. In fish fed 10 µg QD/day, QDs or their degradation products traversed the intestinal epithelia and accumulated in the liver. Less than 0.01% of the QD's cadmium was retained in the liver or intestinal tissues. This compares to 0.9% and 0.5% of the cadmium in the intestine and liver, respectively of fish fed a CdCl2 diet. Cadmium was also detected in the eggs from parents fed 10 µg QD/day. No significant changes in hepatic total glutathione, lipid peroxidation, or expression of genes involved in metal metabolism or oxidative stress were observed. While QDs in the diet are minimally bioavailable, unusual levels of vitellogenin transcription in male fish as well as declining fecundity require further investigation to determine if endocrine disruption is of environmental concern.

Microfluidic mixing and the formation of nanoscale lipid vesicles.

We investigate the formation of unilamellar lipid vesicles (liposomes) with diameters of tens of nanometers by controlled microfluidic mixing and nanoparticle determination (COMMAND). Our study includes liposome synthesis experiments and numerical modeling of our microfluidic implementation of the batch solvent injection method. We consider microfluidic liposome formation from the perspective of fluid interfaces and convective-diffusive mixing, as we find that bulk fluid flow parameters including hydrodynamically focused alcohol stream width, final alcohol concentration, and shear stress do not primarily determine the vesicle formation process. Microfluidic device geometry in conjunction with hydrodynamic flow focusing strongly influences vesicle size distributions, providing a coarse method to control liposome size, while total flow rate allows fine-tuning the vesicle size in certain focusing regimes. Although microfluidic liposome synthesis is relatively simple to implement experimentally, numerical simulations of the mixing process reveal a complex system of fluid flow and mass transfer determining the formation of nonequilibrium vesicles. These results expand our understanding of the microfluidic environment that controls liposome self-assembly and yield several technological advances for the on-chip synthesis of nanoscale lipid vesicles.

Liposome-templated supramolecular assembly of responsive alginate nanogels.

Nanosized gel particles (nanogels) are of interest for a variety of applications, including drug delivery and single-molecule encapsulation. Here, we employ the cores of nanoscale liposomes as reaction vessels to template the assembly of calcium alginate nanogels. For our experiments, a liposome formulation with a high bilayer melting temperature (Tm) is selected, and sodium alginate is encapsulated in the liposomal core. The liposomes are then placed in an aqueous buffer containing calcium chloride, and the temperature is raised up to Tm. This allows permeation of Ca2+ ions through the bilayer and into the core, whereupon these ions gel the encapsulated alginate. Subsequently, the lipid bilayer covering the gelled core is removed by the addition of a detergent. The resulting alginate nanogels have a size distribution consistent with that of the template liposomes (ca. 120-200 nm), as confirmed by transmission electron microscopy and light scattering. Nanogels of different average sizes can be synthesized by varying the template dimensions, and the gel size can be further tuned after synthesis by the addition of monovalent salt to the solution.

Microfluidic directed formation of liposomes of controlled size.

A new method to tailor liposome size and size distribution in a microfluidic format is presented. Liposomes are spherical structures formed from lipid bilayers that are from tens of nanometers to several micrometers in diameter. Liposome size and size distribution are tailored for a particular application and are inherently important for in vivo applications such as drug delivery and transfection across nuclear membranes in gene therapy. Traditional laboratory methods for liposome preparation require postprocessing steps, such as sonication or membrane extrusion, to yield formulations of appropriate size. Here we describe a method to engineer liposomes of a particular size and size distribution by changing the flow conditions in a microfluidic channel, obviating the need for postprocessing. A stream of lipids dissolved in alcohol is hydrodynamically focused between two sheathed aqueous streams in a microfluidic channel. The laminar flow in the microchannel enables controlled diffusive mixing at the two liquid interfaces where the lipids self-assemble into vesicles. The liposomes formed by this self-assembly process are characterized using asymmetric flow field-flow fractionation combined with quasi-elastic light scattering and multiangle laser-light scattering. We observe that the vesicle size and size distribution are tunable over a mean diameter from 50 to 150 nm by adjusting the ratio of the alcohol-to-aqueous volumetric flow rate. We also observe that liposome formation depends more strongly on the focused alcohol stream width and its diffusive mixing with the aqueous stream than on the sheer forces at the solvent-buffer interface.