Browsing by Author "López, Gabriel P"

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

Biofouling, the accumulation of biomolecules, cells, organisms and their deposits on submerged and implanted surfaces, is a ubiquitous problem across various human endeavors including maritime operations, medicine, food industries and biotechnology. Since several decades, there have been substantial research efforts towards developing various types of antifouling and fouling release approaches to control bioaccumulation on man-made surfaces. In this work we hypothesized, investigated and developed dynamic change of the surface area and topology of elastomers as a general approach for biofouling management. Further, we combined dynamic surface deformation of elastomers with other existing antifouling and fouling-release approaches to develop multifunctional, pro-active biofouling control strategies.

This research work was focused on developing fundamental, new and environment-friendly approaches for biofouling management with emphasis on marine model systems and applications, but which also provided fundamental insights into the control of infectious biofilms on biomedical devices. We used different methods (mechanical stretching, electrical-actuation and pneumatic-actuation) to generate dynamic deformation of elastomer surfaces. Our initial studies showed that dynamic surface deformation methods are effective in detaching laboratory grown bacterial biofilms and barnacles. Further systematic studies revealed that a threshold critical surface strain is required to debond a biofilm from the surface, and this critical strain is dependent on the biofilm mechanical properties including adhesion energy, thickness and modulus. To test the dynamic surface deformation approach in natural environment, we conducted field studies (at Beaufort, NC) in natural seawater using pneumatic-actuation of silicone elastomer. The field studies also confirmed that a critical substrate strain is needed to detach natural biofilm accumulated in seawater. Additionally, the results from the field studies suggested that substrate modulus also affect the critical strain needed to debond biofilms. To sum up, both the laboratory and the field studies proved that dynamic surface deformation approach can effectively detach various biofilms and barnacles, and therefore offers a non-toxic and environmental friendly approach for biofouling management.

Deformable elastomer systems used in our studies are easy to fabricate and can be used as complementary approach for existing commercial strategies for biofouling control. To this end, we aimed towards developed proactive multifunctional surfaces and proposed two different approaches: (i) modification of elastomers with antifouling polymers to produce multifunctional, and (ii) incorporation of silicone-oil additives into the elastomer to enhance fouling-release performance.

In approach (i), we modified poly(vinylmethylsiloxane) elastomer surfaces with zwitterionic polymers using thiol-ene click chemistry and controlled free radical polymerization. These surfaces exhibited both fouling resistance and triggered fouling-release functionalities. The zwitterionic polymers exhibited fouling resistance over short-term (∼hours) exposure to bacteria and barnacle cyprids. The biofilms that eventually accumulated over prolonged-exposure (∼days) were easily detached by applying mechanical strain to the elastomer substrate. In approach (ii), we incorporated silicone-oil additives in deformable elastomer and studied synergistic effect of silicone-oils and surface strain on barnacle detachment. We hypothesized that incorporation of silicone-oil additive reduces the amount of surface strain needed to detach barnacles. Our experimental results supported the above hypothesis and suggested that surface-action of silicone-oils plays a major role in decreasing the strain needed to detach barnacles. Further, we also examined the effect of change in substrate modulus and showed that stiffer substrates require lower amount of strain to detach barnacles.

In summary, this study shows that (1) dynamic surface deformation can be used as an effective, environmental friendly approach for biofouling control (2) stretchable elastomer surfaces modified with anti-fouling polymers provides a pro-active, dual-mode approach for biofouling control, and (3) incorporation of silicone-oils additives into stretchable elastomers improves the fouling-release performance of dynamic surface deformation technology. Dynamic surface deformation by itself and as a supplementary approach can be utilized biofouling management in biomedical, industrial and marine applications.

The design and application of effective drug carriers is a fundamental concern in the delivery of therapeutics for the treatment of cancer and other vexing health problems. Traditionally utilized chemotherapeutics are limited in efficacy due to poor bioavailability as a result of their size and solubility as well as significant deleterious effects to healthy tissue through their inability to preferentially target pathological cells and tissues, especially in treatment of cancer. Thus, a major effort in the development of nanoscopic drug delivery vehicles for cancer treatment has focused on exploiting the inherent differences in tumor physiology and limiting the exposure of drugs to non-tumorous tissue, which is commonly achieved by encapsulation of chemotherapeutics within macromolecular or supramolecular carriers that incorporate targeting ligands and that enable controlled release. The overall aim of this work is to engineer a hybrid nanomaterial system comprised of protein and silica and to characterize its potential as an encapsulating drug carrier. The synthesis of silica, an attractive nanomaterial component because it is both biocompatible as well as structurally and chemically stable, within this system is catalyzed by self-assembled elastin-like polypeptide (ELP) micelles that incorporate of a class of biologically-inspired, silica-promoting peptides, silaffins. Furthermore, this methodology produces near-monodisperse, hybrid inorganic/micellar materials under mild reaction conditions such as temperature, pH and solvent. This work studies this material system along three avenues: 1) proof-of-concept silicification (i.e. the formation and deposition of silica upon organic materials) of ELP micellar templates, 2) encapsulation and pH-triggered release of small, hydrophobic chemotherapeutics, and 3) selective silicification of templates to potentiate retention of peptide targeting ability.

There are over 30 million Foley urinary catheters used annually, and the greatest problem with Foley catheters is catheter-associated urinary tract infections (CAUTIs). CAUTIs are the number one cause of hospital-acquired infections and make up to 40% of nosocomial infections. Biofilms on urinary catheters are critical to the progression of symptomatic CAUTIs, but are difficult to treat due to the protective effect of the biofilm matrix against antibiotics. The anti-fouling catheter technology proposed and demonstrated herein uses a mechanical, non-antibiotic approach to physically remove biofilms and thereby provide an appealing option for potentially stopping the progression of symptomatic infections. Additionally, because the anti-fouling technology is mechanical, it can circumvent the persistent failings of chemical and biological approaches that have failed to address catheter-associated urinary tract infections for the last 50+ years since Foley catheters were introduced.

