Biocompatible Microfabrication of 3D Isolation Chambers for Targeted Confinement of Individual Cells and Their Progeny

We describe a technique to physically isolate single/individual cells from their surrounding environment by fabricating three-dimensional microchambers around selected cells under biocompatible conditions. Isolation of targeted cells is achieved via rapid fabrication of protein hydrogels from a biocompatible precursor solution using multiphoton lithography, an intrinsically 3D laser direct write microfabrication technique. Cells remain chemically accessible to environmental cues enabling their propagation into well-defined, high density populations. We demonstrate this methodology on gram negative (<i>E. coli</i>), gram positive (<i>S. aureus</i>), and eukaryotic (<i>S. cerevisiae</i>) cells. The opportunities to confine viable, single/individual-cells and small populations within user-defined microenvironments afforded by this approach should facilitate the study of cell behaviors across multiple generations

Biocompatible Microfabrication of 3D Isolation Chambers for Targeted Confinement of Individual Cells and Their Progeny

We describe a technique to physically isolate single/individual cells from their surrounding environment by fabricating three-dimensional microchambers around selected cells under biocompatible conditions. Isolation of targeted cells is achieved via rapid fabrication of protein hydrogels from a biocompatible precursor solution using multiphoton lithography, an intrinsically 3D laser direct write microfabrication technique. Cells remain chemically accessible to environmental cues enabling their propagation into well-defined, high density populations. We demonstrate this methodology on gram negative (<i>E. coli</i>), gram positive (<i>S. aureus</i>), and eukaryotic (<i>S. cerevisiae</i>) cells. The opportunities to confine viable, single/individual-cells and small populations within user-defined microenvironments afforded by this approach should facilitate the study of cell behaviors across multiple generations

Enlarged Pore Size in Mesoporous Silica Films Templated by Pluronic F127: Use of Poloxamer Mixtures and Increased Template/SiO₂ Ratios in Materials Synthesized by Evaporation-Induced Self-Assembly

Although evaporation-induced self-assembly (EISA) has proven to be a convenient method for synthesizing nanoporous silica films (and particles), accessing material structures with pore sizes larger than ca. 10 nm remains experimentally inconvenient. The use of pore swelling agents (SAs), commonly used during the hydrothermal synthesis of mesoporous silicas, results in little or no pore size expansion due to evaporation or phase separation. Moreover, diblock copolymer templates can yield large pores but are quite expensive and generally require the addition of strong organic cosolvents. Here, we hypothesized that pores templated by the Pluronic triblock polymer F127 could be successfully enlarged, without phase separation, by using a chemically similar, nonvolatile, secondary Pluronic polymer (P103) as the SA. We find pore size increased up to 15 nm for a spherical pore morphology, with a phase transition to a multilamellar vesicle (MLV)-based nanostructure occurring as the P103/F127 ratio is further increased. This MLV phase produces even larger pore sizes due to the collapse of concentric silica shells upon template removal. Remarkably, F127 alone exhibits expansion of pore size (up to ca. 16 nm) as the template/silica ratio is increased. We find appearance of the MLV phase is due to geometric packing considerations, with expansion of F127 micelle size being a result of favorable intermolecular interactions driven by the large poly­(ethylene oxide) content of F127. Other Pluronic polymers with this feature also exhibit variable pore size based on the template/silica ratio, enabling the synthesis of mesoporous films with 3D pore connectivity and truly variable pore size of ca. 4.5 to almost 20 nm

Three-Dimensional Encapsulation of <i>Saccharomyces cerevisiae</i> in Silicate Matrices Creates Distinct Metabolic States as Revealed by Gene Chip Analysis

