Anuj Chauhan, Ph.D. - US grants

Abstract


Proposal Title: DNA Separation on a Chip by Lateral Electric Fields
Proposal Number: CTS-0302271
Principal Investigator: Anuj Chauhan
Institution: University of Florida


The objective of this project is to separate DNA molecules of different sizes in microfabricated channels through the combination of pressure-driven flow through the channel and electric fields orthogonal to the flow. The electric field will concentrate the charged DNA molecules near the wall, while the axial flow will tend to transport the smaller molecules with higher diffusivities more rapidly down the channel. Operation in a cyclic manner should lead to an effective separation of DNA molecules by size. Several variations on this method are proposed to increase the efficiency of the separation. The current, standard method for the separation of DNA molecules is through capillary electrophoresis, which is effective for smaller molecules up to about 20,000 base pairs. As new methods for sequencing larger molecules become available, the most difficult step will be the isolation of DNA. The proposed separation method may be applicable to molecules in the megabase pair range. In terms of the broader impacts, separation of DNA fragments on the basis of their lengths represents an important step in a number of areas of molecular biology, especially DNA sequencing for applications in medical diagnostics and drug delivery. The proposed strategy does not require any gels, and it could be implemented in lab-on-a-chip devices. In the educational area DNA separation topics will be included in transport courses, and outreach will include visits to middle and high schools for demonstration e

ABSTRACT - 0426327

Dispersion of Nanoparticles in Hydrogels for Ophthalmic Drug Delivery
Approximately 90% of all ophthalmic drug formulations are now applied as eye-drops1. While eye-drops are convenient and well accepted by patients, a majority of the drug contained in the drops is lost due to tear drainage. The drops mix with the tear fluid, and subsequently, about 95% of the drug flows through the upper and the lower canaliculi2. Eventually, a major portion of the drug is absorbed in the nasolacrymal duct, and enters the blood stream. This can lead to serious side effects. For instance, absorption of Timolol, a beta-blocker used to treat glaucoma, has harmful effects on the heart3. Furthermore, topical ophthalmic drug delivery results in a relatively high drug concentration in the tear film followed by a rapid decline. This causes sharp variations in the drug delivery rates to the cornea that reduces the efficacy of ophthalmic drugs4.

Intellectual Merit: To reduce drug loss, eliminate systemic side effects, and improve drug efficacy, we propose to develop disposable soft contact lenses as a new vehicle for ophthalmic drug delivery. The essential idea is to encapsulate the ophthalmic drug formulations in nanoparticles, and to disperse these drug-laden particles in the lens material. This work focuses on soft lenses made of poly 2-hydroxyethyl methacrylate (HEMA) hydrogel. The hydrogel matrix of HEMA lenses is synthesized by bulk or solution polymerization of HEMA monomers in the presence of a cross linker such as ethylene glycol di-methacrylate (EGDMA)5. Addition of drug-laden particles in the polymerizing medium results in the formation of a particle-dispersion in the hydrogel matrix. If a contact lens made of this material is placed on an eye, the drug diffuses from the particles, and through the lens matrix, and enters the thin tear film trapped between the cornea and the lens.

The three specific objectives of the study are, (i) encapsulate ophthalmic drugs in nanosized colloidal particles and study the stability of these particles, (ii) incorporate the drug-laden particles in the hydrogel matrix during the polymerization process, and study the microstructure and the physical properties of the particle-laden gel, and (iii) measure and model the drug release rates from the particles and the hydrogel to determine the controlling mechanism, and eventually control the release rates from the hydrogel by manipulating the particle and/or gel properties.

We have successfully fabricated transparent gels loaded with two different types of particles: microemulsion drops and liposomes. We have also established that contact lenses fabricated from the particle laden gels can deliver ophthalmic drugs at therapeutic rates for a few days. The proposed future work focuses on advancing the fundamental knowledge of particle entrapment, aggregation, segregation, controlling mechanisms of drug transport, and fluid mechanics of the human eye. In addition we wish to increase drug loading and improve the drug release profiles to obtain zero order delivery rates, and also develop the optimal systems for various ophthalmic drugs.

