Gas-liquid Microfluidic Reactors Research Articles

Effects of Microfluidic Shear on the Plasmid DNA Structure: Implications for Polymeric Gene Delivery Vectors.

Microfluidic manufacturing of advanced gene delivery vectors necessitates consideration of the effects of microfluidic shear forces on the structural integrity of plasmid DNA (pDNA). In this paper, we expose pDNA to variable shear forces in a two-phase, gas-liquid microfluidic reactor and apply gel electrophoresis to analyze the products of on-chip shear-induced degradation. The effects of shear rate, solvent environment, pDNA size, and copolymer complexation on shear-induced degradation are investigated. We find that small naked pDNA (pUC18, 2.7 kb) exhibits shear rate-dependent shear degradation in the microfluidic channels in a mixed organic solvent (dioxane/water/acetic acid; 90/10/<0.1 w/w/w), with the extents of both supercoil isoform relaxation and complete fragmentation increasing as the maximum shear rates increase from 4 × 105 to 2 × 106 s-1. However, over the same range of shear rates, the same pDNA sample shows no evidence of microfluidic shear-induced degradation in a pure aqueous environment. Quiescent control experiments in the same mixed organic solvent prove that a combination of solvent and shear forces is involved in the observed shear-induced degradation. Furthermore, we show that shear degradation effects in mixed organic solvents can be significantly attenuated by complexation of pDNA with the block copolymer polycaprolactone-block-poly(2-vinylpyridine) prior to exposure to microfluidic shear. Finally, we demonstrate that medium (pDSK519, 8.1 kb) and large (pRK290, 20 kb) naked pDNA are more sensitive to shear-induced microfluidic degradation in the mixed organic solvent environment than small pDNA, with both plasmids showing complete fragmentation even at the lowest shear rate, although we found no evidence of shear-induced damage in water for the largest investigated naked pDNA even at the highest flow rate. The resulting understanding of the interplay of the solvent and shear effects during microfluidic processing should inform microfluidic manufacturing routes to new gene therapy formulations.

Microfluidic Shear Processing Control of Biological Reduction Stimuli-Responsive Polymer Nanoparticles for Drug Delivery

We demonstrate microfluidic manufacturing of glutathione (GSH)-responsive polymer nanoparticles (PNPs) with controlled in vitro pharmacological properties for selective drug delivery. This work leverages previous fundamental work on microfluidic control of the physicochemical properties of GSH-responsive PNPs containing cleavable disulfide groups in two different locations (core and interface, DualM PNPs). In this paper, we employ a two-phase gas-liquid microfluidic reactor for the flow-directed manufacturing of paclitaxel-loaded or DiI-loaded DualM PNPs (PAX-PNPs or DiI-PNPs, where DiI is a fluorescent drug surrogate dye). We find that both PAX-PNPs and DiI-PNPs exhibit similar flow-tunable sizes, morphologies, and internal structures to those previously described for empty DualM PNPs. Fluorescent imaging of DiI-PNP formulations shows that microfluidic manufacturing greatly improves the homogeneity of drug dispersion within the PNP population compared to standard bulk microprecipitation. Encapsulation of PAX in DualM PNPs significantly increases its selectivity to cancerous cells, with various PAX-PNP formulations showing higher cytotoxicity against cancerous MCF-7 cells than against non-cancerous HaCaT cells, in contrast to free PAX, which showed similar cytotoxicity in the two cell lines. In addition, the characterization of DualM PNP formulations formed at various microfluidic flow rates reveals that critical figures of merit for drug delivery function-including encapsulation efficiencies, GSH-triggered release rates, rates of cell uptake, cytotoxicities, and selectivity to cancerous cells-exhibit microfluidic flow tunability that mirrors trends in PNP size. These results highlight the potential of two-phase microfluidic manufacturing for controlling both structure and drug delivery function of biological stimuli-responsive nanomedicines toward improved therapeutic outcomes.

Microfluidic encapsulation of SN-38 in block copolymer nanoparticles: effect of hydrophobic block composition on loading and release properties

A gas–liquid microfluidic reactor was used to prepare polymer nanoparticles (PNPs) containing the drug 7-ethyl-10-hydroxy camptothecin (SN-38) from a series of poly(methyl caprolactone-co-caprolactone)-b-poly(ethylene oxide) (P(MCL-co-CL)-b-PEO) amphiphilic block copolymers with variable MCL content in the hydrophobic block. All three copolymers formed spheres with ∼20 nm core diameters by TEM, although some rigid rod-like aggregates were also formed by the PMCL-50 and PMCL-75 copolymers. SN-38 encapsulation efficiencies (EE = 2.7%–3.0%) and loading levels (DL = 2.0%–2.9%) were similar for the three copolymers. In vitro release kinetics became significantly slower as the MCL content increased, with release half times increasing monotonically from 3.4 to 6.2 h as the MCL content of the hydrophobic block increased from 50% to 100%. The ability to systematically tune release half times via controlled variation in the hydrophobic block composition, while maintaining constant PNP size and loading levels, represents an intriguing chemical handle for the optimization of SN-38 nanomedicines.

