Research

Point-of-Care Tests for HPV

It is widely established that human papilloma virus (HPV) is the root cause of cervical cancer in women. Current methods of detection are time consuming since they involve collecting samples from women in a physician’s office and sending the sample for analysis to a centralized clinical laboratory. To overcome the delay in obtaining results, we are developing a rapid isothermal point-of-care diagnostics test for the 14 high risk genotype strains of HPV. The test is easy to perform and accurately diagnoses HPV in less than 15 min. This novel rapid assay will dramatically simplify cervical cancer screening for women and health providers in under-served communities and nations. 

Point-of-Care Tests for SARS-CoV-2

Rapid on-site diagnosis of SARS-CoV-2 is key for therapy and to prevent further spreading of the virus. Thus, there is a need for rapid PoC SARS-CoV-2 molecular screening tests that are selective, sensitive, reliable, and easily integrated in different settings around the world. We are developing a simple, rapid (<30 min), and inexpensive test specific for SARS-CoV-2 and its variants that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers that facilitate detection with paper-based microfluidics and visual inspection. We envision our test platform as an on-site primary screening tool for local hospitals, doctors’ offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

Point-of-Care Tests for Zika Virus

We are developing novel molecular Point of Care (PoC) methods required for detection of emerging pathogens (e.g., Flavi Virus (Zika)) that are low-cost and do not require extensive equipment. Simple sample collection and purification is followed by isothermal amplification (e.g., rolling circle amplification (RCA)), after which the amplified product is captured and colorimetrically detected by lateral flow assay. Since these assays are based on the genomic sequence of these pathogens, different pathogen strains and related pathogens (e.g., Dengue versus Zika) can be distinguished.

Point-of-Care Test for Bacterial Vaginosis

Bacterial vaginosis (BV) is characterized by the imbalance of the bacteria naturally present in the vaginal microbiome and is the most common genital condition among reproductive aged women. The potential outcomes of BV negatively impact the patient’s quality of life due to the potential physical discomfort, embarrassment, and social isolation as a result of undiagnosed BV. BV is also associated with poor reproductive health outcomes and an increased risk of sexually transmitted infections (STIs) and human immunodeficiency virus (HIV) acquisition. Clinical and laboratory diagnosis is challenging, and there is a need for an accurate, affordable, and simple point of care test to identify BV. One of the pathogenic bacteria associated with BV is Gardnerella vaginalis, which produces the toxin vaginolysin (VLY). This project involves design of a diagnostic test for the presence of VLY as a first step to develop a point-of-care test for BV. We have developed a method for the expression, purification, and detection of vaginolysin via an ELISA, which was then translated into a point-of-care device by adapting it into a lateral flow assay.

Point-of-Care Tests for Food Pathogens

This project is in collaboration with Dr. Sapna Deo

Point-of-Care Tests for Urinary Tract Infections

This project is in collaboration with Dr. Sapna Deo

Deployable Microneedles for Localized Treatment of Cancer

We are working on the development of a novel drug delivery platform with the aim of successfully releasing a chemotherapeutic active compound into its site of action. Microneedles are delivery systems created out of biocompatible and biodegradable polymers that allow us to modulate the release rate of drugs. We have created drug-loaded microneedles that can be easily implanted in the desired location to release the chemotherapeutic agent in a controlled manner, achieving a sustained therapeutic effect and avoiding potential side effects.

Development and Fabrication of Graphene-Based Solid-State Gas Sensors

Electronic gas sensors have long been used as a method to identify surrounding gaseous species accurately and in real time. They can be applied in environmental sampling for continuous monitoring of quality of surrounding air or can be used as an electronic nose. Commercial solid-state gas sensors, while having small size, low power and low cost have problems with long term use and limited accuracy. Graphene is one atom thick plane of carbon packed in a honeycomb structure and it packs some unique mechanical and electronic properties. Graphene has been used frequently in sensor applications including gas sensors, electronic, biological and chemical sensors. A graphene nanomesh-based chemiresistor was conceived as the initial prototype to build. Chemiresistor has an active sensing layer that interacts with the target chemical. It has the following advantages: it is easy to fabricate, uses a small amount of active materials, wide variety of sensitive materials and linear sensor profile. The advantage of using graphene to make sensors is due to its high thermo-electric conduction, huge surface area to volume ratio, and high resistance to mechanical and chemical stress. The high surface area leads to higher sensitivity. In addition to this, graphene nanomesh gives an additional advantage which is being able to tune the bandgap, thus reducing the noise from thermal flow of electrons.

Biomaterials for On-Site Controlled Delivery of Anticancer Drugs

Transarterial chemoembolization (TACE) is a procedure where the blood vessels going into the tumor cells are blocked using microbeads containing anti-cancer drugs. However, current TACE materials release their chemotherapeutic agents in a relatively fast rate. To improve the release kinetics and achieve slow controlled release of the chemotherapeutic drug doxorubicin, we designed a pH-sensitive doxorubicin-loaded microspheres and used this formulation for chemoembolization purposes. The doxorubicin is immobilized on the beads via the pH-labile hydrazine moiety between doxorubicin and N-methacryloglycylglycine. When the microspheres are in the low pH microenvironment of the tumor, the linkage will be hydrolyzed releasing doxorubicin to the tumor environment. The beads synthesis was optimized with respect to their percent composition, polymerization rate, temperature, and their pH dependent drug release kinetics. Currently, we are in the process of studying their release profiles and they will soon be tested, in vivo, for their embolization efficacy.

Fast-Acting Biomaterials for Control of Homeostasis

Hemostasis comprises all physiological mechanisms whose ultimate aim is to stop the bleeding from an injury. After specific surgical procedures, patients have a potential risk of suffering hemorrhages that could have severe consequences and compromise their well-being. Therefore, we are working to produce fast-acting biocompatible materials that can be implanted in the wound tract of the patient that will aid the hemostasis process and avoid severe hemorrhagic complications.

