Browsing by Subject "Drug delivery"

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Characterization of biological hydrogel barriers
(2014-12) Kaliki, Srimahitha; Smyth, Hugh D.C.,; Barr, Ronald; Milner, Thomas; Dunn, Andrew; Marek, Stephen
Biological hydrogel barriers include mucus, bacterial biofilms, fungal biofilms, and others. Biofilms are polysaccharide hydrogels. Biofilms are commonly found in the lungs of cystic fibrosis patients. Cystic fibrosis (CF) patients are susceptible to these types of chronic infections because their mucus barrier is abnormal. A common bacterial infection in these patients is caused by the bacterium Pseudomonas aeruginosa. While it is found that the bacteria can infect CF patients easily, the treatment of such infections by drugs had been found to be quite inefficient due to the structure of the biofilm itself and formidable mucus barrier. Mucus is a hydrogel which protects the gastrointestinal, genitor-urinal and respiratory tracts from pathogens and external environments. In our preliminary studies, topically applied nanoparticles disrupted these hydrogel barriers and resulted in the increase in permeability to solutes. The long term goal of this proposal is to understand and quantify the effects of the interaction between nanoparticles and biological hydrogel barriers. Discovering how nanoparticles disrupt the hydrogel barriers is important for understanding the health risks. The hypothesis of this research is that nanoparticles result in disruption of the hydrogel barrier structure that leads to increased exposures to co-deposited solutes. Quantifying the structural changes and diffusivity of such solutes using different novel techniques is the central object of my thesis. Bulk Rheological studies were performed using mucin samples treated with nanoparticles. It was noticed that the viscosities showed a negative trend with regards to the nanoparticle sizes which seemed to be contradictory to Einstein’s prediction. A possible mechanism of action was explained. Multiple particle tracking was performed to quantify viscosities of nanoparticles in mucin solution. Subsequently, drug diffusion studies were performed on similar samples to provide a relationship between the nanoparticle size and the drug permeability. Atomic force microscopy was performed in liquid cell using force mode on biofilms when treated with different sized nanoparticles. Micro-elasticity of these biofilms was calculated and compared.
Composite nanogels for the triggerable release of chemotherapeutics
(2015-12) Peters, Jonathan Thomas; Peppas, Nicholas A., 1948-; Freeman, Benny; Johnston, Keith; Sanchez, Isaac; Zoldan, Janeta
The development of external stimuli responsive nanoparticles has progressed greatly since its inception in the seventies. However, apart from some clinical success for slow release delivery via liposomes, the technology has stalled for the delivery of chemotherapeutics due to a myriad of problems with cytocompatibility and premature diffusion of drug payload. The solution to cytocompatibility has been the coating of the system with polyethylene glycol. New methods have been developed to attach polyethylene glycol (PEG) tethers to the surface of otherwise unreactive particles. Surface hydrolysis of acrylamide containing polymers can be used to produce carboxylic acid functional groups near the surface of the polymeric nanoparticles. These nanoparticles can then be functionalized with PEG via EDC/NHS chemistry. The use of surface hydrolysis not only allows for reaction with these neutral polymers, but also provides greater control of PEG localization and leads to an unintrusive method to add the much needed stealth coating. In order to address the issue with premature release, new polymer systems have been developed. These systems are based around theory of hydrophobic interaction in order to improve the polymer/drug interaction in order to limit the unwanted diffusional release of drug payload. This interaction was addressed in a number of ways, focusing on both compartmentalization and copolymerization in order to develop nanogels that can entrap and withhold more drug from the surrounding area. An in depth look into the interactions that encourage drug uptake in these systems was performed by altering the copolymer chosen for these systems. This work looks into effects on phase transition, functional groups, hydrophobicity, and any structural changes that occur as a result of the polymerization scheme. After drawing conclusions on the interactions that encourage drug uptake, complex systems were devised to take advantage of these interactions. Core shell systems were designed to take advantage of the convective release of lower critical solution systems while still utilizing the mechanisms that improve drug retention. These systems were synthesized by two methods, emulsion polymerization and micelle crosslinking. These systems have been showed to improve the drug interaction and retention of doxorubicin as a model chemotherapeutic.
Degradable poly(ethylene glycol) based hydrogels for pulmonary drug delivery and in vitro T cell differentiation applications
(2013-08) Fleury, Asha Tarika; Roy, Krishnendu
Hydrogels, defined as three-dimensional, hydrophilic networks, offer extensive biomedical applications. The areas of application are heavily concentrated in drug delivery and tissue engineering because of the hydrogels’ ability to mimic extracellular matrixes of tissue while maintaining a high level of biocompatibility. Specifically, poly(ethylene glycol) (PEG) is a well-established biomaterial in hydrogel applications due to its high water-solubility, low toxicity, high biocompatibility, and stealth properties. This thesis discusses two applications of PEG-based degradable hydrogels. The first is the targeted, site-specific, controlled release of biologic drugs administered by inhalation. There are many challenges to designing a pulmonary delivery system for inhalation of biologic drugs such as low respirable fractions and short resident time in the lungs. In this report, the hydrogel microcarriers for encapsulated drugs were formed by cross-linked PEG and peptide sequences synthesized during a mild emulsion process. The microgels underwent freeze-drying in the presence of cryoprotectants and formulated for dry powder inhalation. The microgels displayed swelling properties to avoid local macrophage clearance in the lungs and exhibited triggered release and degradation in response to enzyme for disease specific release. Dry formulations were tested for aerosolization properties and indicated ability to be delivered to the deep lung by a dry powder inhaler. Lastly, microgels were successfully delivered to mice lungs via intratracheal aerosol delivery. This thesis also discusses the use of PEG-based hydrogel as a biomaterial microenvironment for encapsulated stem cells as a means of in vitro T cell differentiation. A 3D hydrogel system creates a biomimetic reconstruction of the cell’s natural microenvironment and allows us to adjust factors such as ligand density and mechanical properties of the hydrogel in order to promote cells differentiation. This report utilizes hydrogels of cross-linked hyaluronic acid and PEG to encapsulate mice bone marrow hematopoietic progenitor cells in the presence of notch ligands, displayed through stromal cells, magnetic microbeads, or immobilized within the hydrogel matrix. Mechanical properties of the hydrogels were tested and the release of encapsulated cells was performed by enzymatic degradation or dissolution. The differentiation data obtained indicated successful differentiation of stem cells into early T cells through the hydrogel system.