Broader Impact: Drug delivery is rapidly becoming a very important research area due to its enormous societal impact. This field has the potential to significantly improve the quality of life, save lots of lives, and offer improved treatment for a number of diseases. Our proposed research will help deliver ophthalmic drugs in an efficient and controlled manner that could potentially reduce drug wastage, improve compliance, minimize side effects and maximize the efficacy of currently available drugs. Drug delivery through contact lenses could be very useful for patients suffering from glaucoma because use of beta-blockers to treat this disease has serious side effects on heart. Furthermore due to the rapidly increasing importance of drug delivery, it is important to expose students to this area of research as early in their careers as possible. To achieve this objective, the PI proposes to include gel fabrication and drug delivery through gels in Transport and Interfacial Phenomena classes and involve undergraduate students in the research.

Intellectual Merit of the Proposed Activity

Microemulsions have received considerable attention due to the numerous applications in a wide variety of areas such as separations, reactions, drug delivery, and detoxification. In all the applications listed above, the process of mass transfer across the surfactant-covered interface plays a key role. In spite of the importance of the transport across the microemulsion surface, the detailed mechanisms of this process are not clearly understood.
The goal of the proposed effort will be to utilize a multiscale simulation technique based on coarse grained molecular dynamics and kinetic Monte Carlo simulations to elucidate the solute transport mechanisms and their dependence on such parameters as surfactant length, surface coverage and curvature, and solute size and shape. The following two competing mechanism are anticipated: the transient channel formation and the solubility-diffusion mechanism. In the first case, a transient channel in the surfactant-covered interface is formed, connecting the oil and water phases, and the solute diffuses through this channel. In the second case, the solute first solubilizes within the surfactant layer at the interface and then undergoes diffusion, which takes place either through hopping between voids in the microstructure of the interface or through a bulk-like Brownian diffusion process.
The first stage of the theoretical modeling will be aimed at detailed understanding of the internal structure and dynamics of the microemulsion interface, as characterized by void sizes, shapes, lifetimes, and dynamics of void and channel formation. Dynamics of relatively small voids will be investigated using coarse-grained molecular dynamics (MD) simulations. However, formation of larger voids and channels and the solute transport between the voids are expected to occur at timescales that are out of reach of direct MD simulations. Therefore, the proposed study will use the umbrella sampling technique to investigate these slow dynamics. The second stage of the proposed work will utilize the obtained void distributions and the rates of formation and destruction of voids to develop a kinetic Monte Carlo scheme to predict the solute transport rates. The combined MD and kinetic Monte Carlo simulations will be used to probe the dependency of the transport rates on various surfactant and solute parameters.
The parameters of the coarse-grained molecular model will be optimized by matching the simulation results to experimental data for surface tension isotherms, bulk diffusivities, and phase transitions. Effects of the interfacial curvature will be investigated by varying the size of microemulsion droplets and by considering flat oil-water-surfactant interfaces. Theoretical predictions for the dependence of the solute transport rates on the microemulsion properties will be compared with experimental data.

Broader Impacts of the Proposed Activity

Microemulsions are commonly used in a wide variety of areas and a detailed understanding of the transport of molecules across the surface will help in development of better products, such as more efficient drug delivery vehicles, that could have a large societal impact. Since this project is a combination of fundamental and applied studies, it will provide a good learning experience for the undergraduate and the graduate students that will work on this project. Also the PIs propose to incorporate various aspects of this project into graduate and undergraduate classes.

COLLABORATIVE RESEARCH: DNA AMPLIFICATION IN A NOVEL INTEGRATED MICROCHIP PLATFORM WITH TEMPORAL THERMAL CONTROL

Implementing precise time and space dependent heating and cooling in a microchip is potentially useful in a wide array of areas including reactions, separation, detection, etc. This project aims to develop such a microchip, and understand the fundamental implications of the temperature changes on fluid flow and heat and mass transfer, while developing a chip that can perform rapid DNA amplification.