We designed and optimized urinary catheter prototypes capable of on-demand removal of biofilms from the previously-inaccessible main drainage lumen of catheters. The concept uses pressure-actuated chambers in elastomer constructs to generate regio-selective strain and thereby remove biofilms. We first grew mature Proteus mirabilis crystalline biofilms on flat silicone elastomer substrates, and showed that application of strain to the substrate debonded the biofilm, and that increasing the strain rate increased biofilm detachment. A quantitative relationship between the applied strain rate and biofilm debonding was found through an analysis of the biofilm segment length and the calculated driving force for debonding. We then constructed proof-of-concept prototypes of sections of anti-fouling catheter shafts using silicone and 3D printed reverse molding in methods akin to those used for soft robotics. The proof-of-concept prototypes demonstrated release of mature P. mirabilis crystalline biofilms from their strained surfaces, and prompted our development of more advanced multi-lumen prototypes. The multi-lumen prototypes were designed and optimized using successive rounds of finite element modeling to adjust the number and postion of intra-wall inflation lumens. We then constructed prototypes based on the optimized design with clinically relevant dimensions and showed they were able to generate greater than 30% strain on the majority of the luminal surface, and along their full length. Those catheter prototypes were able to on-demand, and repeatedly, remove greater than 80% of a mixed community biofilm of P. mirabilis and E. coli. In summary, this study shows (1) strain in the elastomeric substrate actively debonds crystalline biofilms in vitro (2) modeling based on characterization of biofilm properties and understanding of substrate strain informs and facilitates prototype catheter design (3) urinary catheter prototypes utilizing inflation-induced substrate strain are capable of on-demand and repeatedly removing biofilms in vitro.

Surfaces with switchable properties in response to external stimuli (e.g., temperature and pH) have attracted substantial research interest because of their ability to modulate biomolecule activity, protein immobilization, and cell adhesion. These stimuli-responsive substrates offer versatile platforms for developing biosensors, cell culture substrates, diagnostic systems, and drug delivery systems. In this work, we controllably functionalized substrates with genetically engineered polypeptides to fabricate thermally responsive surfaces for various bioanalytical applications. Genetically engineered elastin-like polypeptides (ELPs) are one class of thermally responsive biopolymers that are characterized by their lower critical solution temperature (LCST) phase behavior in water; ELPs at a given concentration in aqueous solvent phase separate to form protein-rich coacervates above the cloud point transition temperature (Tt). ELPs present an attractive alternative to synthetic, stimuli-responsive polymers due to their biocompatibility, monodispersity, and controlled physicochemical properties.

To fabricate ELP-modified surfaces with desired structure and functionality, we first investigated the adsorption behavior of ELP homopolymers and ELP block copolymers onto silica surfaces. We provided an in-depth understanding of adsorption kinetics, mechanism and surface conformation for the “canonical” ELP sequence (Val-Pro-Gly-Val-Gly), which enabled precise conformational control of the adsorbed ELPs. We also showed that genetically incorporating the silaffin R5 peptides into ELP chains significantly enhanced the binding affinity of ELPs to silica surfaces, leading to thicker ELP layers with a higher surface coverage. To extend this work, we also explored the adsorption behavior of ELP block copolymers onto silica surfaces using theoretical and experimental approaches. Our results showed that the silaffin tag not only enhanced the binding of ELP block copolymers to silica surfaces, but also directed micelle adsorption, leading to close-packed micellar arrangements dissimilar to the sparse and patchy arrangements observed for ELP micelles lacking a silaffin tag. In addition, the surface-grafted ELP unimers exhibited interfacial phase transition behavior, while the adsorbed ELP micelles were no longer thermally-responsive. These studies provided insight into the design of ELP based smart surfaces with controlled structure-architecture-function relationship.

After achieving programmable adsorption of ELPs onto surfaces, we exploited these thermally responsive surfaces for several bioanalytical applications, including cell culture and diagnostic assays. We first developed a simple approach to pattern cells on gold patterned silicon substrates using ELPs with cell- and gold-binding domains. Cell patterning was achieved by exploiting orientation of the adsorbed ELP to either enhance (gold regions) or impede (silicon oxide regions) cell adhesion at particular locations on the patterned surface. Along a similar vein, we fabricated a thermoresponsive cell culture substrate using rationally designed ELP coatings with precisely spaced cell-adhesive motifs. The reversible swelling and collapse of ELPs thermally modulated the accessibility of cell-binding domains to enable cell adhesion at T > Tt and efficient cell recovery at T < Tt.

In addition, we have utilized ELP-modified particles to develop smart diagnostics. We demonstrated proof-of-concept for an acoustofluidic, chip-based method that enables the rapid capture and isolation of biomarkers from blood for off-chip quantification. We showed that biomarkers were rapidly immobilized onto the surfaces of ELP-modified particles via co-aggregation, and continuously separated from the blood cells using an acoustofluidic device. The captured biomarkers can then be quantified using flow cytometry, or released from the surfaces of particles for further analysis. By designing ELP fusion proteins that can capture target bioactive materials, this platform system can be readily extended to separate a range of biological materials (e.g., cells, viruses and cell-free DNA) from complex biofluids.

In summary, we achieved controlled adsorption of ELP homopolymers and block copolymers onto surfaces with tailored architecture and functionality. These ELP-modified smart surfaces have been utilized to create cellular patterns, a thermoresponsive cell culture substrate, and a biomarker separation and detection platform.