Design, development and optimization of methacrylic acid-based pH-responsive hydrogels for the oral delivery of model protein therapeutics
(2016-05) Steichen, Stephanie Danelle; Peppas, Nicholas A., 1948-; Baker, Aaron; Suggs, Laura; Watts, Alan; Yeh, Tim
Protein therapeutics have tremendous potential to treat a variety of debilitating diseases, however, due to their large size and low stability, their administration has been limited to injections. An orally delivered carrier capable of protecting the protein during its transit through the gastrointestinal tract could potentially overcome the limits of protein stability, improve oral bioavailability, and provide a more patient tolerated means of protein administration. A pH-responsive hydrogel system composed of methacrylic acid, N-vinyl pyrrolidone, and poly(ethylene glycol), designated as P((MAA-co-NVP)-g-EG), was developed. The effects of crosslinking lengths and density and incorporation of poly(ethylene glycol) tethers on swelling behavior and loading capability of protein therapeutics were evaluated. It was shown that P((MAA-co-NVP-g-EG) swelled to a greater extent than P(MAA-co-NVP) in equilibrium studies at neutral pH and showed a faster rate of swelling in dynamic conditions. Mesh sizes ranged from 400-650 Å, which was deemed sufficient for drug loading and release. Three therapeutic proteins were evaluated for their loading capability: insulin, porcine growth hormone (pGH), and ovalbumin. The weight loading efficiencies of the proteins were optimized by varying pH and ionic strength and ranged from 8-9% for insulin, 6-7% for pGH, and 4-5% for ovalbumin. There was no discernible effect of crosslinking density and length on loading. The incorporation of PEG tethers showed a significant improvement in loading of human GH, increasing weight-loading efficiencies from 4.5 to 6.4%. When exposed to simulated gastrointestinal conditions, the loaded microparticles released ~10% of their hGH payload at intestinal pH. The hydrogels were also evaluated for their safety in vitro and in vivo. No cytotoxicity was observed in two model intestinal cell lines at dosages less than 2.5 mg/mL. When exposed to C57Bl/6 mice at 66.7 mg/kg dose, the hydrogels triggered no inflammatory response and no deleterious effects on organ function after acute and long-term administration. In model intestinal epithelial layers, both P((MAA-co-NVP)-g-EG) and P(MAA-co-NVP) hydrogels significantly improved transport of hGH over hGH alone. In vivo studies performed in Sprague Dawley rats confirmed that hGH-loaded hydrogels yielded hGH bioavailabilities ranging from 0.46-4.8%. Therefore, these pH-responsive hydrogels show great promise for oral protein delivery.
Designing chimeric transmembrane proteins to improve therapeutic delivery and to understand receptor trafficking
(2019-11-07) Zhao, Chi (Ph. D in biomedical engineering); Stachowiak, Jeanne Casstevens; Georgiou, George; Sakiyama-Elbert, Shelly; Zoldan, Janet; Cui, Zhengrong
Transmembrane proteins represent a key class of macromolecules involved in many cellular processes. Therefore, incorporating these sophisticated machineries into therapeutic delivery platforms has the potential to confer unique material functions. In addition, understanding the trafficking of transmembrane proteins has the potential to reveal novel mechanisms for regulating the plasma membrane content. Towards engineering materials with functional transmembrane proteins, I first report the development of cell-derived plasma membrane vesicles with engineered transmembrane targeting proteins. These engineered vesicles precisely target cells on the basis of surface receptor expression level. In further engineering these vesicles for intracellular delivery, I take advantage of the gap junctions, a network of transmembrane proteins consisting of connexins. Gap junctions play important roles in facilitating molecular exchange between adjacent cells and therefore provide direct access to the cytoplasm. Upon incorporating connexins and targeting proteins into my delivery vehicles, I report the development of targeted Connectosomes, plasma membrane vesicles with high concentrations of connexins. These vesicles reduced the therapeutically effective dose of doxorubicin by 1000-fold compared to conventional liposomal formulations. Building on this platform, I demonstrate the feasibility of using Connectosomes as delivery vehicles for small interfering RNAs into the cytoplasm, enabling effective gene silencing. These results address many longstanding challenges in achieving efficient intracellular delivery by capitalizing on the unique capabilities of gap junction transmembrane proteins. Towards further understanding the mechanisms of transmembrane protein trafficking, I then investigate the impact of receptor heterodimerization on endocytic uptake. My findings suggest that receptor uptake depends on a delicate balance between the opposing influences of collaboration and competition. Specifically, the endocytic uptake of a weakly-internalized receptor increases substantially upon binding to a strongly-internalized receptor. However, clathrin-coated structures have a limited capacity to accommodate receptors. Therefore, competition starts to dominate when the two receptors in the heterodimer are either both strongly-internalized or have very low binding affinity for each other. These findings suggest that cells achieve precise control over signaling events originating at the plasma membrane by regulating multiple pools of receptors simultaneously. Collectively, my thesis work has demonstrated the power of chimeric transmembrane proteins to address translational and fundamental research problems.
