Angela K. Pannier

Fabrication and characterization of DNA-loaded zein nanospheres

BackgroundParticulates incorporating DNA are promising vehicles for gene delivery, with the ability to protect DNA and provide for controlled, localized, and sustained release and transfection. Zein, a hydrophobic protein from corn, is biocompatible and has properties that make it a promising candidate material for particulate delivery, including its ability to form nanospheres through coacervation and its insolubility under physiological conditions, making it capable of sustained release of encapsulated compounds. Due to the promise of this natural biomaterial for drug delivery, the objective of this study was to formulate zein nanospheres encapsulating DNA as the therapeutic compound, and to characterize size, charge, sustained release, cell cytotoxicity and cellular internalization of these particles.ResultsZein nanospheres encapsulating DNA were fabricated using a coacervation technique, without the use of harsh solvents or temperatures, resulting in the preservation of DNA integrity and particles with diameters that ranged from 157.8 ± 3.9 nm to 396.8 ± 16.1 nm, depending on zein to DNA ratio. DNA encapsulation efficiencies were maximized to 65.3 ± 1.9% with a maximum loading of 6.1 ± 0.2 mg DNA/g zein. The spheres protected encapsulated DNA from DNase I degradation and exhibited sustained plasmid release for at least 7 days, with minimal burst during the initial phase of release. Zein/DNA nanospheres demonstrated robust biocompatibility, cellular association, and internalization.ConclusionsThis study represents the first report on the formation of zein particles encapsulating plasmid DNA, using simple fabrication techniques resulting in preservation of plasmid integrity and tunable sizes. DNA encapsulation efficiencies were maximized to acceptable levels at higher zein to DNA ratios, while loading was comparable to that of other hydrophilic compounds encapsulated in zein and that of DNA incorporated into PLGA nano- and microspheres. The hydrophobic nature of zein resulted in spheres capable of sustained release of plasmid DNA. Zein particles may be an excellent potential tool for the delivery of DNA with the ability to be fine-tuned for specific applications including oral gene delivery, intramuscular delivery, and in the fabrication of tissue engineering scaffolds.

MR elastography monitoring of tissue-engineered constructs

The objective of tissue engineering (TE) is to create functional replacements for various tissues; the mechanical properties of these engineered constructs are critical to their function. Several techniques have been developed for the measurement of the mechanical properties of tissues and organs; however, current methods are destructive. The field of TE will benefit immensely if biomechanical models developed by these techniques could be combined with existing imaging modalities to enable noninvasive, dynamic assessment of mechanical properties during tissue growth. Specifically, MR elastography (MRE), which is based on the synchronization of a mechanical actuator with a phase contrast imaging pulse sequence, has the capacity to measure tissue strain generated by sonic cyclic displacement. The captured displacement is presented in shear wave images from which the complex shear moduli can be extracted or simplified by a direct measure, termed the shear stiffness. MRE has been extended to the microscopic scale, combining clinical MRE with high‐field magnets, stronger magnetic field gradients and smaller, more sensitive, radiofrequency coils, enabling the interrogation of smaller samples, such as tissue‐engineered constructs. The following topics are presented in this article: (i) current mechanical measurement techniques and their limitations in TE; (ii) a description of the MRE system, MRE theory and how it can be applied for the measurement of mechanical properties of tissue‐engineered constructs; (iii) a summary of in vitro MRE work for the monitoring of osteogenic and adipogenic tissues originating from human adult mesenchymal stem cells (MSCs); (iv) preliminary in vivo studies of MRE of tissues originating from mouse MSCs implanted subcutaneously in immunodeficient mice with an emphasis on in vivo MRE challenges; (v) future directions to resolve current issues with in vivo MRE in the context of how to improve the future role of MRE in TE. Copyright

Combined optical and acoustical method for determination of thickness and porosity of transparent organic layers below the ultra-thin film limit