While microfluidic DNA amplification devices have been fabricated, their use in practical applications is nonexistent due to small throughputs. Here we propose a new paradigm for PCR in a microchannel that is based on temporal temperature cycling. To accomplish this objective, we propose a new chip design for implementing precise and accurate temperature gradients (both spatial and temporal). Furthermore, we propose a synergistic approach that leverages the strengths of both the PI's by combining modeling and experiments to develop a clear understanding of fundamental issues relevant to the proposed device. These issues include contribution of thermal expansion-contraction, and reactions on dispersion and amplification efficiency, and the effect of various design and operating parameters on the fluid-flow and mass transfer in the device. Such an understanding is crucial for designing an optimal microfluidic flow reactor for DNA amplification with a high throughput.
The intellectual merit of the program is manifested in the goals of the project that include (i) development of a Smart Thermal Microchip (STM) for precise temperature modulation, (ii) an improved quantitative understanding of transport in microscale systems that are subjected to temporally changing temperatures; (iii) development of a numerical and analytical tools to analyze heat transfer in the Smart Thermal Microchip and to predict the dispersion and amplification of DNA samples using various input parameters such as the channel dimensions, number and frequency of the temperature cycles, dispersion coefficient of the DNA and the initial plug size; (iv) development of a novel idea for continuous and high throughput polymerase chain reaction on a microfluidic chip without an imposed pressure driven flow. The results of this proposal will improve our understanding of mass transfer in systems with reactions and temporal temperature gradients, and in particular lead to a thorough understanding of the transport processes involved in the DNA amplification by polymerase chain reaction on a microchip. These results will lead to a rational design and operation of these chips.
The results of this research will have broader impacts in a number of areas. The Smart Thermal Microchip will find applications in other areas related to reactions, separations and detection. Furthermore, the amplification process is an integral part of DNA analysis and the importance of DNA analysis cannot be overstated. It is already important in various areas such as analysis of clinical samples, identification of mutations, detecting cancer, testing safety of genetically modified foods, forensic analysis and applications of DNA analysis are only expected to grow considerably. It is envisioned that the results of this study will enable optimization of amplification devices and additionally lead to development of a novel high throughput device. The educational program couples core skills of thermal and mass transport to reaction kinetics. The research will be integrated with the curriculum development of the Chemical Engineering Department of the University of Florida and of Brown University in the division of engineering and the chemical and biochemical engineering program. The program supports development of new courses in transport processes. The program offers excellent opportunities to new undergraduate laboratories exploring microfluidics. Students also apply their skills in transport phenomena to unveil methods for detecting microbial threats. The program is truly interdisciplinary and invites opportunities for collaborations. Strong ties are promoted between the fundamental engineering research and assay development in biotechnology and nanotechnology industries. Lastly, the research program unites the interests of the two PIs and will foster significant collaborations and exchange of ideas between the two research groups.

The objective of this research project is to develop particle-laden soft contact lenses as a new vehicle for ophthalmic drug delivery in order to reduce drug loss, eliminate systemic side effects, and improve drug efficacy and compliance. Nanoparticles are dispersed in hydrogels in several nanomanufacturing processes for improving mechanical, electrical, optical, and transport properties of hydrogels. In this project, the PIs plan to develop a new approach for preparing nanoparticles, explore the mechanisms of particle entrapment in hydrogels and evolution of microstructure, and the effect of the particle entrapment on physical and transport properties of the gels, when the particles are added to the polymerizing medium. Here, the project will focus on the specific application of controlling the drug release properties of the hydrogels to develop extended wear contact lenses for delivering ophthalmic drugs. The three specific aims of the research are: (i) Develop drug loaded highly crosslinked nanoparticles and understand the mechanism of particle formation and drug transport in the nanoparticles; (ii) Polymerize silicone-hydrogels in presence of the highly crosslinked nanoparticles to fabricate contact lenses loaded with drug encapsulated highly crosslinked nanoparticles; (iii) Characterize the particle loaded contact lenses to understand microstructure, drug transport, and all other properties of the gels relevant to the use of these materials for contact lenses. The research will combine both modeling and experiments to explore fundamentals of nanoparticle and lens preparation while focusing on the eventual goal of developing contact lenses for drug delivery. Currently, approximately 90 percent of all ophthalmic drug formulations are applied as eye-drops. While eye-drops are convenient and well accepted by patients, these suffer from low bioavailability (<5 percent), side-effects due to systemic uptake, and low compliance. The compliance could be lower than 50 percent and further smaller when multiple eye drops are required each day, which contributes to worsening of the ophthalmic disease even when treatments are available. The bioavailability increases to about 50 percent when ophthalmic drugs are delivered through contact lenses because of the increase in the residence time of the drugs in the tear film. The increased bioavailability (>50 percent) results in lower side effects, and will likely lead to higher compliance because of the continuous drug delivery for about 2-weeks with a contact lens. In preliminary results a novel approach has been developed of making ultrasmall (~4 nm) particles without utilizing any surfactant that release drugs for over 15 days. This novel approach of making nanoparticles without using surfactants is scalable to industrial standards, and it could be useful in several areas of nanomanufacturing, particularly when addition of surfactant is undesirable or too expensive. This concept has also been proven by fabricating transparent particle-laden gels loaded with novel nanoparticles containing a glaucoma drug timolol. It has also been established that contact lenses containing the highly crosslinked particles can deliver timolol at therapeutic rates for 2-3 weeks.