Development and characterization of microencapsulated nanoparticle systems for oral vaccination by protein-antigens
(2017-05) Sharpe, Lindsey Anne; Peppas, Nicholas A., 1948-; Croyle, Maria; Maynard, Jennifer; Suggs, Laura; Zoldan, Janeta
A composite platform strategy for oral vaccination with subunit antigens was developed to improve i) ease of administration and distribution; and ii) induction of mucosal immunity. The platform is referred to as Polyanhdyride-Releasing MicroParticle Technology, or PROMPT. In its core, polyanhydride nanoparticles based on 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) served simultaneously as adjuvant and delivery vehicle of subunit antigens, while microencapsulation by pH-responsive polymers based on poly(ethylene glycol) (PEG) and poly(methacrylic acid) (PMAA) enabled targeted intestinal delivery of the nanoparticle payload. PROMPT formulations were synthesized by pH-mediated self-assembly to encapsulate nanoparticles. The reversible pH-responsive transition of these formulations coincided with the pH transition experienced during intestinal delivery, such that particles dissociated to release nanoparticles above pH 5. The physicochemical characteristics of the composite microgels were evaluated by Fourier transform infrared spectroscopy, electron microscopy, and confocal microscopy. PROMPT formulations demonstrated pH-dependent burst release of the encapsulated model antigen, ovalbumin, and then sustained release thereafter in both neutral pH and simulated gastrointestinal conditions. The biocompatibility and immunostimulatory capabilities of PROMPT formulations were evaluated in relevant cell lines to identify lead candidates for in vivo immunization experiments. PROMPT composite formulations demonstrated greater than 85% viability at microgel concentrations less than 1mg/mL, as indicated by cellular proliferation and membrane integrity. PROMPT microgels also demonstrated the ability to activate bone marrow-derived dendritic cells in vitro by stimulating cell surface marker expression and cytokine secretion. Finally, the ability of lead formulations to elicit immune responses was assessed in vivo by administering PROMPT formulations to BALB/c mice by oral gavage. PROMPT formulations induced measurable ovalbumin-specific IgA and IgG in mucosal fluids and blood serum, respectively, while soluble antigen and nanoparticles alone did not. This work shows that microencapsulation of nanoparticles for oral vaccine administration is a promising platform for developing safe, effective subunit-based vaccines.
Development and evaluation of enzymatically-degradable hydrogel microparticles for pulmonary delivery of nanoparticles and biologics
(2012-12) Wanakule, Prinda 1985-; Roy, Krishnendu
The emerging class of biologic drugs, including proteins, peptides, and gene therapies, are widely administered by injection, despite potential systemic side effects. Rational design of targeted carriers that can be delivered non-invasively, with reduced side effects, is essential for the success of these therapies, as well as for the improvement of patient compliance and quality of life. One potential approach is to take advantage of specific physiological cues, such as enzymes, which would trigger drug release from a drug carrier. Enzymatic cleavage is highly specific and could be tailored for certain diseased tissues where specific enzymes are up regulated. Enzymatically-degradable hydrogels, which incorporate an enzyme- cleavable peptide into the network structure, have been extensively reported for releasing drugs for tissue engineering applications. These studies showed that a rapid response and corresponding drug release occurs upon enzyme exposure, whereas minimal degradation occurs without enzyme. Recently, Michael addition reactions have been developed for the synthesis of such enzymatically-degradable hydrogels. Michael addition reactions occur under mild physiological conditions, making them ideally suited for polymerizing hydrogels with encapsulated biologic drugs without affecting its bioactivity, as in traditional polymerization and particle synthesis. The focus of my research was to create enzymatically-degradable hydrogel microparticles, using Michael addition chemistry, to evaluate for use as an inhalable, disease-responsive delivery system for biologic drugs and nanoparticles. In this dissertation, I utilize bioconjugation and Michael addition chemistries in the design and development of enzymatically-degradable hydrogels, which may be tailored to a multitude of disease applications. I then introduce a new method of hydrogel microparticle, or microgel, synthesis known as the Michael Addition During Emulsion (MADE) method. These microgel carriers were evaluated in vitro, and found to exhibit triggered release of encapsulated biologic drugs in response to enzyme, no significant cytotoxic effects, and the ability the avoid rapid clearance by macrophages. Lastly, in vivo studies in mice were conducted, and microgels were found to exhibit successful delivery to the deep lung, as well as prolonged pulmonary retention after intratracheal aerosol delivery. In conclusion, a new class of enzymatically-degradable microgels were successfully developed and characterized as a versatile and promising new system for pulmonary, disease-responsive delivery of biologic drugs.