Materials Synthesis for Molecular Recognition of Polyaromatic Compounds

Electronic gas sensors have long been used as a method to identify surrounding gaseous specie accurately and in real time. They can be applied in environmental sampling for continuous monitoring of quality of surrounding air or can be used as an electronic nose. Commercial solid-state gas sensors, while having small size, low power and low cost have problems with long term use and limited accuracy. Graphene is one atom thick plane of carbon packed in a honeycomb structure and it packs some unique mechanical and electronic properties. Graphene has been used frequently in sensor applications including gas sensors, electronic, Biological and chemical sensors. A graphene nanomesh based chemiresistor was conceived as the initial prototype to build. Chemiresistor has an active sensing layer that interacts with the target chemical. It has the following advantages: it is easy to fabricate, uses a small amount of active materials, wide variety of sensitive materials and linear sensor profile. The advantage of using graphene to make sensors is due to its high thermo-electric conduction, huge surface area to volume ratio, and high resistance to mechanical and chemical stress. The high surface area leads to higher sensitivity. In addition to this, graphene nanomesh gives an additional advantage which is being able to tune the bandgap, thus reducing the noise from thermal flow of electrons.

Development of a Theranostic Gold Nanoparticle to Enhance Prostate Cancer Imaging and Treatment

This is an interdisciplinary project with Dr. Alan Pollack, which aims to develop gold nanoparticles that target prostate-specific membrane antigens (PSMA) and are conjugated with HDAC inhibitors. The proposed transdisciplinary project brings together expertise in androgen receptor (AR) signaling in prostate cancer management (Pollack), gold x-ray fluorescence for imaging (Ford), and nanoparticle mediated drug delivery (Daunert). The foundations of our approach are based on the hypotheses that (i) anti-PSMA antibody conjugated to gold nanoparticles (PTGNPs) will sensitize prostate cancer to RT; (ii) gold x-ray fluorescence from irradiated PTGNPs results in high levels of localization and quantification that is comparable or better than that achieved by PSMA targeted PET scans; (iii) the addition of an HDAC inhibitor (HDACi) to PTGNPs, either directly (Hi-PTGNPs) or using dendrimers to increase HDACi loading (Hi(D)-PTGNPs) will enhance radiosensitization; and (iv) PTGNPs with or without dendrimer HDACi loading (Hi(D)-PTGNPs) will enhance cell killing from ADT in part through ATF3 upregulation and AR downregulation. Prior work from our group has established that ATF3 upregulation interrupts AR signaling and potentiates the inhibition of prostate tumor growth from ADT. Preliminary data indicate that some of the HDACi’s upregulate ATF3 in androgen sensitive LNCaP cells. Preliminary data form our team also has demonstrated the feasibility of preparing antibody-conjugated nanoparticles as well as conjugated dendrimer nanocarriers for theranostic and targeted drug delivery applications.

Implantable Microneedles for Prevention of Hearing Loss

Inner ear drug delivery techniques addressing hearing loss are challenging to develop due to the inherent complexity of the cochlear anatomy, which limits molecular transportation. A promising solution is the use of biodegradable polymers because the continuous release of bioactive molecules without introducing foreign compounds is highly desirable. This project involves the development and placement of drug infused polymeric microneedles into the cochlea to minimize hearing loss over extended periods of time.

Community Behavior of the Microbiome

Much focus has been put into research of the human microbiomes with investigators working to determine specific bacterial species that are present and correlate them with disease state. However, one must always take into account that bacteria do not function independently and often coordinate their behavior across their community via quorum sensing (QS), a complex system of gene regulation dependent upon small chemical molecules known as quorum sensing molecules (QSMs). QS controls a plethora of bacterial processes across differing species, most notably biofilm formation and production of virulence factors. QS has been shown to be a form of interkingdom signaling, with bacterial QSMs being able to directly interact with receptors on cells in its human host, potentially even initiating oncogenic pathways in mammalian cells.

Thus, my research group has shifted its focus on the microbiome beyond the metagenomics approach to also include investigations into bacterial QSMs as a way to better understand the community behavior.

Biosensors for Quorum Sensing Molecules

Bacterial quorum sensing (QS), a cell-cell communication system based on small molecules known as quorum sensing molecules (QSMs), controls processes reliant on the synchronized behaviour of large communities of bacteria. QS hinges on the timely production, release, and detection of QSMs. As bacterial density increases, so does the concentrations of QSMs; once a threshold of QSMs is surpassed molecular cascades are triggered, resulting in population-wide gene regulation. A recent study identified a novel QSM, 3,5-dimethylpyrazin-2-ol (DPO) employed by pathogenic Vibrio cholerae to inhibit biofilm formation. DPO is synthesized by threonine dehydrogenase (Tdh). Tdh is highly conserved across bacteria, archaea, and eukarya, suggesting a possible role for DPO in other unidentified QS circuits. Studies that have evaluated microbiome composition and function in the context of health and disease have thus far relied on the use of costly metagenomic sequencing, metabolomic profiling, and bioinformatic analysis. These investigations have resulted in our understanding that the healthy microbiome is highly diverse and that this community of microbes is in an altered state in a variety of disorders. The transition into an altered state with profound effects on the host physiology is not fully understood, however it is known that these massive shifts in bacterial populations involve a complex interplay between distinct members of the community and their environment, implicating QS in population-wide gene regulation. Our group is interested in expanding our understanding of the microbiome beyond metagenomic and metabolomic sequencing by enabling the study of bacterial communication systems as it relates to host-microbiome interactions.

There is currently an unmet need for tools enabling quantification of QSM’s, and whole-cell biosensors (WCB’s) can help to fill that void. WCBs integrate molecular recognition with signal generating proteins to create a sensing cell that can be integrated in a device to detect a target molecule(s). The goal of this proposal is to investigate DPO’s currently unexplored relevance to host health and disease by developing a new biosensing tool capable of the specific and selective quantitative detection of DPO in biological samples. The central hypothesis of this project is that the development of a WCB for detection of the novel AI, DPO, will provide insight into and highlight the relevance of bacterial communication systems within the microbiome in the context of health and disease states.

Quorum Sensing as a Biomarker in Inflammatory Bowel Disease

Crohn’s Disease (CD), a type of Inflammatory Bowel Disease (IBD), is an immune-mediated, chronic inflammatory disease affects approximately 565,000 individuals in the United States. Although considered a disease of the Western world, CD incidence is on the rise in the developing world as well as in younger populations; thus, there is increasing need for better diagnosis and management of CD. The field continues to rely on colonoscopy (see image below of a healthy and inflamed colon) for a diagnosis of CD and to assess mucosal healing in response to therapy. Unfortunately, this is an invasive procedure carrying risk and is unpleasant for patients to have performed frequently.

