Gold Nanoparticles Coated Optical Fiber for Real-time Localized Surface Plasmon Resonance Analysis of In-situ Light-Matter Interactions

In situ measurement of analytes for in vivo or in vitro systems has been challenging due to the bulky size of traditional analytical instruments. Also, frequent in vitro concentration measurements rely on fluorescence-based methods or direct slicing of the matrix for analyses. These traditional approaches become unreliable if localized and in situ analyses are needed. In contrast, for in situ and real-time analysis of target analytes, surface-engineered optical fibers can be leveraged as a powerful miniaturized tool, which has shown promise from bio to environmental studies. Herein, we demonstrate an optical fiber functionalized with gold nanoparticles using a dip-coating process to investigate the interaction of light with molecules at or near the surface of the optical fiber. Localized surface plasmon resonance from the light-matter interaction enables the detection of minute changes in the refractive index of the surrounding medium. We used this principle to assess the in situ molecular distribution of a synthetic drug (methylene blue) in an in vitro matrix (agarose gel) having varying concentrations. Leveraging the probed Z-height in diffused analytes, combined with its in silico data, our platform shows the feasibility of a simple optofluidic tool. Such straightforward in situ measurements of analytes with optical fiber hold potential for real-time molecular diffusion and molecular perturbation analyses relevant to biomedical and clinical studies

Optimisation of a chronically implanted catheter for intraparenchymal delivery of therapeutics to the brain

Delivering drugs to the brain to treat degenerative diseases and other conditions is challenging because of the blood brain barrier, which acts as a filtration system preventing over 99 % of large, therapeutic molecules from entering the brain from the blood. A first-in-man study, delivering drugs directly through chronically implanted catheters inserted deep into the brain, formed the basis of this project. The short and long-term distribution data from this clinical study provided the direction of this research. Surgical planning guidelines were created which provide device specific, numerical values to optimise retention of infusate within target neuroanatomy. Optimisation of these implanted catheters was assessed through device characterisation, material investigation, development of miniaturised delivery systems for in vivo investigations and the creation of a finite element model of infusions into porous ‘brain’ matter. Despite dissimilar mechanical properties to brain tissue, agarose gel has superior permeability and optical properties over composite hydrogels for the characterisation of a recessed step catheter. In vitro experiments varying catheter features and infusion regimes identified significant changes in the distribution patterns of infused fluids which propagate through porous substrates, such as gels or the brain. By adjusting the catheter step length and peak volumetric flow rate, optimisation of implanted catheters could maximise coverage of target neuro anatomy. Gliosis around the implanted catheter was anticipated as a result of the immune response to injury. Through experimentation gliosis was shown not to be exacerbated by intermittent infusions. The extent of injury during implantation plays a greater role. Changes in clinical infusion distribution patterns may have been linked to observations of lower gliosis levels around the same time as test infusions occur clinically (1month post implant). Longer recovery periods could provide improved reliability of test infusions to inform users ahead of setting prescriptions for extended infusion regimes

An arborizing, multiport catheter for maximizing drug distribution in the brain via convection enhanced delivery

Glioblastoma (GBM) is a high-grade malignant glioma with a mortality rate that exceeds 95% despite over eight decades of medical research dedicated to improve outcomes. GBM is extremely difficult to treat and practically incurable with standard treatment involving surgical resection, radiation, concomitant and/or adjuvant chemotherapy. Therefore, convection enhanced delivery (CED) was developed to improve therapeutic outcomes. CED involves intraparenchymal delivery of drugs into diseased tissue via a small catheter. CED has proven to bypass the blood brain barrier and achieve better drug distribution than diffusion-based therapies. Nevertheless, the large volumes necessary to target entire tumors and peritumor volumes have been previously unachievable with currently-available catheters. This dissertation describes the development of a multiport, arborizing catheter designed specifically for improving drug distribution in the brain. The performance of early-stage arborizing catheter prototypes was compared to single-port catheters in infusion studies using agarose brain phantoms. Volume dispersed (V [subscript d]) and mean distribution ratios (V [subscript d] :V [subscript i]) were quantified and compared between the two catheters. The arborizing catheter produced higher V [subscript d] values; however, it did not exhibit the greatest V [subscript d] :V [subscript i], likely due to overlapping distribution volumes from the multiple individual ports. Following infusion in brain phantoms, a biotransport study of the arborizing catheter was conducted using a multiphasic finite element framework. The model was used to predict dispersion volume of a solute in a permeable hyperelastic solid matrix as a function of separation distance between adjacent ports. Results show that increasing port distance can increase V [subscript d]; however, infusion time also increases significantly with greater port distance. One way to mitigate increased infusion times is to employ higher infusion flow rates. Finally, the performance of improved arborizing catheters was compared to reflux-preventing single-port catheters in excised pig brains. CT scans were used to quantify V [subscript d] and V [subscript d] :V [subscript i] of infused iohexol (contrast-enhancing agent). The average volume dispersed for the arborizing catheter was 5.8 times greater than the single-port catheter. Mean distribution ratios for both catheters were similar. Using the multiple ports of the arborizing catheter, high V [subscript d] was achieved at a low infusion rate with negligible reflux. Given that previous attempts of CED reported poor drug distribution, the arborizing catheter may help overcome the limitations of CED.Biomedical Engineerin