Development and optimization of pH-responsive oral delivery systems for protein therapeutics
(2020-12-09) Miller, Matthew Kyle; Peppas, Nicholas A., 1948-; Croyle, Maria; Maynard, Jennifer; Rosales, Adrianne
A pH-responsive anionic linear polymer was synthesized via controlled radical polymerization using reversible addition-fragmentation chain transfer (RAFT) polymerization. The linear polymer was used to form pH-responsive self-assembled nanoparticles via the nanoprecipitation technique capable of loading antibodies with high efficiency at high weight loading percentages. The linear polymer system and the nanoprecipitation process were optimized using a Design of Experiments approach, resulting in a system capable of maintaining high antibody loading while exhibiting favorable release characteristics in simulated gastric, intestinal, and circulatory conditions. The optimized polymer and nanoparticle system was able to achieve a loading efficiency of 94% with a final weight loading of 32.9% protein compared to polymer. This optimized system showed minimal release in simulated gastric and intestinal conditions, while exhibiting high release at physiological pH, which is ideal for a nanocarrier designed to transit the intestinal epithelium before releasing its payload. The optimized polymer and nanoparticle system were then evaluated in a series of in vitro models to determine their cytocompatibility and transport capabilities. The linear polymer showed no cytotoxicity at concentrations up to 1 mg/mL in two model cell lines using two different assays, which is higher than any concentration likely to be achieved in vivo. Nanoparticles were conjugated with Fc ligands to evaluate if targeting the neonatal Fc receptor (FcRn) on intestinal enterocytes could enhance transport of nanoparticles across the epithelial cell barrier. The nanoparticles were then evaluated in a transepithelial transport model using Caco-2 and HT29-MTX model cell lines. Transepithelial transport was analyzed using an ELISA assay as well as confocal microscopy of fluorescently labeled antibodies. Nanoparticles conjugated with Fc ligands resulted in transepithelial protein transport up to 25x higher than unconjugated nanoparticles or unencapsulated protein based on ELISA results. Confocal microscopy proved inconclusive in differentiating transport between groups, likely due to fluorescence quenching of the FITC probe in acidic intracellular vesicles combined with high levels of background fluorescence. Finally, cell penetrating peptides (CPPs) were evaluated as an alternative means of increasing transepithelial transport of proteins. CPPs were evaluated in a pH-responsive complexation hydrogel system delivering insulin. The crosslinking density and crosslinker type were found to have a strong effect on the loading and release characteristics of CPP-insulin dual loaded microparticles. In an in situ closed-loop intestinal model in rats, the CPP L-PenetraMax showed a promising ability to increase transepithelial transport of insulin, resulting in a decrease in blood glucose levels compared to microparticles loaded with insulin alone. Future work will likely benefit from a combination of CPPs with Fc-conjugated nanoparticle delivery systems.
Development of a multiphysics model for blood flow and drug transport with application to patient-specific coronary artery flow
(2007-05) Brasher, Nathan Francis; Hughes, Thomas J.R.
A multiphysics model of blood flow and drug dispersion has been constructed for a section of coronary artery. Using NURBS-based isogeometric technology to describe the geometry, I model the release of a liquid drug into the bloodstream as well as its transport and deposition into the arterial lumen. This model includes fluid-structure interaction between the blood and the arterial wall, a porous-media model governing the penetration of fluid into the arterial lumen, and a scalar advection-diffusion equation describing the drug. I present a numerical method for coupling the fluid and porous-media equations. I use this methodology to simulate patient-specific geometries with a set of physically relevant parameters.
Development of multifunctional electrospun wraps for bone healing
(2020-11-17) Buie, Taneidra Walker; Cosgriff-Hernandez, Elizabeth; Suggs, Laura; Zoldan, Janeta; Laverty, David
The Masquelet technique is a two-staged procedure that uses an induced biological membrane and bone graft to reconstruct critical-sized bone defects. However, unpredictable clinical outcomes result due to the variable durability and the transient vascular network of the induced membrane, as well as high incidences of osteomyelitis. To this end, we have engineered a resorbable multifunctional electrospun wrap that guides formation of the induced membrane with improved durability and enhanced angiogenesis while simultaneously preventing infection. We achieve this by developing and combining an antimicrobial poly(lactic-co-glycolic) acid (PLGA) mesh and an angiogenic crosslinked gelatin mesh. We first confirmed the ability of electrospun PLGA to provide sustained release of gentamicin sulfate or gallium maltolate above its minimum inhibitory concentration (MIC). Studies that evaluated antimicrobial activity indicated that osteomyelitis-derived bacteria was not susceptible to released gallium maltolate at the hypothesized MIC and further established the accurate gallium maltolate MIC. The inhibitory concentration of each antimicrobial on osteoblasts was compared to the respective MIC to determine if they were safe and effective at released concentrations. Results concluded that the gentamicin sulfate-loaded PLGA mesh is safer and more effective mesh. Next, the bioactivity retention of vascular endothelial growth factor (VEGF) released from electrospun photo-crosslinked gelatin-methacrylate was confirmed. Subcutaneous implantation of the VEGF-loaded mesh in a rat corroborated resorption and the capacity for sustained release. A multifunctional electrospun wrap was then engineered to prevent osteomyelitis and guide formation of the induced membrane by combining the antimicrobial and angiogenic platforms with co-electrospinning. The combination of the two fiber populations was confirmed microscopically and offered independently tuned bimodal release of gentamicin sulfate and VEGF. Overall, this work provides the fundamentals to advance the development of a multifunctional electrospun wrap that can guide formation of the induced membrane and prevent osteomyelitis for improved clinical outcomes with the Masquelet technique. This work offers a substrate that can recruit and support cellular adhesion, provide a template for matrix deposition and tissue remodeling, and enable bimodal release of bioactive agents. These studies also enhance the capacity of electrospun platforms to serve as stand-alone therapies or combinatorial therapies in various bone regeneration applications.
