Wyatt N. Vreeland

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.

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.

Effects of temperature, acyl chain length, and flow-rate ratio on liposome formation and size in a microfluidic hydrodynamic focusing device

Microfluidic hydrodynamic focusing of an alcohol–lipid mixture into a narrow fluid stream by two oblique buffer streams provides a controlled and reproducible method of forming phospholipid bilayer vesicles (i.e., liposomes) with relatively monodisperse and specific size ranges. Previous work has established that liposome size can be controlled by changing the relative and absolute flow rates of the fluids. In other previous work, a kinetic (non-equilibrium) theoretical description of the detergent dilution liposome formation method was developed, in which planar lipid bilayer discs aggregate until they become sufficiently large to close into spherical liposomes. In this work, we show that an approximation of the kinetic theory can help explain liposome formation for our microfluidic method. This approximation predicts that the liposome radius should be approximately proportional to the ratio of the membrane bending elasticity modulus to the line tension of the hydrophobic edges of the lipid bilayer disc. In combination with very fast microfluidic mixing, this theory enables a new method to measure the ratio of the elasticity modulus to the line tension of membranes. The theory predicts that the temperature should change the liposome size primarily as a result of its effect on the ratio of the membrane bending elasticity modulus to the line tension, in contrast to previous work on microdroplet and microbubble formation, which showed that the effect of temperature on droplet/bubble size was primarily due to viscosity changes. In agreement with theory, most membrane compositions form larger liposomes close to or below the gel-to-liquid crystalline phase transition temperature, where the membrane elasticity modulus is much larger, and they have a much smaller dependence of size on temperature far above the transition temperature, where the membrane elasticity modulus is relatively constant. Other parameters modulated by the temperature (e.g., viscosity, free energy, and diffusion coefficients) appear to have little or no effect on liposome size, because they have counteracting effects on the lipid aggregation rate and the liposome closure time. Experiments are performed using phospholipids with varying hydrophobic acyl chain lengths that have phase transition temperatures ranging from −1 °C to 55 °C, so that the temperature dependence is examined below, above, and around the transition temperature. In addition, the effect of IPA stabilizing the edges of the bilayer discs can be examined by comparing the liposome sizes obtained at different flow-rate ratios. Finally, polydispersity is shown to increase as the median liposome size increases, regardless of whether the change in size is due to changing temperature or flow-rate ratio.

Microfluidic Synthesis of PEG- and Folate-Conjugated Liposomes for One-Step Formation of Targeted Stealth Nanocarriers

ABSTRACTPurposeA 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.MethodsControlled 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.ResultsPEG 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.ConclusionsLiposomes 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.

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.

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 QDs 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.

Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines. Theoretical model.

We recently demonstrated that the molecular mass distribution of an uncharged polymer sample can be analyzed using free-solution capillary electrophoresis of DNA-polymer conjugates. In these conjugates, the DNA is providing the electromotive force while the uncharged polydisperse polymer chains of the sample retard the DNA engine with different amounts of hydrodynamic drag. Here we present a theoretical model of this new analytical method. We show that for the most favourable, diffusion-limited electrophoresis conditions, there is actually an optimal DNA size to achieve the separation of a given polymer sample. Moreover, we demonstrate that the effective friction coefficient of the polymer chains is related to the stiffness of the two polymers of the conjugate, thus offering a method to estimate the persistence length of the uncharged polymer through mobility measurements. Finally, we compare some of our predictions with available experimental results.

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.

Liposome-based delivery of a boron-containing cholesteryl ester for high-LET particle-induced damage of prostate cancer cells: A boron neutron capture therapy study

Abstract Purpose: The efficacy of a boron-containing cholesteryl ester compound (BCH) as a boron neutron capture therapy (BNCT) agent for the targeted irradiation of PC-3 human prostate cancer cells was examined. Materials and methods: Liposome-based delivery of BCH was quantified with inductively coupled plasma-mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC). Cytotoxicity of the BCH-containing liposomes was evaluated with neutral red, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and lactate dehydrogenase assays. Colony formation assays were utilized to evaluate the decrease in cell survival due to high-linear energy transfer (LET) particles resulting from 10B thermal neutron capture. Results: BCH delivery by means of encapsulation in a lipid bilayer resulted in a boron uptake of 35.2 ± 4.3 μg/109 cells, with minimal cytotoxic effects. PC-3 cells treated with BCH and exposed to a 9.4 × 1011 n/cm2 thermal neutron fluence yielded a 20–25% decrease in clonogenic capacity. The decreased survival is attributed to the generation of high-LET α particles and 7Li nuclei that deposit energy in densely ionizing radiation tracks. Conclusion: Liposome-based delivery of BCH is capable of introducing sufficient boron to PC-3 cells for BNCT. High-LET α particles and 7Li nuclei generated from 10B thermal neutron capture significantly decrease colony formation ability in the targeted PC-3 cells.

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.