Polymer Micro- and Nanofluidic Systems for In Vitro Diagnostics: Analyzing Single Cells and Molecules

Polymer micro- and nanofluidic systems, with their critical dimensions, offer a potential to outperform conventional analysis techniques and diagnostic methods by enhancing speed, accuracy, sensitivity and specificity. In this work, applications of microfluidics have been demonstrated to address the existing challenges in stroke diagnosis, by mRNA expression profiling from whole blood within \u3c20 min. A brief overview of various biomarkers for stroke diagnosis is given in chapter 1 followed by design and testing of individual microfluidic modules (chapter 2 and 3) required for the development of POC diagnostic strategy for stroke. We have designed and evaluated the performance of polymer microfluidic devices for the isolation of leukocyte subsets, known for their differential gene expression in the event of stroke. Target cells (T-cells and neutrophils) were selected from with greater purities, from 50 µl whole human blood by using affinity based capture in COC devices within a 6.6 min processing time. In addition, we have also demonstrated the ability to isolate and purify total RNA by using UV activated polycarbonate solid phase extraction platform. Polymer-based nanofluidic devices were used to study the effects of surface charge on the electrodynamic transport dynamics of target molecules. In this work, we report the fabrication of mixed-scale micro- and nanofluidic networks in poly(methylmethacrylate), PMMA, using thermal nanoimprint lithography using a resin stamp and surface modification of polymer nanoslits and nanochannels for the assessment of the associated electrokinetic parameters – surface charge density, zeta potential and electroosmotic flow. This study provided information on possible routes that can be adopted to engineer proper wall chemistry of polymer nanochannels for the enhancement or reduction of solute/wall interactions in a variety of relevant single-molecule studies

Formulation development of a stable, orally delivered live human neonatal rotavirus(rv3-bb) vaccine candidate

Rotavirus is the most common cause of gastroenteritis among children under 5 years of age leading to ~200,000 deaths in 2013.1 Rotavirus-attributed mortality can be significantly reduced by promoting global implementation of rotavirus vaccination by vaccine dosage cost reduction and optimizing vaccine efficacy in low-resource countries. Furthermore, a rotavirus vaccine administered at birth could prevent neonatal mortality and reduce the risk of intussusception 2. An oral human neonatal rotavirus vaccine candidate (RV3-BB) has been developed from the human neonatal rotavirus strain RV3 (G3P[6]) 2 , and a recently published Phase IIb clinical trial showed RV3-BB was efficacious in preventing severe rotavirus gastroenteritis via a neonatal or infant schedule in Indonesia2. The overall goals of this project are to develop and implement commercially viable bulk and drug product manufacturing processes of a stable liquid formulation for oral delivery (without pre-neutralization) that is affordable in the developing world (Fig. 1). The consortium working on this program is sponsored by the Bill and Melinda Gates Foundation between Batavia Biosciences, Murdoch Children’s Research Institute, BioFarma, and The University of Kansas. Please click Additional Files below to see the full abstract

Effect of Formulation Variables on the Stability of a Live, Rotavirus (RV3-BB) Vaccine Candidate using in vitro Gastric Digestion Models to Mimic Oral Delivery

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 °C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil®) or antacid (Mylanta®) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acid-induced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2–8 °C storage. Finally, a statistical Design -of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery

Formulation development of a live attenuated human rotavirus (RV3-BB) vaccine candidate for use in low- and middle-income countries

Formulation development was performed with the live, attenuated, human neonatal rotavirus vaccine candidate (RV3-BB) with three main objectives to facilitate use in low- and middle- income countries including (1) a liquid, 2–8°C stable vaccine, (2) no necessity for pre-neutralization of gastric acid prior to oral administration of a small-volume dose, and (3) a low-cost vaccine dosage form. Implementation of a high-throughput RT-qPCR viral infectivity assay for RV3-BB, which correlated well with traditional FFA assays in terms of monitoring RV3-BB stability profiles, enabled more rapid and comprehensive formulation development studies. A wide variety of different classes and types of pharmaceutical excipients were screened for their ability to stabilize RV3-BB during exposure to elevated temperatures, freeze-thaw and agitation stresses. Sucrose (50–60% w/v), PEG-3350, and a solution pH of 7.8 were selected as promising stabilizers. Using a combination of an in vitro gastric digestion model (to mimic oral delivery conditions) and accelerated storage stability studies, several buffering agents (e.g., succinate, adipate and acetate at ~200 to 400 mM) were shown to protect RV3-BB under acidic conditions, and at the same time, minimize virus destabilization during storage. Several optimized RV3-BB candidate formulations were identified based on negligible viral infectivity losses during storage at 2–8°C and −20°C for up to 12 months, as well as by relative stability comparisons at 15°C and 25°C (up to 12 and 3 months, respectively). These RV3-BB stability results are discussed in the context of stability profiles of other rotavirus serotypes as well as future RV3-BB formulation development activities