Drug delivery strategies to treat Pseudomonas aeruginosa biofilm infections
(2018-05) Bahamondez-Canas, Tania Francisca; Smyth, Hugh D. C.; Cui, Zhengrong; Ghosh, Debadyuti; Davies, Bryan W.
Bacteria growing as biofilms have gained recognition as an important therapeutic challenge due to their relation with the chronicity of infectious diseases. Biofilms are microbial communities that grow within a self-secreted polymeric matrix and represent the predominant growth form of bacteria in nature, as compared to free-floating bacteria (known as the planktonic growth). Biofilms can be thousands of times more resistant to antimicrobials than planktonic bacteria, which is explained, in part, by the shielding effect of the extracellular matrix and by the slow growth of subpopulation within the biofilm (also known as persister bacteria). Pseudomonas aeruginosa is an opportunistic pathogen and is reported to be the main organism responsible for the recurrent lung infections in cystic fibrosis (CF) patients. P. aeruginosa biofilms have been isolated from CF mucus samples, but also from chronic wounds and chronic rhinosinusitis. The objective of the work presented in this document was to investigate strategies to improve the activity of antibiotics against P. aeruginosa biofilm infections using drug delivery, pharmaceutical and material science approaches. First, we investigated via a high-throughput screening, potential excipients that could enhance the activity of tobramycin (Chapter 2). We then selected illustrative excipients that improved the activity of tobramycin against P. aeruginosa biofilms to develop three prototype dry powder formulations for pulmonary delivery (Chapter 3). These formulations reduced significantly the survival of P. aeruginosa biofilms in vitro under a treatment schedule that simulated tobramycin concentrations found in the airways after pulmonary delivery. In another approach, we tested PEG-conjugated antibiotics in an in vitro model that comprised two relevant barriers for drug delivery in lung infections: P. aeruginosa growing as biofilms and a CF-like mucus barrier. We found that the conjugation of tobramycin to PEG (Tob-PEG) (Chapter 4) and colistin to PEG (Col-PEG) (Chapter 5) significantly improved their antimicrobial activities against P. aeruginosa biofilms growing the in the mucus barrier model. Finally, we explored the complexation of ciprofloxacin with copper (CIP-Cu) as a strategy to reduce the lung-to-blood ciprofloxacin permeability and sustain high local concentrations after lung delivery. CIP-Cu retained the antimicrobial activity against P. aeruginosa biofilms in vitro (Appendix A) and the in vivo evaluation of CIP-Cu resulted in significant reduction of P. aeruginosa survival in a chronic lung infection model (Appendix B). Overall, these strategies have addressed different aspects of biofilms resistance to antibiotics with promising therapeutic potential for further development
The effect of particle size and shape on margination and adhesion propensity
(2011-08) Jurney, Patrick Levi; Shi, Li, Ph. D.; Roy, Krishnendu
This thesis presents an experimental study of the effect that particle size and shape have on nanoparticle magination and adhesion propensity in micro-capillaries. With the use of half elliptical cross-section microfluidic channels that were fabricated using photolithography as well as wet and dry etching techniques and geometrically mimetic of human microcirculation, particles ranging from 93 to 970 nm were flown and imaged individually adhering to the channel walls. The results show a significant increase in particle adhesion below 200 nm as well as the emergence of a critical particle diameter above which no particle adherence was observed. The volume delivery efficiency was also shown to increase below 200 nm, providing insight for the rational design of nanocarriers for targeted cancer therapeutics.
Efficacy of hyper-osmotic agent (100% anhydrous glycerol) in tissue and light-activated micro-pattern drug delivery device in in vivo rabbit eye
(2011-05) Zaman, Raiyan Tripti; Rylander, H. Grady (Henry Grady), 1948-; Welch, Ashley J.; Milner, Thomas E.; Zhang, Xiaojing; Vargas, Gracie
My PhD research involves multi-disciplinary areas of study such as measuring perfusion of blood vessels in hamster dorsal skin using laser speckle imaging technique. In this study the changes were measured in blood flow velocity and diameters of micro vasculatures after the influence of glycerol application. The second study identifies the changes in morphology and optical properties of eye tissue after applying hyper-osmotic agent such as 100% anhydrous glycerol. Further investigation on the reversal process was performed without any application of 0.9% saline. The third study identified the variation in fluorescence in hamster dorsal skin tissue and enucleated porcine eyes with temperature. This study investigated the variation in fluorescence intensity with temperatures starting at 14°C and compared in vivo and in vitro results for consistency. The fourth study investigated an implantable drug delivery package that was fabricated using PMMA and implanted between the sub-conjunctival and super-scleral space and release the content of the device by either mechanical pressure or light-activated ophthalmic Nd:YAG laser after optically clearing the eye tissue by topical application of a hyper-osmotic agent, 100% anhydrous glycerol. A hyper-osmotic agent creates a transport region in the conjunctiva and sclera to get visual access of the compartments in the drug delivery package. This new technology would provide the option to the patient of one time implantation of the carrier system containing the drug. Each time the patient requires medication a ND-YAG or other laser beam will propagate through the cleared eye tissue to release the drug in measurable doses at the discretion of the doctor from the package directly in to the vitreous humor. In this study we have measured half-life of the dye in the vitreous humor or posterior chamber and biocompatibility. The last study had drawn distinction between the fluorescence signals based on the location (anterior or posterior chamber) of the 10% Na fluorescence dye in the in vivo rabbit and ex vivo pig eyes.