Microfluidic Manufacturing of SN-38-Loaded Polymer Nanoparticles with Shear Processing Control of Drug Delivery Properties.

Two-phase gas-liquid microfluidic reactors provide shear processing control of SN-38-loaded polymer nanoparticles (SN-38-PNPs). We prepare SN-38-PNPs from the block copolymer poly(methyl caprolactone- co-caprolactone)- block-poly(ethylene oxides) (P(MCL- co-CL)- b-PEO) using bulk and microfluidic methods and at different drug-to-polymer loading ratios and on-chip flow rates. We show that, as the microfluidic flow rate ( Q) increases, encapsulation efficiency and drug loading increase and release half times increase. Slower SN-38 release is obtained at the highest Q value ( Q = 400 μL/min) than is achieved using a conventional bulk preparation method. For all SN-38-PNP formulations, we find a dominant population (by number) of nanosized particles (<50 nm) along with a small number of larger aggregates (>100 nm). As Q increases, the size of aggregates decreases through a minimum and then increases, attributed to a flow-variable competition of shear-induced particle breakup and shear-induced particle coalescence. IC25 and IC50 values of the various SN-38-PNPs against MCF-7 cells show strong flow rate dependencies that mirror trends in particle size. SN-38-PNPs manufactured on-chip at intermediate flow rates show both minimum particle sizes and maximum potencies with a significantly lower IC25 value than the bulk-prepared sample. Compared to conventional bulk methods, microfluidic shear processing in two-phase reactors provides controlled manufacturing routes for optimizing and improving the properties of SN-38 nanomedicines.

Microfluidic Processing Approach to Controlling Drug Delivery Properties of Curcumin-Loaded Block Copolymer Nanoparticles.

We apply gas-liquid microfluidic reactors containing flow-variable, high-shear "hot spots" to produce curcumin-loaded polymer nanoparticles (CUR-PNPs) comprised of poly(caprolactone)- block-poly(ethylene oxide) (PCL- b-PEO) block copolymers at various flow rates and CUR loading ratios. CUR-PNPs prepared using the conventional nanoprecipitation method (bulk method) showed decreased encapsulation efficiency and increased drug precipitation as the loading ratio increased. However, CUR-PNPs prepared by microfluidic manufacturing showed both increased encapsulation efficiency and increased drug loading as either the flow rate or the loading ratio increased. This enabled microfluidic CUR loading percentages of up to 30% to be achieved in this study, which to our knowledge is a record for block copolymer PNPs. As well, it is shown that increased flow rate of microfluidic manufacturing leads to decreased mean CUR-PNP sizes (down to ∼50 nm) and narrower size distributions, along with significantly different CUR release kinetics compared to CUR-PNPs prepared at slower flow rates. In vitro antiproliferation experiments against MDA-MB-231 cells give an average IC50 value of 24 μM for CUR-PNPs compared to 13 μM for free CUR at the same incubation time of 72 h. Compared to conventional bulk and single-phase microfluidic strategies, this unique two-phase reactor represents an exciting manufacturing platform for optimizing polymeric CUR nanomedicines though flow-directed shear processing.

Effects of chemical and processing variables on paclitaxel-loaded polymer nanoparticles prepared using microfluidics

For paclitaxel (PAX)-loaded polymeric nanoparticles PNPs prepared in a two-phase gas-liquid microfluidic reactor, the effects of microfluidic flow rate on the multiscale structure, loading efficiency and release rate are determined for three different copolymer compositions and two orders of magnitude variation in the PAX loading ratio. All experiments are carried out in the limit of low drug-to-polymer loading ratios (r≤0.01, w/w). In this range of r, PCL crystallinity, loading efficiency and release rate are not significantly affected by the amount of PAX dissolved in the core. These results are in sharp contrast to microfluidic PNPs prepared in a range of high loading ratios (r≥0.1), where the amount of added PAX has a strong influence on the multiscale structure and properties of drug delivery PNPs. For the case of r=0.01, we show that flow rate strongly affects PNP morphologies for all three block copolymer compositions. For the shortest and longest PCL block lengths, the relative number of cylindrical morphologies increases and then decreases with increasing flow rate, whereas for the intermediate PCL block length, the number of cylinders steadily increases as the flow rate increases. Internal PCL crystallinities and PAX loading efficiencies show similar trends, both parameters increasing and decreasing with increasing flow rate for the extreme PCL block lengths and steadily increasing for the intermediate PCL block length. PAX release profiles indicate a marked slowing of PAX release as either the PCL block length or the microfluidic flow rate increase. Working in the limit of low loading ratio, this work provides clarity on separating the relative effects of copolymer composition and processing along with perturbations caused by the molecular cargo on the structure and function of drug delivery PNPs. These critical insights thus inform controlled microfluidic preparation of more medically-relevant PNPs at higher therapeutic loading levels.