Developing a manufacturing process to deliver a cost effective and stable liquid human rotavirus vaccine

Despite solid evidence of the success of rotavirus vaccines in saving children from fatal gastroenteritis, more than 82 million infants worldwide still lack access to a rotavirus vaccine. The main barriers to global rotavirus vaccine coverage include cost, manufacturing capacity and suboptimal efficacy in low- and lower-middle income countries. One vaccine candidate with the potential to address the latter is based on the novel, naturally attenuated RV3 strain of rotavirus, RV3-BB vaccine administered in a birth dose strategy had a vaccine efficacy against severe rotavirus gastroenteritis of 94% at 12 months of age in infants in Indonesia. To further develop this vaccine candidate, a well-documented and low-cost manufacturing process is required. A target fully loaded cost of goods (COGs) of ≤$3.50 per course of three doses was set based on predicted market requirements. COGs modelling was leveraged to develop a process using Vero cells in cell factories reaching high titers, reducing or replacing expensive reagents and shortening process time to maximise output. Stable candidate liquid formulations were developed allowing two-year storage at 2–8 °C. In addition, the formulation potentially renders needless the pretreatment of vaccinees with antacid to ensure adequate gastric acid neutralization for routine oral vaccination. As a result, the formulation allows small volume dosing and reduction of supply chain costs. A dose ranging study is currently underway in Malawi that will inform the final clinical dose required. At a clinical dose of ≤6.3 log10 FFU, the COGs target of ≤$3.50 per three dose course was met. At a clinical dose of 6.5 log10 FFU, the final manufacturing process resulted in a COGs that is substantially lower than the current average market price, 2.44 USD per dose. The manufacturing and formulation processes were transferred to BioFarma in Indonesia to enable future RV3-BB vaccine production

Parallel Affinity-Based Isolation of Leukocyte Subsets Using Microfluidics: Application for Stroke Diagnosis

We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection. As an example of the performance of these devices, CD4+ T-cells and neutrophils were selected from whole blood as these cells are known to express genes found in stroke-related expression profiles that can be used for the diagnosis of this disease. CD4+ T-cells and neutrophils were simultaneously isolated with purities >90% using affinity-based capture in cyclic olefin copolymer (COC) devices with a processing time of ∼3 min. In addition, sufficient quantities of the cells could be recovered from a 50 μL whole blood input to allow for reverse transcription-polymerase chain reaction (RT-PCR) following cell lysis. The expression of genes from isolated T-cells and neutrophils, such as S100A9, TCRB, and FPR1, was evaluated using RT-PCR. The modification and isolation procedures demonstrated here can also be used to analyze other cell types as well where multiple subsets must be interrogated

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits

Mixed-scale nano-and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 mu m in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: similar to 6% for PMMA and similar to 9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 +/- 0.7 x 10(-4) cm(2) V(-1) s(-1) and 7.6 +/- 0.6 x 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for lambda-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).close171

Discrete microfluidics for the isolation of circulating tumor cell subpopulations targeting fibroblast activation protein alpha and epithelial cell adhesion molecule

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges

UV activation of polymeric high aspect ratio microstructures: Ramifications in antibody surface loading for circulating tumor cell selection

The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of ???3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymer&apos;s damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the device&apos;s cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.close9

Formulation Development and Improved Stability of a Combination Measles and Rubella Live-Viral Vaccine Dried for Use in the Nanopatch(TM) Microneedle Delivery System

Measles (Me) and rubella (Ru) viral diseases are targeted for elimination by ensuring a high level of vaccination coverage worldwide. Less costly, more convenient MeRu vaccine delivery systems should improve global vaccine coverage, especially in low – and middle – income countries (LMICs). In this work, we examine formulating a live, attenuated Me and Ru combination viral vaccine with Nanopatch™, a solid polymer micro-projection array for intradermal delivery. First, high throughput, qPCR-based viral infectivity and genome assays were established to enable formulation development to stabilize Me and Ru in a scaled-down, custom-built evaporative drying system to mimic the Nanopatch™ vaccine coating process. Second, excipient screening and optimization studies identified virus stabilizers for use during the drying process and upon storage in the dried state. Finally, a series of real-time and accelerated stability studies identified eight candidate formulations that met a target thermal stability criterion for live vaccines (<1 log10 loss after 1 week storage at 37°C). Compared to −80°C control samples, the top candidate formulations resulted in minimal viral infectivity titer losses after storage at 2–8°C for 6 months (i.e., <0.1 log10 for Me, and ~0.4 log10 for Ru). After storage at 25°C over 6 months, ~0.3–0.5 and ~1.0–1.4 log10 titer losses were observed for Me and Ru, respectively, enabling the rank-ordering of the stability of candidate formulations. These results are discussed in the context of future formulation challenges for developing microneedle-based dosage forms containing stabilized live, attenuated viral vaccines for use in LMICs