Engineering liposomal particles for direct, intracellular delivery of therapeutic molecules
(2021-07-28) Trementozzi, Andrea Nga-Kay; Stachowiak, Jeanne Casstevens; Cui, Zhengrong; Smyth, Hugh; Suggs, Laura; Kumar, Manish
Nanoparticles have been developed in the last few decades in effort to improve delivery of chemotherapeutics and reduce off-target effects. Despite longer circulation times and improved tumor localization, chemotherapeutics still often lack improved efficacy with nanoparticle delivery compared to delivery of free drug. Many nanoparticles enter cells by endocytosis, where degradation in lysosomes or immediate exocytosis often limits cytoplasmic drug release, ultimately reducing bioavailability of drugs within the cell. Towards overcoming these barriers, for my thesis work, I have developed two different approaches for novel liposomal nanoparticles that improve efficiency of intracellular delivery. The first approach relies on the permeability of lipid phase boundaries in ternary lipid compositions to promote effective release. Exploiting this property, I demonstrated delivery of doxorubicin using ternary liposomes that have the potential to undergo membrane phase separation upon contact with the cell surface. My results revealed that ternary, phase separating vesicles improve the performance of doxorubicin by up to 5-fold in comparison to delivery of conventional liposomal doxorubicin. The second approach I examined for improving efficient intracellular delivery of therapeutics harnesses the cellular gap junction network. Gap junctions allow direct, intracellular diffusion of molecules from one cell to another. Our lab has developed connectosomes, cell-derived vesicles that contain gap junction transmembrane proteins enabling the formation of gap junction channels with cells. Doxorubicin delivery via connectosomes dramatically reduced the minimum lethal dose, in vitro, in comparison to conventional liposomal doxorubicin. In my graduate work, I demonstrated that connectosomes also improve doxorubicin delivery to cellular targets in vivo in mouse models by up to 16-fold compared to conventional liposomal doxorubicin. Further, I investigated the potential for connectosomes to deliver not just small molecule therapeutics, but macromolecular therapeutics to cells. I show that polymer-like macromolecules of 10 kDa can diffuse through gap junction channels, and further that they exhibit 95% loading into connectosomes through gap junction hemichannel pores. Overall, my work reveals two novel approaches for improving delivery efficiency to intracellular targets for therapeutic molecules. These two approaches demonstrate mechanisms to overcome the barriers of endocytosis by enabling direct delivery of molecular cargo to the cellular interior.
Inhaled mycophenolate mofetil formulations for the prevention of lung allograft rejection
(2012-08) Dugas, Helene Laurence; Williams, Robert O., 1956-; Cui, Zhengrong; McConville, Jason T.; McGinity, James W.; Peters, Jay I.
The use of lung transplantation, a life saving intervention, has been increasing over the last thirty years with a disappointing median survival of only 4.8 years. Despite the progress made in immunosuppressive therapies, allograft rejection following transplantation is the leading cause of death. As part of the immunosuppressive therapy, mycophenolate mofetil (MMF), the ester prodrug of mycophenolic acid (MPA) has proven its efficacy among heart, liver, kidney as well as lung transplanted patients. However, due to its rapid excretion, high daily doses are necessary and lead to serious side effects, forcing the patient to stop and change their course of treatment. Administration of drugs to the lungs is known to minimize local and systemic side effects by employing a lower amount of drug, to increase patient compliance and to improve the efficacy of the treatment. Therefore, developing novel MMF formulations for targeted delivery to the lungs will broaden the therapeutic options against lung transplant rejection. Within the framework of this dissertation, the development of an inhaled formulation of MMF was investigated. MMF must be metabolized by carboxylesterases to become active and its metabolism suffers from high inter- and intra-patient variability. The first objective of this dissertation was to investigate the occurrence of MMF hydrolysis in the lung. The second objective was to study the in vivo deposition,metabolism and distribution in rats, of an inhaled micron-size MMF suspension in comparison to inhaled IV Cellcept® and oral Cellcept®, the currently marketed products. According to the in vitro results, MMF is metabolized in human lung cells by carboxylesterases. The in vivo results showed an incomplete metabolism of MMF when delivered as a suspension due to the limited dissolution of the drug in the lungs. Following inhalation, the MMF suspension achieved higher and more prolonged concentration of the total drug in the lungs and lymphoid tissues as compared to the inhaled IV Cellcept®. The pulmonary delivery of the MMF suspension was able to achieve similar levels of drug in the lungs, higher levels in the lymphoid tissues and significantly lower levels in the systemic circulation when compared to the levels obtained from the oral gavage of oral Cellcept®. Ultimately, this dissertation demonstrated that the administration of micron-size MMF suspension offers a great potential for pulmonary administration.
