Stephen Kennedy

An Integrated Microrobotic Platform for On‐Demand, Targeted Therapeutic Interventions

The presented microrobotic platform combines together the advantages of self-folding NIR light sensitive polymer bilayers, magnetic alginate microbeads, and a 3D manipulation system, to propose a solution for targeted, on-demand drug and cell delivery. First feasibility studies are presented together with the potential of the full design.

Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells.

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 mus pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvettes electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.

Biphasic Ferrogels for Triggered Drug and Cell Delivery

Ferrogels are an attractive material for many biomedical applications due to their ability to deliver a wide variety of therapeutic drugs on-demand. However, typical ferrogels have yet to be optimized for use in cell-based therapies, as they possess limited ability to harbor and release viable cells. Previously, an active porous scaffold that exhibits large deformations and enhanced biological agent release under moderate magnetic fields has been demonstrated. Unfortunately, at small device sizes optimal for implantation (e.g., 2 mm thickness), these monophasic ferrogels no longer achieve significant deformation due to a reduced body force. A new biphasic ferrogel, containing an iron oxide gradient, capable of large deformations and triggered release even at small gel dimensions, is presented in this study. Biphasic ferrogels demonstrate increased porosity, enhanced mechanical properties, and potentially increased biocompatibility due to their reduced iron oxide content. With their ability to deliver drugs and cells on-demand, it is expected that these ferrogels will have wide utility in the fields of tissue engineering and regenerative medicine.

3D Printed Microtransporters: Compound Micromachines for Spatiotemporally Controlled Delivery of Therapeutic Agents

Functional compound micromachines are fabricated by a design methodology using 3D direct laser writing and selective physical vapor deposition of magnetic materials. Microtransporters with a wirelessly controlled Archimedes screw pumping mechanism are engineered. Spatiotemporally controlled collection, transport, and delivery of micro particles, as well as magnetic nanohelices inside microfluidic channels are demonstrated.

Experimental Studies of Persistent Poration Dynamics of Cell Membranes Induced by Electric Pulses

Experiments have been conducted that investigate the plasma membrane response of HL-60 human promyelocytic leukemia cells to pulsed electric fields (PEFs) with pulsewidths ranging from 125 ns to 1 ms. Both single and trains of monopolar PEFs were delivered to HL-60 cells while suspended in microcuvettes where they were monitored in real-time via optical microscopy. By monitoring the uptake of trypan blue (TB) into the cell cytoplasm, the formation of pores that persist long after the electric stimulus was recorded as a function of pulsewidth, pulse amplitude, and interpulse spacing. Results qualitatively agree with previous experimental observations, as well as theoretical predictions on pulse accumulation effects due to persistence of large pores, and rapid relaxation times of transient small pores. The resealing or relaxation time was estimated to be approximately 250 ns for the transient pores and 1 s or more for the persistent pores. The presence of calcium and magnesium in the cell suspension fluid impeded persistent pore formation, particularly at shorter pulsewidths. Finally, the authors report the first experimental measurement of two fundamental membrane poration parameters: the pore diffusivity and the pore-perimeter line-tension-energy coefficient

Rapid and extensive collapse from electrically responsive macroporous hydrogels

Electrically responsive hydrogels are created with interconnected macropores, which greatly enhance their ability to rapidly undergo volumetric collapse when subjected to moderate electric fields. When optimized, these electrogels are easily integrated into arrays capable of rapid configurational and chromatic optical modulations, and when loaded with drugs, are able to coordinate the delivery profile of multiple drugs.

Sequential Release of Nanoparticle Payloads from Ultrasonically Burstable Capsules

In many biomedical contexts ranging from chemotherapy to tissue engineering, it is beneficial to sequentially present bioactive payloads. Explicit control over the timing and dose of these presentations is highly desirable. Here, we present a capsule-based delivery system capable of rapidly releasing multiple payloads in response to ultrasonic signals. In vitro, these alginate capsules exhibited excellent payload retention for up to 1 week when unstimulated and delivered their entire payloads when ultrasonically stimulated for 10-100 s. Shorter exposures (10 s) were required to trigger delivery from capsules embedded in hydrogels placed in a tissue model and did not result in tissue heating or death of encapsulated cells. Different types of capsules were tuned to rupture in response to different ultrasonic stimuli, thus permitting the sequential, on-demand delivery of nanoparticle payloads. As a proof of concept, gold nanoparticles were decorated with bone morphogenetic protein-2 to demonstrate the potential bioactivity of nanoparticle payloads. These nanoparticles were not cytotoxic and induced an osteogenic response in mouse mesenchymal stem cells. This system may enable researchers and physicians to remotely regulate the timing, dose, and sequence of drug delivery on-demand, with a wide range of clinical applications ranging from tissue engineering to cancer treatment.

Switchable Release of Entrapped Nanoparticles from Alginate Hydrogels

Natural biological processes are intricately controlled by the timing and spatial distribution of various cues. To mimic this precise level of control, the physical sizes of gold nanoparticles are utilized to sterically entrap them in hydrogel materials, where they are subsequently released only in response to ultrasound. These nanoparticles can transport bioactive factors to cells and direct cell behavior on-demand.

Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

Background The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired.

Work in progress - a first-year introduction-to-engineering course on Society’s Engineering Grand Challenges

A new cross-disciplinary first-year course, ldquoIntroduction to Societypsilas Engineering Grand Challenges,rdquo has been developed as part of a college-wide initiative at the University of Wisconsin-Madison to transform undergraduate engineering education for 2010 and beyond. The inspiration for developing this new course was the National Academy of Engineeringpsilas ldquoEngineering Grand Challengesrdquo project. By emphasizing humanitarian applications in an introductory engineering course, we expect to not only inspire future generations of engineers and show students how the skills they will be learning can have a positive impact on quality of life, but also encourage more women to pursue engineering degrees. The course consists of an introductory module followed by two theme-based modules of the studentpsilas choosing, selected from the following themes: ldquoEngineering challenges that impact our lives on a personal scale,rdquo ldquoEngineering for the developing world,rdquo ldquoEngineering the megacity,rdquo ldquoGlobal engineering challenges,rdquo and ldquoEngineering beyond Planet Earth.rdquo The team-taught course is being offered for the first time in the spring of 2008. This paper presents an overview of the course, the expected outcomes, and the preliminary results from a survey-based assessment tool.