Hydrogel Microparticle-Templated Anti-Solvent Crystallization of Small-Molecule Drugs.

Conventional formulation strategies for hydrophobic small-molecule drug products frequently include mechanical milling to decrease active pharmaceutical ingredient (API) crystal size and subsequent granulation processes to produce an easily handled powder. A hydrogel-templated anti-solvent crystallization method is presented for the facile fabrication of microparticles containing dispersed nanocrystals of poorly soluble API. Direct crystallization within a porous hydrogel particle template yields core-shell structures in which the hydrogel core containing API nanocrystals is encased by a crystalline API shell. The process of controllable loading (up to 64% w/w) is demonstrated, and tailored dissolution profiles are achieved by simply altering the template particle size. API release is well described by a shrinking core model. Overall, the approach is a simple, scalable and potentially generalizable method that enables novel means of independently controlling both API crystallization and excipient characteristics, offering a "designer" drug particle system.

Single-cell assays using integrated continuous-flow microfluidics.

The recent maturation of continuous-flow microfluidic technologies has coincided with transformative new methods to profile single cells, including their genetic types, protein expression and enzyme activities. Continuous-flow high-throughput single-cell screening and sorting can reveal relationships across cellular phenotypes (e.g., enzyme activity and secretion) and genetic fingerprints. This technology provides unique opportunities, as well as experimental and computational challenges, for integrative approaches that can process large amounts of single-cell data. In this chapter, we discuss recent advances in integrated continuous-flow microfluidic approaches with a focus on measurements and statistical analysis of single-cell enzyme activity and their applications in quantitative biology, synthetic biology, and diagnosis.

Plasmonic droplet screen for single-cell secretion analysis.

Single-cell secretion analysis technologies are needed to elucidate the heterogeneity of cellular functionalities. Although ligand binding assays in microwells provide a promising approach for measuring single-cell secretions, their throughput is limited. Recently, droplet assays have been developed for high-throughput single-cell screening. However, because washing steps are difficult to perform with droplets, there are still challenges in measuring secretions using droplet assays. In this study, a plasmonic droplet screen approach is developed for one-step washing-free multiplex detection of single-cell secretions. Individual cells are encapsulated with antibody-conjugated gold nanorods (AuNRs) in droplets to evaluate their secretion levels. The shift in the plasmon resonance peak reflects the amount of secreted protein without needing additional indicator and washing steps. The plasmonic signals from a continuous flow of single-cell droplets are collected by dark-field spectroscopy (∼100-150 cells min-1). This platform is tested by screening interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) secreted from suspended leukemia cells and adherent breast cancer cells. Overall, this novel strategy shows the potential and flexibility of high-efficiency multiplex single-cell secretion analysis.

Nano-in-Micro Smart Hydrogel Composite for a Rapid Sensitive Immunoassay.

Immunoassays are an important tool in various bioanalytical settings, such as clinical diagnostics, biopharmaceutical analysis, environmental monitoring, and food testing. An enzyme-linked immunosorbent assay (ELISA) is usually used to amplify immunoassay signals; however, it requires labor-intensive and time-consuming procedures, which hinders its application to rapid cytokine detection. In this study, a nano-in-micro composite system, where immunosensing polystyrene beads (≈320 nm) are incorporated within a stimuli-responsive microgel matrix (≈40 µm) via microfluidics, is investigated. The intrinsic volume phase-transition change properties of the smart microgels allows an enzyme-free enhanced immunoassay, enabling instant enhancement in signal-to-noise ratios of ≈5-fold. This nano-in-micro hydrogel composite offers a simple yet highly effective method for sensitive and multiplexed cytokine analysis without complex enzyme-based signal amplification steps, greatly benefitting advanced immune medicine.

Smart Hydrogel Microfluidics for Single-Cell Multiplexed Secretomic Analysis with High Sensitivity.

Secreted proteins determine a range of cellular functionalities correlated with human health and disease progression. Because of cell heterogeneity, it is essential to measure low abundant protein secretions from individual cells to determine single-cell activities. In this study, an integrated platform consisting of smart hydrogel immunosensors for the sensitive detection of single-cell secretions is developed. A single cell and smart hydrogel microparticles are encapsulated within a droplet. After incubation, target secreted proteins from the cell are captured in the smart hydrogel particle for immunoassay. The temperature-induced volume phase transition of the hydrogel biosensor allows the concentration of analytes within the gel matrix to increase, enabling high-sensitivity measurements. Distinct heterogeneity for live cell secretions is determined from 6000 cells within 1 h. This method is tested for low abundant essential secretions, such as interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 secretions of both suspended cells (HL60) and adherent cells (MCF7 and MDA-MB-231). This platform is highly flexible and can be used to simultaneously measure a wide range of clinically relevant cellular secretions; it thus represents a novel tool for precise biological assays.

Sustained release of hydrophobic drugs by the microfluidic assembly of multistage microgel/poly (lactic-co-glycolic acid) nanoparticle composites.

The poor solubility of many newly discovered drugs has resulted in numerous challenges for the time-controlled release of therapeutics. In this study, an advanced drug delivery platform to encapsulate and deliver hydrophobic drugs, consisting of poly (lactic-co-glycolic acid) (PLGA) nanoparticles incorporated within poly (ethylene glycol) (PEG) microgels, was developed. PLGA nanoparticles were used as the hydrophobic drug carrier, while the PEG matrix functioned to slow down the drug release. Encapsulation of the hydrophobic agents was characterized by fluorescence detection of the hydrophobic dye Nile Red within the microgels. In addition, the microcomposites prepared via the droplet-based microfluidic technology showed size tunability and a monodisperse size distribution, along with improved release kinetics of the loaded cargo compared with bare PLGA nanoparticles. This composite system has potential as a universal delivery platform for a variety of hydrophobic molecules.

Perfusion enhanced polydimethylsiloxane based scaffold cell culturing system for multi-well drug screening platform.

Conventional two-dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver-specific functions and their cuboidal morphology over longer term culture. In this study, we present a three-dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver-specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N-Acetyl-Para-Amino-Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold-bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS-based scaffold system provides a more conducive microenvironment with better cell-to-cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing.

Computational fluid model incorporating liver metabolic activities in perfusion bioreactor.

The importance of in vitro hepatotoxicity testing during early stages of drug development in the pharmaceutical industry demands effective bioreactor models with optimized conditions. While perfusion bioreactors have been proven to enhance mass transfer and liver specific functions over a long period of culture, the flow-induced shear stress has less desirable effects on the hepatocytes liver-specific functions. In this paper, a two-dimensional human liver hepatocellular carcinoma (HepG2) cell culture flow model, under a specified flow rate of 0.03 mL/min, was investigated. Besides computing the distribution of shear stresses acting on the surface of the cell culture, our numerical model also investigated the cell culture metabolic functions such as the oxygen consumption, glucose consumption, glutamine consumption, and ammonia production to provide a fuller analysis of the interaction among the various metabolites within the cell culture. The computed albumin production of our 2D flow model was verified by the experimental HepG2 culture results obtained over 3 days of culture. The results showed good agreement between our experimental data and numerical predictions with corresponding cumulative albumin production of 2.9 × 10(-5) and 3.0 × 10(-5) mol/m(3) , respectively. The results are of importance in making rational design choices for development of future bioreactors with more complex geometries.