Intelligent nanoscale hydrogels for the oral delivery of hydrophobic therapeutics
(2015-05) Puranik, Amey Shreekant; Peppas, Nicholas A., 1948-; Contreras , Lydia; Sanchez , Isaac; Stachowiak , Jeanne; Yeh, Hsin-Chih
In this work, novel oral drug delivery formulations were developed for the administration of hydrophobic therapeutics, with the overarching goal of improving their solubility and permeability in the gastrointestinal tract. We have developed a set of four nanoscale hydrogels, formulated by incorporating different hydrophobic monomer components, and screen them for optimal physicochemical properties, drug loading and release, and ability to modulate intestinal permeability and P-glycoprotein related drug efflux. Here, we employ an evolved paradigm of in vitro tests to gauge the potential of these novel nanoscale carriers for the specific application of improving oral solubility and permeability of poorly water-soluble and less permeable therapeutics. All the responsive nanoscale hydrogels are capable of undergoing a transition in size in response to change in pH. We capitalize on the interplay between the incorporated hydrophobic monomer choices and screened resulting physicochemical properties to determine an optimal nanoscale formulation. Depending upon the selection of the hydrophobic monomer, the sizes of the nanoparticles vary widely from 120 nm to about 500 nm at pH 7.4. We also evaluate cytocompatibility of the nanoparticle formulations in vitro in the presence of an intestinal epithelial cell mode to find that all formulations are reasonably cytocompatible. Subsequently, we discuss some of the key findings and results of characterization studies that validate the success of achieving desired molecular architecture and physicochemical properties of the formulation. We then confirm the capacity of the nanocarrier to be able to load and release hydrophobic therapeutics in gastrointestinally relevant environments. Further, the ability of the nanocarriers to transport the hydrophobic therapeutic doxorubicin is determined by evaluating permeability of doxorubicin with intestinal epithelial cell monolayers. Furthermore, demonstrate functional abilities desired from a therapeutically relevant, oral delivery system is tested. Specifically, to overcome problems associated with P-glycoprotein related efflux and reduced drug permeability in the small intestine, we evaluated the ability of the nanoformulation to achieve therapeutic success in relevant and characteristic in vitro cancer cell lines. Finally, we make concluding remarks on the ability of the nanoparticles to function as improved formulations of hydrophobic therapeutics capable of performing and achieving the end-goal of delivering hydrophobic therapeutics orally for the treatment of cancer.
Investigating the stimuli responsiveness of reversible thia-conjugate addition crosslinked hydrogels
(2022-12-01) FitzSimons, Thomas Miyamoto; Rosales, Adrianne M.; Bonnecaze, Roger T; Anslyn, Eric V; Lynd, Nathaniel A; Page, Zachariah A
Dynamic hydrogels have recently demonstrated utility as versatile biomaterials. Characterized by the spontaneous rearrangement of crosslinks at ambient conditions, dynamic hydrogels more closely mimic the native extracellular matrix better than their static hydrogel counterparts, and the wide parameter space allows for a high degree of control for drug release applications. Despite the promising applicability that these hydrogels demonstrate, the structure-property relationships that govern the effects of the hydrogel composition on the mechanics remain poorly understood. An ideal system for investigating both the relationship of crosslinking on hydrogel mechanics in addition to biomaterial applications are hydrogels crosslinked through reversible covalent crosslinks. A number of reversible covalent bonds have well defined mechanisms that allow for facile determination of their rate constants via kinetic model fitting. Oscillatory rheometry also provides a robust mechanical method to characterize both the overall stiffness and dynamics of the dynamic hydrogels. In order to investigate the structure-property relationships in reversible covalently crosslinked hydrogels, this work first incorporated a reversible thia-conjugate addition reaction into a hydrogel. Previous small molecule research established this reaction as a reversible covalent reaction under standard biological conditions, and the first part of this project functionalized macromers and analyzed how chemical changes to the functional groups influenced the kinetics and mechanics of the resulting hydrogel. The accepted mechanism of this reaction reveals a deprotonation step in the forward and reverse direction which lead to the second area of research, wherein pH values from 3 to 7 were used to alter the kinetics and mechanics of a reversible thia-conjugate addition crosslinked hydrogels. Additionally, a gastrointestinal molecular release study was performed to demonstrate the drug delivery applications of this hydrogel. Finally, the temperature dependence of the crosslinking reaction was analyzed, and reaction parameters were determined from the mechanical analysis. In summary, three stimuli; chemistry, pH, and temperature, were used to investigate the structure-property relationships in reversible thia-conjugate addition crosslinked hydrogels with additional focus placed on the biomaterial applications of these materials.
Mathematical modeling of coupled drug and drug-encapsulated nanoparticle transport in patient-specific coronary artery walls
(2009-12) Hossain, Shaolie Samira; Hughes, Thomas J. R.
A vast majority of heart attacks occur due to rapid progression of plaque buildup in the coronary arteries that supply blood to the heart muscles. The diseased arteries can be treated with drugs delivered locally to vulnerable plaques—ones that may rupture and release emboli, resulting in the formation of thrombus, or blood clot that can cause blockage of the arterial lumen. In designing these local drug delivery devices, important issues regarding drug distribution and targeting need to be addressed to ensure device design optimization as physiological forces can cause the local concentration to be very different from mean drug tissue concentration estimated from in vitro experiments and animal studies. Therefore, the main objective of this work was to develop a computational tool-set to support the design of a catheter-based local drug delivery system that uses nanoparticles as drug carriers by simulating drug transport and quantifying local drug distribution in coronary artery walls. Toward this end, a three dimensional mathematical model of coupled transport of drug and drug-encapsulated nanoparticles was developed and solved numerically by applying finite element based isogeometric analysis that uses NURBS-based techniques to describe the artery wall geometry. To gain insight into the parametric sensitivity of drug distribution, a study of the effect of Damkohler number and Peclet number was carried out. The tool was then applied to a three-dimensional idealized multilayered model of the coronary artery wall under healthy and diseased condition. Preliminary results indicated that use of realistic geometry is essential in creating physiological flow features and transport forces necessary for developing catheter-based drug delivery design procedures. Hence, simulations were run on a patient-specific coronary artery wall segment with a typical atherosclerotic plaque characterized by a lipid pool encased by a thin fibrous cap. Results show that plaque heterogeneity and artery wall inhomogeneity have a considerable effect on drug distribution. The computational tool-set developed was able to successfully capture trends observed in local drug delivery by incorporating a multitude of relevant physiological phenomena, and thus demonstrated its potential utility in optimizing drug design parameters including delivery location, nanoparticle surface properties and drug release rate.