The microbiome plays a critical role in the initiation and perpetuation of intestinal inflammation in Crohn's disease patients with studies of the microbiota in CD patients revealing a decrease in overall diversity. With such changes in the composition of the bacterial community, in collaboration with Dr. Maria T. Abreu, Director of the Crohn’s and Colitis Center at the University of Miami Miller School of Medicine, we wished to determine how this impacts bacterial quorum sensing (QS), the community communication system employed by bacteria. As such, we determined the levels of specific classes of QSMs in the blood serum of individuals with CD and active intestinal inflammation. Our results, shown below, indicate that bacterial QSMs act as a serum marker for intestinal inflammation in individuals with CD and may prove a biomarker of disease state.

The Role of Quorum Sensing in Colon Cancer

Dysbiosis is a consistent finding in IBD and colitis associated cancer (CAC), however, actionable strategies to reduce inflammation and/or CAC risk through microbiome manipulation are not yet available. Quorum sensing molecules (QSMs) are secreted by bacteria and are responsible for bacterial communication, biofilm formation, and activation of virulence systems. Recently, specific QSMs have been shown to stimulate oncogenic pathways in mammalian cells including pancreatic cancer and colonic mucosa.

To determine how bacterial QSM levels in serum correlate with CAC development, in collaboration with Dr. Maria T. Abreu, we assessed a sub-population of individuals at high risk for development of CAC. Individuals selected had been diagnosed with Ulcerative Colitis (UC) >10 years at the point of evaluation and either had active colonic inflammation or quiescent disease. As a comparison, we selected individuals who had been diagnosed with UC for <5 years and either had active or quiescent disease. As shown below, we found that patients with UC >10 years and active disease had elevated levels of certain classes of QSMs compared to the other groups, indicating their potential role as a biomarker or underlying initiator of CAC.

Host-Microbiome Interactions in Spinal Cord Injury

In the United States each year approximately 11-12,000 new spinal cord injuries (SCI) occur resulting in a spectrum of neurological impairments and disabilities. Currently, there are approximately 250,000 people living in the US with the devastating neurological deficits and somatic and autonomic reflexes that develop with chronic SCI. Chronic SCI causes interrelated secondary complications that significantly compromise health and quality of life after injury, including pathological dysfunction in the autonomic nervous system, immune system, and gastrointestinal tract.

Work by our group, in collaboration with Dr. Dalton Dietrich, Scientific Director of the Miami Project to Cure Paralysis and the University of Miami Miller School of Medicine, has determined that SCI causes short- and long-term alterations to intestinal inflammation, the gut microbiome, and bacterial QSM levels in a rat model of SCI. Ongoing studies in our laboratory are working to translate these findings into individuals living with SCI, with the intent to determine the molecular mechanisms underlying the GI dysfunction within these individuals and thus potentially develop therapeutics to restore function. As shown in the figure below, alterations to the gut microbiome (beta diversity) have been determined in individuals living with SCI as compared to control.

Effect of the Microbiome in the Gut-Brain Axis

Key to unlocking the role of the gut microbiome is to understand the interactions with its environment. A potential link between the gut and the brain is the mammalian neurotransmitter serotonin. More than 90% of the body’s monoamine serotonin is synthesized by gut enterochromaffin cells. However, the molecular mechanism that dictates the levels of serotonin produced and its metabolism is not fully elucidated. This is of utmost importance given that gut-derived serotonin is responsible for regulation of functions such as bone development, immune responses, gut motility, and platelet aggregation. Perhaps more interesting is the role that dysregulation of serotonin plays in the pathogenesis of certain intestinal diseases such as irritable bowel syndrome (IBS).

To this end, our research has demonstrated that serotonin can act as a bacterial communication molecule for pathogenic bacteria, specifically Pseudomonas aeruginosa. Serotonin increases Pseudomonas virulence factor and biofilm production in vitro and enhances virulence in infected mice. These findings are beneficial in understanding the impact of the microbiome on human health and may open the door for novel treatments for human disease.

The Role of Quorum Sensing in Depression

Major depressive disorder is a debilitating disease with a lifetime incidence of ~20% with current treatments often lacking efficacy and taking several weeks to be effective. Recent evidence has pointed toward a role of the gut microbiome composition in exacerbating psychiatric disorders, including major depression. The bidirectional communication between the gut and the brain is thought to involve neural, hormonal, and immunological routes, including the sympathetic and parasympathetic arms of the autonomic nervous system and the enteric nervous system. Dysregulation of this communication often leads to pathophysiological effects.

As part of ongoing studies in our group, in collaboration with Dr. Eleonore Beurel, we have investigated alterations in the microbiome and quorum sensing molecules in a murine model of depressive-like behaviors (learned helplessness) and found that the specific quorum sensing molecule, autoinducer-2 (AI-2), can induce depressive-like behaviors. Further, these effects can be alleviated through inhibition of the AI-2 pathway. Continued investigations will allow us to better understand the role bacterial quorum sensing plays in depression and potentially lead to development of therapeutic interventions.

Examining the Association between the reproductive tract Microbiome and successful implantation of embryos

In collaboration with Dr. Jonah Bardos, we are researching whether the microbiome inside a woman’s reproductive tract (vaginal, cervical, and uterine) have an effect on the success of an in vitro fertilization (IVF) cycle. In addition, we are evaluating a new method to see if we can predict the success or failure of a cycle before we even transfer the embryo. We are sampling discarded instruments after they are used as well as obtaining samples from the lower reproductive tract in order to determine the presence and levels of bacterial quorum sensing molecules (QSMs). This is very exciting and cutting-edge research that has the potential to change the way IVF is practiced. Currently, we have confirmed the presence of a category of QSMs within cervical swabs from healthy individuals as shown below.

Understanding the Role of the Microbiome in Endometrial Cancer

Endometrial cancer (EC) is the most common gynecologic malignancy in the United States, yet there is no screening test for it. The incidence of this disease has increased, such that it is now one of the leading causes of cancer death in women. Metagenomics, or the comprehensive evaluation of the microbial genome, is a novel way to understand the vaginal microbial environment and has revealed differences in diversity and phyla abundance between benign and tumor conditions, and across tumor grades, which also correlated with tumor genomics. As such, in collaboration with Drs. Matthew Schlumbrecht, Vice Chair of Global and Community Health at the University of Miami Miller School of Medicine, and Sophia George, we have begun investigations into bacterial quorum sensing (QS) in EC. As shown below, classes of quorum sensing molecules from endometrial swabs were found to have different levels depending upon low-grade (LG) EC, high-grade (HG) EC, or from healthy controls.

