Eric Freeman

Modeling the proton sponge hypothesis: examining proton sponge effectiveness for enhancing intracellular gene delivery through multiscale modeling

Dendrimers have been proposed as therapeutic gene delivery platforms. Their superior transfection efficiency is attributed to their ability to buffer the acidification of the endosome and attach to the nucleic acids. For effective transfection, the strategy is to synthesize novel dendrimers that optimize both of these traits, but the prediction of the buffering behavior in the endosome remains elusive. It is suggested that buffering dendrimers induce an osmotic pressure sufficient to rupture the endosome and release nucleic acids, which forms to sequestrate most internalized exogenous materials. Presented here are the results of a computational study modeling osmotically driven endosome burst or the ‘proton sponge effect.’ The approach builds on previous cellular simulation efforts by linking the previous model with a sponge protonation model, then observing the impact on endosomal swelling and acidification. Calibrated and validated using reported experimental data, the simulations offer insights into defining the properties of suitable dendrimers for enhancing gene delivery as a function of polymer structure.

High Energy Density Nastic Materials: Parameters for Tailoring Active Response

The engineered active material considered mimics bulk deformation similar to nastic movements in the plant kingdom. Controlled transport of charge and fluid across a selectively permeable membrane employing biological processes is employed to achieve bulk deformation. The membrane may contain biological ion pumps, ion channels, and ion exchangers surrounding a spherical inclusion in a polymer matrix; in this study only ion pumps and exchangers are considered. This work examines parameters of significance to designs employing active materials, including free strain, blocked stress, impedance matched work, and rate of response. The effects of varying material design parameters, such as system geometry and membrane permeability are considered to aid in the custom design of an active material appropriate to a given application. Predictions suggest that the rate of initial response may be on the order of msec with impedance matched work in excess of 200 kJ/m3.

Sensitivity and directionality of lipid bilayer mechanotransduction studied using a revised, highly durable membrane-based hair cell sensor

A bioinspired, membrane-based hair cell sensor consists of a planar lipid bilayer formed between two lipid-coated water droplets that connect to an artificial hair. This assembly enables motion of the hair caused by mechanical stimuli to vibrate the bilayer and produce a capacitive current. In this work, the mechanoelectrical transduction mechanism and sensing performance is experimentally characterized for a more-durable, revised hair cell embodiment that includes a cantilevered hair rooted firmly in the surrounding solid substrate. Specifically, this study demonstrates that the revised membrane-based hair cell sensor produces higher time rates of change in capacitance (0.8–6.0 nF s−1) in response to airflow across the hair compared to the original sensor (45–60 pF s−1) that did not feature a cantilevered hair. The 10-fold to 100-fold increase in the time rate change of capacitance corresponds to greater membrane bending and, thus, higher sensing currents. Membranes in the revised sensor exhibit changes in area due to bending on the order of 0.2–2.0%, versus 0.02% for the original sensor. Experiments also reveal that the bilayer displays highest sensitivity to mechanical perturbations normal to the plane of the bilayer, a membrane can transduce hair motion at frequencies below the hairs characteristic frequency, and bilayers formed between polymerized hydrogel volumes exhibit a higher sensing currents than those formed between liquid aqueous volumes. Finally, measurements of sensitivity (5–35 pA m−1 s−1) and minimum (4.0−0.6 m s−1) and maximum (28−13 m s−1) sensing thresholds to airflow are performed for the first time, and we observe maximum electrical power (~65 pW) in the membrane occurs for combinations of slower airflow and higher voltage. These results highlight that along with the dimensions of the hair and the compositions of the aqueous volumes, sensing performance can be tuned with applied voltage.

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein

Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a β-fibrillizing peptide (βFP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a βFP that forms β-sheet fibrils at high salt concentrations. The IgG-binding, fluorogenic film can be injected in vivo through conventional needled syringes. Confocal microscopic images and dose-response titration experiments showed that loading of IgG into the film was mediated by pAG(MG) bound to the FAP. Release of IgG in vitro was significantly delayed by the bioaffinity mechanism; 26% of the IgG were released from films embedded with pAG(MG) after five days, compared to close to 90% in films without pAG(MG). Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAG(MG). When injected into the subcutaneous space of mouse footpads, film-embedded IgG were retained locally, with distribution through the lymphatics impeded. The ability to track IgG binding sites and distribution simultaneously will aid the optimization of local antibody delivery systems.