Controlling Structure and Function of Polymeric Drug Delivery Nanoparticles Using Microfluidics.

We demonstrate control of multiscale structure and drug delivery function for paclitaxel (PAX)-loaded polycaprolactone-block-poly(ethylene oxide) (PCL-b-PEO) polymeric nanoparticles (PNPs) via synthesis and flow-directed shear processing in a two-phase gas-liquid microfluidic reactor. This strategy takes a page from the engineering of commodity plastics, where processing rather than polymer chemistry provides an experimental handle on properties and function. PNPs formed from copolymers with three different PCL block lengths show sizes, morphologies, and loading efficiencies that depend on both the PCL block length and the microfluidic flow rate. By varying flow rate and comparing with a conventional bulk method of PNP preparation, we show that flow-variable shear processing provides control of PNP sizes and morphologies and enables slower PAX release times (up to 2 weeks) compared to bulk-prepared PNPs. Antiproliferative effects against cultured MCF-7 breast cancer cells were greatest for PNPs formed at an intermediate flow rate, corresponding to small and low-polydispersity spheres formed uniquely at this flow condition. Formation and flow-directed nanoscale shear processing in gas-liquid microfluidic reactors provides a manufacturing platform for drug delivery PNPs that could enable more effective and selective nanomedicines through multiscale structural control.

Microfluidic Synthesis of Photoresponsive Spool-Like Block Copolymer Nanoparticles: Flow-Directed Formation and Light-Triggered Dissociation

We demonstrate flow-directed production of nanoparticles of the photoresponsive block copolymer poly(o-nitrobenzyl acrylate)-b-polydimethylacrylamide in a gas–liquid microfluidic reactor. Microfluidic formation produced a variety of morphologies dependent on flow rate, with spheres and short cylinders, then large compound micelles, then unique spooled cylinders appearing as flow rate increased. The higher excess free energy of spooled cylinders relative to the quiescent equilibrium state is evidenced by their faster off-chip relaxation times (∼1 day) as compared to other flow-directed structures. Comparison of light-triggered dissociation of flow-directed nanoparticles prepared at two different flow rates shows faster disruption of nanoparticles prepared at the higher flow rate. The application of variable flow to direct both structure and responsivity suggests interesting possibilities for controlled microfluidic manufacturing of “smart” polymeric colloids.

Multiscale Control of Hierarchical Structure in Crystalline Block Copolymer Nanoparticles Using Microfluidics.

Hierarchical semicrystalline block copolymer nanoparticles are produced in a segmented gas-liquid microfluidic reactor with top-down control of multiscale structural features, including nanoparticle morphologies, sizes, and internal crystallinities. Control of multiscale structure on disparate length scales by a single control variable (flow rate) enables tailoring of drug delivery nanoparticle function including release rates.

Flow-Directed Block Copolymer Micelle Morphologies via Microfluidic Self-Assembly

The self-assembly of amphiphilic block copolymers in a gas-liquid microfluidic reactor produces variable, flow-directed micellar morphologies entirely different from off-chip equilibrium structures. A polystyrene-block-poly(acrylic acid) copolymer, which forms exclusively spheres off-chip, generates kinetic cylinders, Y-junctions, bilayers, and networks by a mechanism of collision-coalescence enabled by strong and localized on-chip shear fields. Variation in the size and relative amount of flow-directed nanostructures is achieved by changing the water content and flow rate. These results demonstrate on-chip processing routes to specific functional colloidal nanostructures.

Rapid Carbon‐11 Radiolabelling for PET Using Microfluidics

Labels all around: A gas–liquid microfluidic reactor is used for the first time to efficiently perform [11C]carbon monoxide carbonylation reactions for the preparation of 11C-radiolabelled amide molecules within a short timeframe (<15 min; see scheme).