Effect of Environmental and Occupational Exposure to PAHs on the Microbiome

The routine exposure of firefighters to numerous toxic agents may affect their reproductive health, which can lead to miscarriages and infertility. An overall challenged reproductive potential has been associated with altered vaginal microbiome composition and dysbiosis. In this project we evaluate the vaginal microbiota of active firefighters and controls via Shotgun Metagenomic sequencing, while also assessing the levels of polycyclic aromatic hydrocarbons in vaginal swabs, with the aim of investigating a potential association between environmental and occupational exposures, and alterations of the microbial profile.

Vanilloid Nanodrugs for Brain-targeted Therapeutic Hypothermia

Therapeutic hypothermia is associated with neuroprotection, as the intentional decrease of temperature can lead to prevention of a surge in neuronal cell death, and can be applied in cases of brain trauma, cardiac arrest, spinal cord injury, and several other acute injuries. The goal of this project is to develop nanodrugs from natural and synthetic compounds, such as vanilloids, which can activate brain thermoreceptors, like TRPV1 receptor, and trigger a localized and controlled cooling effect. Our approach is based on a carrier-free, nanodrug synthesis, which produces self-assembled nanovanilloids that exhibit intrinsic thermoregulatory properties able to induce rapid, sustained, and reversible hypothermia.

Nanocarrier-mediated Targeted Stem Cell Therapy for Liver Tissue Regeneration

Hepatocellular cancer (HCC) is seen in the background of cirrhosis with associated liver dysfunction. In 2018, there were over 40,710 new HCC cases and approximately 28,920 deaths in the US. Globally, there were 841,080 new cases in 2018, and 1,361,836 new cases are expected in 2040. Image-guided Radiofrequency Ablation (RFA) is a minimally invasive treatment, however, patients with severe liver dysfunction are noncandidates due to the risk of complete liver failure from tissue loss. Mesenchymal stem cells (MSC) can regenerate liver tissue, but targeted homing of MSC into the site is challenging.

We have developed a nanocarrier-mediated cell delivery method by coating the surface of the stem cells with dendrimer nanoparticles complexed with adhesion molecules. These nanocarriers function as a ‘GPS’ to navigate the MSC to their destination via recognizing and binding with the counterpart adhesion molecules expressed on the activated endothelial cells (EC) in the injured tissues. Once anchored on the activated endothelium, nanocarrier-coated cells extravasate and home to the target tissues.RFA induces a strong inflammatory response at the margins of the ablation zone. Intercellular adhesion molecule-1 (ICAM-1) is upregulated in EC in response to thermal injury. ICAM-1 interacts with the I-domain of the lymphocyte function-associated antigen-1 (LFA-1). MSC are coated with dendrimer nanoparticles complexed with I-Domain of LFA-1. The primary objective of this study is to test the safety and efficacy of liver tissue regeneration following thermal ablation in porcine livers using nanocarrier mediated MSC therapy.

sE-Selectin based Targeting of Inflamed Tissues

During this project, we will design a delivery nano-platform based on the E-selectin’s ability to bind to its ligands in the inflamed endothelium. Our hypothesis is that coating therapeutic bone marrow-derived cells with E-selectin would lead to a homing effect of these cells in the wound and hence enhance the rate of wound healing. E-selectin, however, is a large and difficult-to-synthesize protein, therefore we will study the motifs responsible for the selectin/ligand interaction to develop a small functional peptide able to fulfill this aim.

Targeted Delivery of Mesenchymal Stem Cells for the Treatment of Inflammatory Bowel Disease

Treatment for inflammatory bowel disease (IBD) continues to be suboptimal in many ways. Traditional therapy is based on inhibition of pro-inflammatory cytokines most notably TNFα and, to a lesser extent, the use of immunomodulators, which are implicated in the development of lymphomas. Some of these malignancies disproportionately affect young people limiting their use. Although the efficacy is high, many patients or parents of affected children are reluctant to use these types of therapies for fear that they will have long-term consequences. Moreover, these types of therapy do not address the structural damage to the gut or in the case of very early onset IBD (veoIBD), the root genetic cause cannot be rectified. Childhood onset IBD makes the issue of structural damage of the gut even more impactful.

Mesenchymal stem cells (MSCs) have been proposed as alternative beneficial therapy for IBD, in particular in Crohn’s disease (CD), because of their anti-inflammatory properties. Although medical therapy is directed at achieving mucosal healing, it is a minority of patients that achieve complete mucosal healing. In order to increase the efficacy of MSCs as a therapeutic in IBD, we have devised a nanocarrier platform to target MSCs to intestinal inflammation. The figure below shows the ability of our platform (MSC-AcG5-LFA1) to specifically target the inflamed intestinal region in a murine model of IBD.

Targeted Delivery of Mesenchymal Stem Cells for the Treatment of Diabetes

Human Umbilical Cord-derived Mesenchymal Stromal Sells (UC-MSC) are emerging as therapeutic candidates because they can modulate immune responses and stimulate tissue repair. These cells have yielded promising results in clinical trials for autoimmune diseases, inflammatory disorders, and COVID-19. UC-MSCs can be safely administered intravenously. When administered this way most cells become trapped in ‘filter organs’ including liver, lung, spleen, and kidneys, and only a small fraction of cells reaches the target inflamed tissues. It would be ideal to deliver UC-MSC specifically to inflamed endothelia. Inflamed endothelia are characterized by an increased expression of intercellular adhesion molecule (ICAM-1). One of the natural binding partners of ICAM-1 is leukocyte function-associated antigen-1 (LFA-1). LFA-1, a member of the integrin family, presents an alpha subunit involved in adhesion. In particular, the LFA-1 alpha subunit I-domain (LFA-1-Id) has a binding site for ICAM-1. The LFA-1 and ICAM-1 interaction enables migration of leukocytes into inflammation sites and could be harnessed to build nanocarriers for the targeted delivery of UC-MSC. We aimed at engineering ‘LFA-1 nanocarriers’, i.e. nanocarriers functionalized with LFA-1-Id, for the targeted delivery of UC-MSC to inflamed endothelia. We have demonstrated that UC-MSCs coated with functionalized LFA-1 nanocarriers adhered more efficiently to inflamed endothelia in vitro. LFA-1 nanocarriers potentiate UC-MSC binding to inflamed endothelia. Future analyses will explore LFA-1 nanocarrier-targeted delivery of UC-MSCs in animal models of autoimmunity, to determine homing and therapeutic effects.