In order to design hybrid cellular/synthetic devices such as sensors and vaccines, it is important to understand how the metabolic state of living cells changes upon physical confinement within three-dimensional (3D) matrices. We analyze the gene expression patterns of stationary phase <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>) cells encapsulated within three distinct nanostructured silica matrices and relate those patterns to known naturally occurring metabolic states. Silica encapsulation methods employed were lipid-templated mesophase silica thin films formed by cell-directed assembly (CDA), lipid-templated mesophase silica particles formed by spray drying (SD), and glycerol-doped silica gel monoliths prepared from an aqueous silicate (AqS+g) precursor solution. It was found that the cells for all three-encapsulated methods enter quiescent states characteristic of response to stress, albeit to different degrees and with differences in detail. By the measure of enrichment of stress-related gene ontology categories, we find that the AqS+g encapsulation is more amenable to the cells than CDA and SD encapsulation. We hypothesize that this differential response in the AqS+g encapsulation is related to four properties of the encapsulating gel: (1) oxygen permeability, (2) relative softness of the material, (3) development of a protective sheath around individual cells (visible in TEM micrographs <i>vide infra</i>), and (4) the presence of glycerol in the gel, which has been previously noted to serve as a protectant for encapsulated cells and can serve as the sole carbon source for <i>S. cerevisiae</i> under aerobic conditions. This work represents a combination of experiment and analysis aimed at the design and development of 3D encapsulation procedures to induce, and perhaps control, well-defined physiological behaviors

Lithographically Defined Macroscale Modulation of Lateral Fluidity and Phase Separation Realized via Patterned Nanoporous Silica-Supported Phospholipid Bilayers

Using lithographically defined surfaces consisting of hydrophilic patterns of nanoporous and nonporous (bulk) amorphous silica, we show that fusion of small, unilamellar lipid vesicles produces a single, contiguous, fluid bilayer phase experiencing a predetermined pattern of interfacial interactions. Although long-range lateral fluidity of the bilayer, characterized by fluorescence recovery after photobleaching, indicates a nominally single average diffusion constant, fluorescence microscopy-based measurements of temperature-dependent onset of fluidity reveals a locally enhanced fluidity for bilayer regions supported on nanoporous silica in the vicinity of the fluid–gel transition temperature. Furthermore, thermally quenching lipid bilayers composed of a binary lipid mixture below its apparent miscibility transition temperature induces qualitatively different lateral phase separation in each region of the supported bilayer: The nanoporous substrate produces large, microscopic domains (and domain-aggregates), whereas surface texture characterized by much smaller domains and devoid of any domain-aggregates appears on bulk glass-supported regions of the single-lipid bilayer. Interestingly, lateral distribution of the constituent molecules also reveals an enrichment of gel-phase lipids over nanoporous regions, presumably as a consequence of differential mobilities of constituent lipids across the topographic bulk/nanoporous boundary. Together, these results reveal that subtle local variations in constraints imposed at the bilayer interface, such as by spatial variations in roughness and substrate adhesion, can give rise to significant differences in macroscale biophysical properties of phospholipid bilayers even within a single, contiguous phase

Three-Dimensional Encapsulation of <i>Saccharomyces cerevisiae</i> in Silicate Matrices Creates Distinct Metabolic States as Revealed by Gene Chip Analysis

In order to design hybrid cellular/synthetic devices such as sensors and vaccines, it is important to understand how the metabolic state of living cells changes upon physical confinement within three-dimensional (3D) matrices. We analyze the gene expression patterns of stationary phase <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>) cells encapsulated within three distinct nanostructured silica matrices and relate those patterns to known naturally occurring metabolic states. Silica encapsulation methods employed were lipid-templated mesophase silica thin films formed by cell-directed assembly (CDA), lipid-templated mesophase silica particles formed by spray drying (SD), and glycerol-doped silica gel monoliths prepared from an aqueous silicate (AqS+g) precursor solution. It was found that the cells for all three-encapsulated methods enter quiescent states characteristic of response to stress, albeit to different degrees and with differences in detail. By the measure of enrichment of stress-related gene ontology categories, we find that the AqS+g encapsulation is more amenable to the cells than CDA and SD encapsulation. We hypothesize that this differential response in the AqS+g encapsulation is related to four properties of the encapsulating gel: (1) oxygen permeability, (2) relative softness of the material, (3) development of a protective sheath around individual cells (visible in TEM micrographs <i>vide infra</i>), and (4) the presence of glycerol in the gel, which has been previously noted to serve as a protectant for encapsulated cells and can serve as the sole carbon source for <i>S. cerevisiae</i> under aerobic conditions. This work represents a combination of experiment and analysis aimed at the design and development of 3D encapsulation procedures to induce, and perhaps control, well-defined physiological behaviors