Metal-polymer nanoparticulate systems for externally-controlled delivery
(2010-12) Gran, Martin Luke; Peppas, Nicholas A., 1948-; Paul, Donald R.; Freeman, Benny D.; Johnston, Keith P.; Emelianov, Stanislav
Metal-polymer nanocomposites consisting of gold nanorods and temperature-responsive hydrogel nanoparticulates were investigated for use in externally-controlled drug delivery systems. Several different thermo-responsive hydrogels including poly(N-isopropyl acrylamide) (PNIPAAm) and poly(N-isopropryl acrylamide-co-acrylic acid) (P(NIPAAm-co-AA)) nanoparticles were synthesized for these nanocomposites using an aqueous dispersion polymerization method. In addition, nanoparticles of interpenetrating polymer networks (IPN) composed of poly(acrylamide) (PAAm) and poly(acrylic acid) (PAA) were synthesized using a water-in-oil emulsion polymerization. Temperature-responsive equilibrium swelling behavior of nanoparticles with varying crosslinking densities was characterized using dynamic light scattering. IPN systems exhibited a positive swelling response upon heating while PNIPAAm and copolymer systems collapsed upon increase in temperature above the transition point. Nanoparticles were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) which demonstrated shape and morphology of polymer particles. Gold-polymer nanocomposites were formed by grafting gold nanorods to the surface of the polymer nanoparticles. Amine-functionalized gold nanorods were coupled to polymers using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) to activate carboxyl groups on the surface of the polymer nanoparticles. TEM confirmed successful formation of the metal-polymer nanocomposites. Loading and release of a model therapeutic were done to assess the potential use of the polymer component of the nanocomposite for drug delivery. Fluorescein, a model for chemotherapeutics, was loaded into P(NIPAAm-co-AA) polymer nanoparticulates. Loading of the compound was shown to be a function of crosslinking density in the polymer network. Maximum loading was achieved using nanoparticles synthesized with a 10 mol% crosslinker feed ratio with entrapment efficiencies of 80.0 % and loading capacities of 12.0 %. Cytotoxicity studies were performed using a NIH/3T3 mouse fibroblast cell model. Cell viabilities in presence of P(NIPAAm-co-AA) nanoparticles were comparable to (not statistically different than) controls at concentrations up to 4 mg/ml. Similarly, gold-polymer composite concentrations up to 0.5 mg/ml caused limited cell death.
Miniaturized antenna and transponder based wireless sensors for internet of things in healthcare
(2014-12) Huang, Haiyu; Akinwande, Deji; Gharpurey, Ranjit; Neikirk, Dean; Hu, Ye; Lu, Nanshu
Future medical and healthcare systems will be largely improved by the wide-spreading of internet of things (IoTs). One of the crucial challenges of IoTs for healthcare is at the wireless sensors. Miniaturization of sensor node profile, minimizing power consumption as well as lowering down design/production cost of antenna, RF circuits and sensor modules have become the key issues for realizing more exciting applications in medical and healthcare fields that never seemed to be possible before. In this dissertation work, we first focus on electrically small antenna (ESA) design and fabrication for medical telemetry. A comprehensive analysis of the radiation properties of a novel electrically small folded ellipsoidal ESA is presented, showing its ability to self-resonate and impedance match without external components. It will benefit various size-restricted applications especially with wireless medical implants. The second focus is on healthcare sensors using ESA as the sensing agent, which saves the power and cost by eliminating the need of extra sensing modules. Specifically, miniaturized helix ESAs are integrated with drug reservoirs to function as wireless transponder sensors for real-time drug dosage monitoring. We also introduce a system level innovation of a passive wireless harmonic transponder/harmonic sniffer/frequency hopped interrogator based sensing system. The μL- liquid level resolution and absolute-accuracy passive sensing is demonstrated in the presence of strong direct coupling, background scatters, distance variance as well as near-filed human body movement interference. Furthermore, we investigate how modern ubiquitous wireless sensor networks could take advantage of sensitive nanostructure materials for enhanced performance. Here we propose a new paradigm of chemically-gated mixed modulation on a single homogeneous graphene device in which the chemical exposure directly modulates an electrical carrier signal. To make the device ubiquitously reusable, a method of precisely tuning the charge neutrality point (Vcnp) is introduced by electrochemical calibration with gate voltage pulse sequence. Such chemically gated graphene modulator can be potentially used in a harmonic transponder as a passive ubiquitous sensor node for chemical and bio sensing applications. Overall the research work presented in the dissertation will help enable cost and power-efficient wireless sensor networks in future healthcare IoTs.