Nanoparticle Based Diagnostics and Therapy (Theranostics)

This project is in collaboration with Dr. Sapna Deo

Carbon Nano-Dots for Imaging and Drug Delivery

Carbon-based materials have become quite significant in many applied sciences. Carbon dots have become the rising star within the carbon family of nanomaterials due to their excellent photophysical properties, high quantum yield, low toxicity, small size, biocompatibility, and abundant low-cost sources. Due to these desirable properties, carbon dots have been used as fluorescent probes for detecting analytes in the environment or in biological systems. Their small size, large specific surface area, and abundant surface functional groups make carbon dots very amenable to be derivatized readily. Most functional groups on carbon dots are pH sensitive therefore the fluorescent properties of the carbon dots are quite responsive to different environments. This sensitivity allows for changes in carbon dots properties; changes such as enhancement/activation and/or quenching of fluorescence. Therefore, carbon dots have become in the forefront for the utility as sensors for the detection of many biological and environmental applications. In our lab, we use carbon dots as labels for analysis of biologically relevant molecules.

Nanocarriers for Gene Therapy

This project is in collaboration with Dr. Sapna Deo

Self-assembled Ivermectin nanodrugs for Zika Virus

The aim of this project is to employ innovative synthetic methods to formulate antiviral agents into new dimensional self-assembled amphiphilic nanostructures. These nanodrugs can be used for the treatment of viral infections caused by flaviviruses, such as ZIKV, thus addressing the unmet need of developing effective new strategies to treat and prevent the further spread of ZIKV.

NanoNaloxone and NanoNaltrexone as Therapy for Stroke Recovery

The US is in the midst of an opioid epidemic that is linked to a number of serious health issues, including an increase in cerebrovascular events, namely stroke. Chronic prescription opioid use exacerbates risk and severity of ischemic stroke, contributing to stroke as the fifth overall cause of death in the US and costing the US health care system over $30 billion annually. Pathologically, opioids challenge the integrity of the blood brain barrier (BBB), resulting in a dysregulation of tight junction (TJ) proteins that are crucial in maintaining barrier homeostasis. Despite this, treatment options for ischemic stroke are limited and there are no pharmacological options to attenuate TJ damage, including in incidents that are linked to opioid use. Herein, we have generated carrier-free, nanodrugs of naloxone and naltrexone with enhanced therapeutic properties compared to the original drugs. The generated nanoforms of both opioid antagonists exhibited successful attenuation of morphine or oxycodone-induced alterations of TJ protein expression and reduced oxidative stress to a greater extent than the parent drugs (non-nano). We then proceeded to evaluate the therapeutic effectiveness of the generated nanodrugs in ischemic stroke in mice exposed to morphine or oxycodone. Our results demonstrate that nano-naloxone protected against stroke severity in mice treated with morphine more potently than naloxone. Overall, this research implements advances in nanotechnology-based repurposing of FDA-approved therapeutics and the obtained results uncover underlying pharmacological mechanisms of opioid antagonists as effective therapeutic agents in ischemic stroke.

NanoCBD as an immunomodulatory therapeutic for Alopecia areata

We have previously reviewed and highlighted the pharmacodynamics attributed to the immune modulatory behavior of cannabidiol (CBD), through modulation of critical immunological signaling pathways, namely Janus kinase/signal transducers and activators of transcription (JAK/STAT). However, the potential enhancement of the existing immunomodulatory effects of CBD by nanotechnology has yet to be explored. We propose a novel synthesis and use of pure, carrier-free, CBD nanodrug formulations to improve the immune-suppressive effects of CBD. Formulation of CBD nanodrugs opens up a novel drug delivery system by addressing the hydrophobicity and poor solubility issues of CBD, as well as reducing the dosage required to exhibit the therapeutic properties of this promising immunomodulatory agent. To date, use of CBD nanoparticles as a therapeutic agent for immunomodulation remains a novel and unexplored therapeutic avenue. Herein, we are the first to synthesize pure, non-toxic, carrier free nanoparticles and show that our CBD nanoparticles dose-dependently induce apoptosis of cytotoxic T-lymphocytes. Additionally, we have shown our CBD nanoformulation is more effective than CBD in non-nano formulation at modulating the expression of JAK/STAT pathway proteins, STAT3 and STAT5, in cytotoxic T-lymphocytes. CBD and CBD nanoparticles were also found to suppress TNF- α, an inflammatory cytokine activator of JAK/STAT signaling, in a dose-dependent manner. These findings reveal a novel therapeutic avenue for future exploration of CBD nanoparticles as efficacious immune modulators.

Clamp peptides for the diagnosis and treatment of SARS-CoV-2

Clamp peptides are computationally designed small molecules that can bind to two different sites of the viral particle and connected with a short bridging peptide sequence. Given their novel design, they can target SARS-CoV-2 virus with high sensitivity and selectivity. Currently, diverse clamp peptides are being created in our lab and are being tested with two different purposes: developing diagnostic platforms that can detect the presence of the viruses and creating formulations that can prevent or reduce their infections.

Clamp peptides for the diagnosis and treatment of Influenza

In this project, we will use different software tools to perform a high-throughput screening of small peptides that will target the influenza viral envelope protein, hemagglutinin. This process includes molecular docking and molecular dynamics simulations that require high computational power; therefore, we will use the University of Miami supercomputer Triton. The goal of this research is to find new molecules for the diagnostic and treatment of seasonal flu. 

Lanmodulin for imaging and diagnostics

Lanthanides are a series of naturally occurring chemical elements that have been in high demand as key components in many new age technologies. Due to their unique properties, such as luminescence, they are used in medical imaging. For example, gadolinium is being used as a contrast agent for Magnetic Resonance Imaging (MRI). The discovery of Lanmodulin, a protein that binds to lanthanides with very high specificity and selectivity, opened an interesting opportunity in the field of therapy and diagnosis. Lanmodulin binds to lanthanides in the picomolar range and can be imaged with MRI and positron emission tomography (PET). When Lanmodulin is genetically fused to targeting molecules, such as antibodies, darpins, and other binding moieties, we can image the targeted events with the aforementioned imaging modalities.