Analysis techniques are needed to determine the quantity and structure of materials composing an organic layer that is below an ultra-thin film limit and in a liquid environment. Neither optical nor acoustical techniques can independently distinguish between thickness and porosity of ultra-thin films due to parameter correlation. A combined optical and acoustical approach yields sufficient information to determine both thickness and porosity. We describe application of the combinatorial approach to measure single or multiple organic layers when the total layer thickness is small compared to the wavelength of the probing light. The instrumental setup allows for simultaneous in situ spectroscopic ellipsometry and quartz crystal microbalance dynamic measurements, and it is combined with a multiple-inlet fluid control system for different liquid solutions to be introduced during experiments. A virtual separation approach is implemented into our analysis scheme, differentiated by whether or not the organic adsorbate and liquid ambient densities are equal. The analysis scheme requires that the film be assumed transparent and rigid (non-viscoelastic). We present and discuss applications of our approach to studies of organic surfactant adsorption, self-assembled monolayer chemisorption, and multiple-layer target DNA sensor preparation and performance testing.

Generalized ellipsometry in-situ quantification of organic adsorbate attachment within slanted columnar thin films

We apply generalized ellipsometry, well-known to be sensitive to the optical properties of anisotropic materials, to determine the amount of fibronectin protein that adsorbs onto a Ti slanted columnar thin film from solution. We find that the anisotropic optical properties of the thin film change upon organic adsorption. An optical model for ellipsometry data analysis incorporates an anisotropic Bruggeman effective medium approximation. We find that differences in experimental data from before and after fibronectin adsorption can be solely attributable to the uptake of fibronectin within the slanted columnar thin film. Simultaneous, in-situ generalized ellipsometry and quartz crystal microbalance measurements show excellent agreement on the amount and rate of fibronectin adsorption. Quantitative characterization of organic materials within three-dimensional, optically anisotropic slanted columnar thin films could permit their use in optical sensor applications.

Fuzzy Modeling of Skin Permeability Coefficients

AbstractPurpose. The purpose of this work was to determine whether a new modeling methodology using fuzzy logic can predict skin permeability coefficients that are given compound descriptors that have been proven to affect percutaneous penetration. Methods. Three fuzzy inference models were developed using subtractive clustering to define natural structures within the data and assign subsequent rules. The numeric parameters describing the rules were refined through the use of an Adaptive Neural Fuzzy Inference System implemented in MatLab. Each model was evaluated using the entire data set. Then predicted outputs were compared to the published experimental data. Results. All databases produced fuzzy inference models that successfully predicted skin permeability coefficients, with correlation coefficients ranging from 0.83 to 0.97. The lowest correlation coefficient resulted from a model using log octanol/water partition coefficient and molecular weight as inputs with two input membership functions evaluated by two fuzzy rules. The correlation coefficient of 0.97 occurred when log octanol/water partition coefficient and hydrogen bond donor activity were used as inputs with three input membership functions evaluated by three fuzzy rules. Conclusions. Fuzzy rule-based models are a realistic and promising tool that can be used to successfully model and predict skin permeability coefficients as well as or better than previous algorithms with fewer inputs

Cellular Arrays for Large-Scale Analysis of Transcription Factor Activity

Identifying molecular mechanisms or therapeutic targets is typically based on large‐scale cellular analysis that measures the abundance of mRNA or protein; however, abundance does not necessarily correlate with activity. We report a method for direct large‐scale quantification of active pathways that employs a cellular array with parallel gene delivery of constructs that report pathway activity. The reporter constructs encode luciferase, whose expression is influenced by binding of transcription factors (TFs), which are the downstream targets of signaling pathways. Luciferase levels are quantified by bioluminescence imaging (BLI), which allows for rapid, non‐invasive measurements. Activity profiles by BLI of 32 TFs were robust, consistent, and reproducible, and correlated with standard cell lysis techniques. The array identified five TFs with differential activity during phorbol‐12‐myristate‐13‐acetate (PMA)‐induced differentiation of breast cancer cells. A system for rapid, large‐scale, BLI quantification of pathway activity provides an enabling technology for mechanistic studies of cellular responses and processes. Biotechnol. Bioeng. 2011;108: 395–403.