If successful, the particle-loaded lenses will lead to a paradigm shift in ophthalmic drug delivery as it will eliminate several deficiencies in current delivery systems including very low bioavailability (<5 percent), potential side-effects, and low compliance. The nanoparticle-loaded lenses will have higher bioavailability (>50 percent), resulting in lower side effects, and will likely lead to higher compliance. This research is inherently multidisciplinary as the project combines expertise in new materials, transport, biomedical engineering, and modeling. Also, this research will likely leads to paradigm shifts in the area of ophthalmic drug delivery, and thus lead to a significant societal impact particularly in the area of glaucoma therapy which affects about 66.8 million people in the world, leaving 6.7 million with bilateral blindness.

This grant supports research that advances the fabrication of contact lenses using nanoparticle-loaded hydrogels with a specific focus on treating ocular cystinosis. Incorporation of nanoparticles in hydrogels through polymerization is a scalable approach to controlling physical properties of the polymer, for example, elasticity, color, refractive index, conductivity and transport properties such as rates of drug release. This project involves incorporation of gold nanoparticles into contact lenses, which are made from either hydrogels or silicone hydrogels. Ocular cystinosis is a disease in which the cornea gets filled with crystals of cystine, which is the oxidized form of amino acid cysteine. The crystal formation eventually leads to many ocular complications including blindness. Typically, the disease is treated by hourly instillation of cysteamine eye drops. The drug reacts with cystine to dissolve the crystals. This grant is based on data that show that gold nanoparticles can bind significant amounts of cystine, thus, if a contact lens containing gold particles is placed on the eye, cystine diffuses from the cornea towards the contact lens, leading to crystal dissolution. The availability of nanostructured contact lenses loaded with gold nanoparticles advances treatment of ocular cystinosis and, thus, impacts the nation's health and welfare, benefiting society. The resulting 'GoldInLens' device could lead to drug-less cystinosis therapy. This research is inter-disciplinary and involves nanomanufacturing, materials science, transport behavior and pharmacy. The multi-disciplinary approach allows for broad training of the undergraduate and graduate students, and broaden participation of underrepresented groups.

The gold particles are incorporated into the contact lens either by addition to the monomer mixture followed by polymerization, or by loading the polymerized lens with chloroauric acid and reduction by sodium citrate to form gold particles in situ. Fundamental studies explore the microstructure evolution during polymerization when nanoparticles are added to the monomer mixture, and the structure of gold aggregates that form in situ. Additionally, the impact of the particle incorporation on critical contact lens properties such as transparency, oxygen permeability and elastic modulus are investigated. Synergistic strategies are explored to maximize cystine capacity including developing contact lens materials with high capacity for cystine, and then increasing that capacity by incorporation of gold nanoparticles. The results from this research potentially have life-changing impact on patients with cystinosis by replacing hourly eye drop therapy with a contact lens worn for just a few hours each day. Additionally, the research unveils interesting fundamentals related to microstructure evolution during polymerization of nano-suspensions and structure of gold nano-aggregates than can form by using a small pore size hydrogel as a scaffold. The project advances several technological areas in nanomanufacturing, nanoscale devices, and particulate dispersions.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.