Bioluminescent Assays for the Detection of Circulating Tumor Cells

Circulating tumor cells (CTCs) are released from a primary tumor into bloodstream and considered as metastatic seeds in the establishment of tumor metastases. Thus, enumeration and characterization of CTCs in metastatic cancer patients provide great potential in cancer management. However, widespread use of CTCs in clinical settings is still challenging. Here we develop a dual-functional assay by combination of antibody-mimetics-tagged bioluminescence proteins with fluorescent dyes, which enables the rapid detection of CTCs followed by either single-cell analysis or biobank of these cells in combining workflow.

Design of an electroactive glucose binding protein variant using global incorporation of non-natural amino acids

Typical protein biosensors employ chemical or genetic labeling of the protein, thus introducing an extraneous molecule to the wild-type parent protein, often changing the overall structure and properties of the protein. While these labeling methods have proven successful in many cases, they also have a series of disadvantages associated with their preparation and function. An alternative route for labeling proteins is the incorporation of unnatural amino acid (UAA) analogues, capable of acting as a label, into the structure of a protein. Such an approach, while changing the local microenvironment, poses less of a burden on the overall structure of the protein. L-DOPA is an analog of phenylalanine and contains a catechol moiety that participates in a quasi-reversible, two-electron redox process, thus making it suitable as an electrochemical label/reporter. The periplasmic glucose/galactose binding protein (GBP) was chosen to demonstrate this detection principle. Upon glucose binding, GBP undergoes a significant conformational change that is manifested as a change in the electrochemistry of L-DOPA. The electroactive GBP was immobilized onto gold nanoparticle-modified, polymerized caffeic acid, screen-printed carbon electrodes (GBP-LDOPA/AuNP/PCA/SPCE) for the purpose of direct measurement of glucose levels and serves as a proof-of-concept of the use of electrochemically-active unnatural amino acids as the label. The resulting reagentless GBP biosensors exhibited a highly selective and sensitive binding affinity for glucose in the micromolar range, laying the foundation for a new biosensing methodology based on global incorporation of an electroactive amino acid into the protein's primary sequence for highly selective electrochemical detection of compounds of interest.

Design of a mediator-free, non-enzymatic electrochemical biosensor for glutamate detection

A mediator-free, non-enzymatic electrochemical biosensor was constructed by covalent immobilization of a genetically engineered periplasmic glutamate binding protein onto gold nanoparticle-modified, screen-printed carbon electrodes (GluBP/AuNP/SPCE) for the purpose of direct measurement of glutamate levels. Glutamate serves as the predominant excitatory neurotransmitter in the central nervous system. As high levels of glutamate are an indicator of many neurologic disorders, there is a need for advancements in glutamate detection technologies. The biosensor was evaluated for glutamate detection by cyclic voltammetry. Binding of glutamate to the immobilized glutamate binding protein results in a conformational change of the latter that alters the microenvironment on the surface of the sensor, which is manifested as a change in signal. Dose–response plots correlating the electrochemical signal to glutamate concentration revealed a detection limit of 0.15 μM with a linear range of 0.1-0.8 μM. Selectivity studies confirmed a strong preferential response of the biosensor for glutamate against common interfering compounds.

Investigation of Tumor-Derived Exosomes as Early Biomarkers for Cancer Diagnosis and Treatment Response Monitoring in Cancers

Exosomes, the secreted nano-sized particles, contain nucleic acid, protein, and lipid cargo specific to the cell of origin and serve as transport vehicles in the intercellular communications under normal and pathologic condition. It is well known that exosomes can be secreted abundantly into peripheral body fluids (e.g. blood and urine). Given that exosomes can be repeatedly obtained from body fluids, they have emerged as a promising non-invasive alternative for tissue biopsies for real-time monitoring disease progression. Thus, the purpose of this project is to investigate tumor-derived exosomes as the early indicator for diagnosing cancer and monitoring treatment response in real time. To achieve this goal, we are developing an innovative multiplex exosome-based diagnostic tool that enables quantitative analysis of circulating exosomes that specifically are derived from tumors.

Exosomes as Drug Delivery Nanocarriers of miRNA Inhibitors of Tumor Progression

Since exosomes play an important role as a transporter in cell-cell communication, using exosomes, a natural product of the body, to deliver therapeutic materials has a potential to enhance therapeutic efficacy and reduce immunogenicity compared with synthetic carriers. Therefore, isolation and successful transfection of exosomes are key processes in utilizing exosomes for cancer therapeutics. Exosome cargo nucleotides integrate into the target cancer cells and result in protein expression alteration, which in turn impacts tumor cell viability and functionality. In this project, we propose to deliver cervical cancer-specific tumor suppressor miRNA-7 via exosomes for improved cervical cancer treatment.

Interdisciplinary investigation of the exposure to polycyclic aromatic hydrocarbons in clinical samples of South Florida firefighters

Polycyclic aromatic compounds (PAHs) are toxic organic compounds, which are usually formed through the incomplete combustion of organic matter, and they have mutagenic and carcinogenic properties. Firefighters are routinely exposed to these ubiquitous contaminants and numerous studies have described a high incidence of various cancer types in firefighters compared to the general population. Aim of the Daunert lab as part of the Sylvester Cancer Center’s Firefighters Cancer Initiative is to develop and apply rapid, non-invasive biotechnological and analytical techniques to qualitatively and quantitatively assess the internalized exposure to PAHs in clinical samples, such as urine and vaginal swabs, collected from firefighters stationed throughout Florida.

Assessment of Aqueous Film-Forming Firefighting Foams’ (AFFFs) toxicity on kidney cells

Firefighters are exposed to per- and polyfluoroalkyl substances (PFAS) through the use of Aqueous Film-Forming Foams (AFFFs) for Class B fire suppression. Since 2015, production and use of AFFFs containing long-chain PFAS (i.e. perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS)) has been phased out due to health concerns. However, little is known for the toxicity of AFFFs with replacement PFAS that are currently in use. The aim of this project is to employ cellular biology and biochemical methods to evaluate the toxicity in kidney-derived cells that are exposed to widely used AFFFs collected from U.S. Fire Departments.