Combined QCM-D/GE as a tool to characterize stimuli-responsive swelling of and protein adsorption on polymer brushes grafted onto 3D-nanostructures

A combined setup of quartz crystal microbalance and generalized ellipsometry can be used to comprehensively investigate complex functional coatings comprising stimuli-responsive polymer brushes and 3D nanostructures in a dynamic, noninvasive in situ measurement. While the quartz crystal microbalance detects the overall change in areal mass, for instance, during a swelling or adsorption process, the generalized ellipsometry data can be evaluated in terms of a layered model to distinguish between processes occurring within the intercolumnar space or on top of the anisotropic nanocolumns. Silicon films with anisotropic nanocolumnar morphology were prepared by the glancing angle deposition technique and further functionalized by grafting of poly-(acrylic acid) or poly-(N- isopropylacrylamide) chains. Investigations of the thermoresponsive swelling of the poly-(N-isopropylacrylamide) brush on the Si nanocolumns proved the successful preparation of a stimuli-responsive coating. Furthermore, the potential of these novel coatings in the field of biotechnology was explored by investigation of the adsorption of the model protein bovine serum albumin. Adsorption, retention, and desorption triggered by a change in the pH value is observed using poly-(acrylic acid) functionalized nanostructures, although generalized ellipsometry data revealed that this process occurs only on top of the nanostructures. Poly-(N-isopropylacrylamide) is found to render the nanostructures non-fouling properties.

Micro- and nanoparticulates for DNA vaccine delivery

DNA vaccination has emerged as a promising alternative to traditional protein-based vaccines for the induction of protective immune responses. DNA vaccines offer several advantages over traditional vaccines, including increased stability, rapid and inexpensive production, and flexibility to produce vaccines for a wide variety of infectious diseases. However, the immunogenicity of DNA vaccines delivered as naked plasmid DNA is often weak due to degradation of the DNA by nucleases and inefficient delivery to immune cells. Therefore, biomaterial-based delivery systems based on micro- and nanoparticles that encapsulate plasmid DNA represent the most promising strategy for DNA vaccine delivery. Microparticulate delivery systems allow for passive targeting to antigen presenting cells through size exclusion and can allow for sustained presentation of DNA to cells through degradation and release of encapsulated vaccines. In contrast, nanoparticle encapsulation leads to increased internalization, overall greater transfection efficiency, and the ability to increase uptake across mucosal surfaces. Moreover, selection of the appropriate biomaterial can lead to increased immune stimulation and activation through triggering innate immune response receptors and target DNA to professional antigen presenting cells. Finally, the selection of materials with the appropriate properties to achieve efficient delivery through administration routes conducive to high patient compliance and capable of generating systemic and local (i.e. mucosal) immunity can lead to more effective humoral and cellular protective immune responses. In this review, we discuss the development of novel biomaterial-based delivery systems to enhance the delivery of DNA vaccines through various routes of administration and their implications for generating immune responses.

Integrating mitosis, toxicity, and transgene expression in a telecommunications packet‐switched network model of lipoplex‐mediated gene delivery

Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms, including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet‐switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP‐induced toxicity can improve transfection efficiency by three‐fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels. Biotechnol. Bioeng. 2014;111: 1659–1671.

Modeling nonviral gene delivery as a macro-to-nano communication system

Abstract The principal role of any communication system is to deliver information from a source to a sink. Since gene delivery systems transport genetic information encoded as DNA to living cells, such systems can be considered as communication systems. Therefore, techniques developed for modeling conventional communication systems should be applicable to model gene delivery systems. The paper describes an approach to model nonviral gene delivery as a macro-to-nano communication system. To facilitate modeling, the gene delivery process is first described in terms of an abstractive layered communication protocol and then processing at each layer is implemented as M/M/ ∞ queues. To validate this approach, the model has been implemented in MATLAB/SIMULINK environment and the simulation results have been compared to experimental data from literature.