Application of proteins in burst delivery systems

Biological proteins embedded in either a biological or an engineered membrane will actively maintain electrochemical balance across that membrane. In this study two applications will be examined. First a system of governing equations will be calibrated for a biological endosome. The endocytosis predictions presented then serve to validate the model. In addition, these predictions introduce new insights into endosome burst, which is of interest for advancing DNA vaccine delivery. The calibrated model is subsequently adapted to an analogous engineering scenario for targeted payload delivery. In the presence of a specific external stimulus, burst release of an arbitrary payload encased in a vesicle akin to an endosome is explored. Control of the process through manipulation of vesicle size, stimulus, and transporters is presented. A case is made for application of proteins as building blocks in the design of targeted response materials.

Multiscale modeling of droplet interface bilayer membrane networks

Droplet interface bilayer (DIB) networks are considered for the development of stimuli-responsive membrane-based materials inspired by cellular mechanics. These DIB networks are often modeled as combinations of electrical circuit analogues, creating complex networks of capacitors and resistors that mimic the biomolecular structures. These empirical models are capable of replicating data from electrophysiology experiments, but these models do not accurately capture the underlying physical phenomena and consequently do not allow for simulations of material functionalities beyond the voltage-clamp or current-clamp conditions. The work presented here provides a more robust description of DIB network behavior through the development of a hierarchical multiscale model, recognizing that the macroscopic network properties are functions of their underlying molecular structure. The result of this research is a modeling methodology based on controlled exchanges across the interfaces of neighboring droplets. This methodology is validated against experimental data, and an extension case is provided to demonstrate possible future applications of droplet interface bilayer networks.

Biologically inspired reversible osmotic actuation through voltage-gated ion channels

This study focuses on the development of a novel high-energy density actuator based on biomimicry principles. The system proposed here draws inspiration from plant motor cells and provides proof of concept for a highly configurable reversible osmotic actuator through the application of voltage-gated ion channels and action potentials. Computational methods are employed to measure the effectiveness of the proposed system in comparison to similar novel actuators.

Applications of Biologically Inspired Membranes

Nastic materials are a novel high energy density engineered membrane based on processes found in the plant kingdom. These membranes are engineered using biomimetic principles using protein transporters extracted from beet cells. These embedded transporters are capable of producing work through pumping fluid across the membrane (dependent on concentrations and fuel availability). Through this process, they may also produce controllable bulk deformation through the establishment of an osmotic gradient.Current experimental testing has been focused largely on a cylindrical setup, where the membrane is stretched across one side of a barrel apparatus. To determine the total potential of the nastic material, a spherical set up must be examined, which is costly to create in a laboratory environment. To determine the worth of the spherical model, a computational model was created using transport principles examined by Endresen et al.This model will be examined and modified to simulate a wide variety of conditions. Through careful evaluation of the results, data for actuation properties and model performance based on inputs and conditions will be obtained. This data will assist in determining the overall capabilities of the nastic material, and will aid future research. Finally, alternate uses for the nastic materials will be explored and discussed.

Parametric Studies of a Coupled Transport/Hyperelastic Model for High Energy Density Nastic Materials

The focus of this research is to optimize the performance of a high energy density active material based upon biological processes. This material uses controlled transport of charge and fluid across a selectively permeable membrane to achieve bulk deformation, similar to nastic movements in the plant kingdom. The membrane utilizes biological ion pumps, ion channels, and ion exchangers surrounding a spherical inclusion in a polymer matrix. This work examines the effect of the geometry of the inclusion and the surrounding matrix on the predictions of the model.Copyright

Encapsulating Networks of Droplet Interface Bilayers in a Thermoreversible Organogel

The development of membrane-based materials that exhibit the range and robustness of autonomic functions found in biological systems remains elusive. Droplet interface bilayers (DIBs) have been proposed as building blocks for such materials, owing to their simplicity, geometry, and capability for replicating cellular phenomena. Similar to how individual cells operate together to perform complex tasks and functions in tissues, networks of functionalized DIBs have been assembled in modular/scalable networks. Here we present the printing of different configurations of picoliter aqueous droplets in a bath of thermoreversible organogel consisting of hexadecane and SEBS triblock copolymers. The droplets are connected by means of lipid bilayers, creating a network of aqueous subcompartments capable of communicating and hosting various types of chemicals and biomolecules. Upon cooling, the encapsulating organogel solidifies to form self-supported liquid-in-gel, tissue-like materials that are robust and durable. To test the biomolecular networks, we functionalized the network with alamethicin peptides and alpha-hemolysin (αHL) channels. Both channels responded to external voltage inputs, indicating the assembly process does not damage the biomolecules. Moreover, we show that the membrane properties may be regulated through the deformation of the surrounding gel.