Passive Sampling Methods for Evaluating Firefighters’ Occupational Exposure to Carcinogens

Compared to the general population, firefighters are known to sustain greater levels of exposure to hazardous compounds despite their personal protective equipment, also known as turnout gear. Among the most significant toxins that firefighters are chronically exposed to are polycyclic aromatic hydrocarbons (PAHs). Additionally, firefighters have also been noted to exhibit an increased incidence of certain types of cancer. Considering a probable link between exposure to PAHs and increased rates of cancer in the fire service, we have aimed to document ambient chemical concentrations in the firefighter work environment using silicone-based wristbands that have the capacity to passively sorb PAHs. Silicone wristbands have been pilot-tested with active-duty firefighters in various firefighter occupational contexts, such as monitoring individual firefighter exposure during active fire situations, monitoring the presence of PAHs within fire stations and off-gassing from used firefighter turnout gear, and monitoring the deposition of PAHs throughout an active fire situation, i.e., in the hot zone, warm zone, and cold zone of a controlled live fire training burn. Profiles of elevated PAH concentrations were documented at various fire stations throughout South Florida for individual firefighters both during their station duties and during active fire response. Additionally, elevated PAH concentrations were observed in the warm zone and cold zone of active fire situations, suggesting that firefighters who are not equipped with contained air may be susceptible to carcinogenic exposure prior to entering the hot zone of an active fire.

Development of solid-state sensors for real time monitoring of polycyclic aromatic hydrocarbons in warm zones during active firefighting

Previous studies of firefighters indicate high rates of exposure to toxic and potentially carcinogenic chemicals, including volatile organic compounds (VOCs) and semi-VOCs (sVOCs). While improvements have been made in the characterization of exposure sustained by first responders, no readily available methods exist for determining the presence of these high-risk compounds in the field in real-time. Modalities that do exist typically are specific to the detection of acute toxins, such as carbon monoxide or hydrogen cyanide. Beginning in October 2018, sensor arrays were deployed during controlled live fire training burns, which are used for instructing new firefighters in firefighting procedures that emulate the environment within an actual fire situation. These controlled training burns employ well-documented fuel sources, allowing for reproducible output of compounds released from the fires. Class A fires employ solid biomass fuels, such as wooden pallets and hay. Class B fires employ liquid or gaseous hydrocarbon fuels, such as propane, diesel, or gasoline. Sensor arrays were designed to allow for multiple sensors specific to target carcinogens to operate in parallel. Additionally, to allow for analysis of volatilized carcinogens within the active fire situation, a mounting system for the sensor apparatus was engineered atop a remotely operated vehicle (ROV) for collection in areas that are closed off to civilians during active fire response operations. Sensor response effectiveness was operationalized as the difference in semi-volatile and volatile organic compounds in ambient air levels prior to and during the controlled live fire training. Sensor array responses measured at varying locations within and outside of the hot zone and warm zone of the controlled burn displayed significant differences in sensor activity consistent with expect semi-volatile and volatile organic compound intensity in the area.

Investigation of First Responders’ Exposures to Carcinogenic Compounds as part of Rescue Operations at the Surfside Condominium Collapse

In the pre-dawn hours of June 24th, 2021, the Champlain Towers South condominium complex collapsed in the town of Surfside, Florida, located north of Miami Beach, killing at least 98 people. As part of rescue efforts, more than 80 Miami-Dade County and City of Miami fire rescue units were deployed at the scene of the collapse. Additionally, urban search and response (USAR) teams from across the United States and from other nations responded to the collapse. These teams deployed from Florida, Ohio, Pennsylvania, Virginia, and Israel, working in alternating 12-hour shifts to search for survivors underneath the rubble. Due to the nature of this catastrophic collapse, significant exposure to carcinogenic compounds was sustained by firefighters and first responders, including from fires breaking out beneath the rubble and suspended particles from controlled demolitions. Silicone-based passive sampling devices were deployed to capture first responders’ exposure to volatilized carcinogens emitted during the collapse, as well as to determine the distance that these compounds migrated from the collapse to the surrounding environment. Following exposure, silicone passive samplers underwent pressurized liquid extraction and gas chromatography-mass spectrometry analysis to determine carcinogen concentrations, finding that first responders exhibited elevated exposures to polyaromatic hydrocarbons, including phenanthrene, fluorene, and pyrene.

Monitoring Redistribution of Persistent Organic Pollutants Originating from Contaminated Sites

In the aftermath of a natural disaster, casualties, loss of life, and damage to property and infrastructure are undoubtedly the most pressing concerns. However, a less visible threat posed by the potential redistribution of persistent organic pollutants (POP) by natural disasters could result in exposure to these chemicals in previously unexposed populations. This raises public health concerns regarding long-term chemical exposure sustained by people within affected areas, including rescue and cleanup teams. Flooding and strong winds characteristic of hurricanes and other powerful storms can facilitate the migration of compounds originating from damaged structures and previously contaminated areas, such as landfills, farms, and EPA Superfund sites. Various sampling methods have been employed by our group to determine the extent to which environmental contaminant redistribution has occurred in the aftermath of Hurricane Irma and Hurricane Michael in 2017 and 2018, respectively. Firefighters responding to the scenes of these storms were given silicone passive sampling wristbands to capture the exposure they sustained during their shifts. Additionally, soil and water samples were taken from various locations within the vicinity of Mexico Beach, a town on the Florida panhandle that was directly impacted by Hurricane Michael and is bordered to the west by Tyndall Air Force Base, which is built upon an EPA Superfund site. Soil and water samples were also taken from the vicinity of EPA Superfund sites in the Tampa and St. Petersburg region of Florida to investigate whether persistent organic pollutants are generally found outside of the bounds of these restricted areas and if they pose a risk to nearby residences and businesses. Samples were processed using pressurized liquid extraction and analyzed using gas chromatography-mass spectrometry. Preliminary results found that a number of toxic compounds, such as chlorinated pesticides, polyaromatic hydrocarbons, and benzene derivatives associated with chemicals in EPA Superfund sites were found outside of the boundaries of the sites.