Influence of Silica Matrix Composition and Functional Component Additives on the Bioactivity and Viability of Encapsulated Living Cells

The remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells is reported. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. These matrices are used to encapsulate living <i>E. coli</i> cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in <i>E. coli</i> bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. <i>E. coli</i> cells encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. This unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states

Mechanistic Modeling Identifies Drug-Uptake History as Predictor of Tumor Drug Resistance and Nano-Carrier-Mediated Response

A quantitative understanding of the advantages of nanoparticle-based drug delivery <i>vis-à-vis</i> conventional free drug chemotherapy has yet to be established for cancer or other diseases despite numerous investigations. Here, we employ first-principles cell biophysics, drug pharmaco-kinetics, and drug pharmaco-dynamics to model the delivery of doxorubicin (DOX) to hepatocellular carcinoma (HCC) tumor cells and predict the resultant experimental cytotoxicity data. The fundamental, mechanistic hypothesis of our mathematical model is that the integrated history of drug uptake by the cells over time of exposure, which sets the cell death rate parameter, and the uptake rate are the sole determinants of the dose response relationship. A universal solution of the model equations is capable of predicting the entire, nonlinear dose response of the cells to any drug concentration based on just two separate measurements of these cellular parameters. This analysis reveals that nanocarrier-mediated delivery overcomes resistance to the free drug because of improved cellular uptake rates, and that dose response curves to nanocarrier mediated drug delivery are equivalent to those for free-drug, but “shifted to the left;” that is, lower amounts of drug achieve the same cell kill. We then demonstrate the model’s general applicability to different tumor and drug types, and cell-exposure time courses by investigating HCC cells exposed to cisplatin and 5-fluorouracil, breast cancer MCF-7 cells exposed to DOX, and pancreatic adenocarcinoma PANC-1 cells exposed to gemcitabine. The model will help in the optimal design of nanocarriers for clinical applications and improve the current, largely empirical understanding of <i>in vivo</i> drug transport and tumor response

Revealing the Interfacial Self-Assembly Pathway of Large-Scale, Highly-Ordered, Nanoparticle/Polymer Monolayer Arrays at an Air/Water Interface

The pathway of interfacial self-assembly of large-scale, highly ordered 2D nanoparticle/polymer monolayer or bilayer arrays from a toluene solution at an air/water interface was investigated using grazing-incidence small-angle scattering at a synchrotron source. Interfacial-assembly of the ordered nanoparticle/polymer array was found to occur through two stages: formation of an incipient randomly close-packed interfacial monolayer followed by compression of the monolayer to form a close-packed lattice driven by solvent evaporation from the polymer. Because the nanoparticles are hydrophobic, they localize exclusively to the polymer–air interface during self-assembly, creating a through thickness asymmetric film as confirmed by X-ray reflectivity. The interfacial self-assembly approach can be extended to form binary NP/polymer arrays. It is anticipated that by understanding the interfacial self-assembly pathway, this simple evaporative procedure could be conducted as a continuous process amenable to large area nanoparticle-based manufacturing needed for emerging energy technologies

Revealing the Interfacial Self-Assembly Pathway of Large-Scale, Highly-Ordered, Nanoparticle/Polymer Monolayer Arrays at an Air/Water Interface

The pathway of interfacial self-assembly of large-scale, highly ordered 2D nanoparticle/polymer monolayer or bilayer arrays from a toluene solution at an air/water interface was investigated using grazing-incidence small-angle scattering at a synchrotron source. Interfacial-assembly of the ordered nanoparticle/polymer array was found to occur through two stages: formation of an incipient randomly close-packed interfacial monolayer followed by compression of the monolayer to form a close-packed lattice driven by solvent evaporation from the polymer. Because the nanoparticles are hydrophobic, they localize exclusively to the polymer–air interface during self-assembly, creating a through thickness asymmetric film as confirmed by X-ray reflectivity. The interfacial self-assembly approach can be extended to form binary NP/polymer arrays. It is anticipated that by understanding the interfacial self-assembly pathway, this simple evaporative procedure could be conducted as a continuous process amenable to large area nanoparticle-based manufacturing needed for emerging energy technologies