Spore-Based Biosensors for Monitoring the Chemical Characteristics of Soil for Environmental and Agriculture Applications

Efficient agriculture and crop production are highly dependent on proper water irrigation and healthy soil, which is characterized by presence of nutrients, metals, and beneficial microorganisms. For optimal plant growth certain ions, such as zinc, copper, phosphate and sulfate should be present in soil at certain concentrations. Several different analytical techniques are required in order to accurately assess ions in soil samples. The ability to analyze biological and environmental samples using portable detection platforms without sample preparation steps is highly desirable, especially in field-based assays. Bacterial whole cell biosensors (WCBs) and, in particular, bacterial spore biosensors, have emerged as excellent tools because they can be prepared in a very reproducible manner, are cost-effective, can survive extreme conditions, and can be regenerated if/when they fail. We have constructed such biosensors using green fluorescence protein to monitor copper and zinc., by inserting an Amino Terminal Copper and Nickel Binding Motif (ATCUN) into a circularly permutated green fluorescent protein, a quencher-based biosensor has been developed for copper. Due to the proximity of the ATCUN motif to the chromophore, upon binding copper dynamically quenches the fluorescence. A dose dependent response was observed in a dynamic range of 0.2 ppm to 150 ppm, with a detection limit of 0.2 ppm. For zinc, a sensor plasmid pSD202 has been developed by inserting smtB-egfp genes into the plasmid pMM1522, where the smtB is a repressor protein that, upon binding zinc, unbinds itself from the O/P region, thus allowing expression of the egfp gene. A dose dependent response was observed in a dynamic range of 1×10^-4 to 1×10^-6M, with a detection limit of 1× 10^-6M.

Engineering Spore-Based Biosensors for Establishing and Monitoring Soil’s Microbial Fingerprint

Many bacteria control expression of specialized genes by producing and responding to extracellular signaling molecules proportional to cell density. This cell-to-cell communication allows a population of bacteria to change the community behavior in a process termed quorum sensing, (QS). When the cell population reaches a critical size reflected by a critical signaling molecule, known as quorum sensing molecules (QSMs). Generally, gram-negative bacteria mostly use N-Acyl-Homoserine lactones (AHLs) as QSMs and gram-positive bacteria use linear and post-translationally modified peptides, while both gram-negative and gram-positive bacteria uses autoinducer-2 (AI-2) and has been proposed as a universal signaling molecule for inter-species communication. AHLs have been detected in environmental samples, such as soil, and both AHL-producing and degrading bacteria have been found in soil ecosystems. This evidence supports the importance of detecting QSMs as microbial molecular markers to study the effect of environmental changes on microbial communities.

Positioning Biosensors

We have constructed biosensor plasmids that allow to monitor bioluminescent gene expression in bacterial biofilms. When these bacteria are grown on agar plates at various densities, they express bioluminescence not only in a density- but also in a position-dependent manner (quorum sensing (QS) and positional sensing (PS), respectively). The use of bioluminescence in QS and PS promises to reveal important regulatory circuits and communication systems relevant for bacterial normal physiology, pathogenicity and virulence.

Identification of Biomarkers of Infection in Mechanically Ventilated Patients in the Intensive Care Unit

Mechanically ventilated patients in the hospital intensive care unit (ICU) are especially vulnerable to hospital-acquired respiratory infections. Ventilator associated pneumonia (VAP) is a major concern in the ICU setting due to difficulties inherent in its timely diagnosis and treatment. The Clinical Pulmonary Infection Score (CPIS) is used by physicians to diagnose VAP using symptoms that the patient is exhibiting; however, this method has been criticized for being too reliant on the physician’s opinion. Gold-standard methods for diagnosing VAP involve the use of invasive and time-consuming techniques such as bronchioalveolar lavage to collect samples from the lower respiratory tract for quantitative culturing. Due to the long turnaround time needed to return results, the patient’s condition may deteriorate before a decision can be made about the type of treatment to render. Broad-spectrum antibiotic treatments to reduce the risk of VAP must be administered conservatively due to their potential impact on the patient and the risk of emergence of antibiotic-resistant bacteria.

There is a clear need for methods that can improve on the shortcoming of current diagnostics for VAP. It is known that bacterial pathologies, including respiratory infections, are associated with observable changes in the profile of volatile and semi-volatile organic compounds (VOCs and SVOCs) in breath and breath condensate samples obtained from human subjects. Various analytical techniques, such as gas chromatography-mass spectrometry (GC-MS) and field-asymmetric ion mobility spectroscopy (FAIMS) can be used for the identification and quantification of biomarkers of infection captured from exhalations of mechanically intubated patients. In comparison to other sampling methods, breath collection can be performed non-invasively and virtually unlimited quantities of breath can be collected for analysis. The determination of chemical fingerprints of infection in the breath of mechanically ventilated patients paves the way for the development of devices for quick, accurate, and non-invasive diagnostics for VAP.

Sensing Breath Biomarkers for Monitoring of Drowsiness

Sleep deprivation in the workplace and behind the wheel have been noted by the National Institute for Occupational Safety and Health as posing a significant public health risk. The operation of a vehicle while sleep-deprived or drowsy increases the risk of an accident, and drowsy driving is estimated to cause thousands of fatal crashes per year and result in billions of dollars in lost revenue. Sleep deprivation in medical personnel has been shown to lead to more errors. Although drowsiness is known to be a major contributor to driver impairment, breath-based chemical parameters and analysis tools for drowsiness are not commercially available as they are for alcohol intoxication, which is determined using blood alcohol content parameters and breathalyzer instruments. Solid-state sensors have been tapped as a potential diagnostic tool for the detection of biomarkers of drowsiness in the breath of drowsy individuals in various scenarios. Initially, field asymmetric ion mobility spectroscopy (FAIMS) was employed to evaluate whether there are changes in the profile of breath of well-rested versus fatigued individuals. Subsequently, to reveal potential biomarkers of fatigue and drowsiness, gas chromatography-mass spectrometry (GC-MS) analysis was conducted. Once initial biomarkers were identified for drowsiness, solid-state sensors were trained and calibrated for the detection of compounds deemed to be potential biomarkers of fatigue and drowsiness; these sensors were tested with breath from drowsy and rested individuals. Sensor responses identified promising signatures with increasing levels of drowsiness as compared to validated measures, such as the Karolinska Sleepiness Scale (KSS), Visual Analog Scale (VAS) and Percentage of Eye Closure (PERCLOS) methodologies.