Glucose diffusivity in tissue engineering membranes and scaffolds: implications for hollow fibre membrane bioreactor

Unlike thin tissues (e.g., skin) which has been successfully grown, growing thick tissues (e.g., bone and muscle) still exhibit certain limitations due to lack of nutrients (e.g., glucose and oxygen) feeding on cells in extracapillary space (ECS) region, or also known as scaffold in an in vitro static culture. The transport of glucose and oxygen into the cells is depended solely on diffusion process which results in a condition where the cells are deprived of adequate glucose and oxygen supply. This condition is termed as hypoxia and leads to premature cell death. Hollow fibre membrane bioreactors (HFMBs) which operate under perfusive cell culture conditions, have been attempted to reduce the diffusion limitation problem. However, direct sampling of glucose and oxygen is almost impossible; hence noninvasive methods (e.g., mathematical models) have been developed in the past. These models have defined that the glucose diffusivity in cell culture medium (CCM) is similar to the diffusivity in water; thus, they do not represent precisely the nutrient transport processes occurring inside the HFMB. In this research, we define glucose as our nutrient specie due to its limited published information with regard to its diffusivity values, especially one that corresponds to cell/tissue engineering (TE) experiments. A series of well-defined diffusion experiments are carried out with TE materials of varying pore size and shapes imbibed in water and CCM, namely, cellulose nitrate (CN) membrane, polyvinylidene fluoride (PVDF) membrane, poly(L-lactide) (PLLA) scaffold, poly(caprolactone) (PCL) scaffold and collagen scaffold. A diffusion cell is constructed to study the diffusion of glucose across these materials. The glucose diffusion across cell-free membranes and scaffolds is investigated first where pore size distribution, porosity and tortuosity are determined and correlated to the effective diffusivity. As expected, the effective diffusivity increases correspondingly with the pore size of the materials. We also observe that the effective glucose diffusivity through the pores of these materials in CCM is smaller than in water. Next, we seeded human osteoblast cells (HOSTE85) on the scaffolds for a culture period of up to 3 weeks. Similar to the first series of the diffusion experiments, we have attempted to determine the effective glucose diffusivity through the pores of the scaffolds where cells have grown at 37°C. The results show that cell growth changes the morphological structure of the scaffolds, reducing the effective pore space which leads to reduced effective diffusivity. In addition, the self-diffusion of glucose in CCM and water has also been determined using a diaphragm cell method (DCM). The results have shown that the glucose diffusivity in CCM has significantly reduced in comparison to the water diffusivity which is due to the larger dynamic viscosity of CCM. The presence of other components and difference in fluid properties of CCM may also contribute to the decrease. We finally employ our experimentally deduced effective diffusivity and self-diffusivity values into a mathematical model based on the Krogh cylinder assumption. The glucose concentration is predicted to be the lowest near the bioreactor outlet, or in the scaffold region, hence this region becomes a location of interest. The governing transport equations are non-dimensionalised and solved numerically. The results shown offer an insight into pointing out the important parameters that should be considered when one wishes to develop and optimise the HFMB design

HYDROGEL: RESPONSIVE STRUCTURES FOR DRUG DELIVERY

Hydrogels are water-swollen 3D networks made of polymers, proteins, small molecules, or colloids. They are porous in structure and entrap/encapsulate large amounts of therapeutic agents and biopharmaceuticals. Their unique properties like biocompatibility, biodegradability, sensitivity to various stimuli, and the ability to be easily conjugated with hydrophilic and hydrophobic drugs with a controlled-release profile make hydrogels a smart drug delivery system. Smart hydrogel systems with various chemically and structurally responsive moieties exhibit responsiveness to external stimuli including temperature, pH, ionic concentration, light, magnetic fields, electrical fields, and chemical and biological stimuli with selected triggers includes polymers with multiple responsive properties have also been developed elegantly combining two or more stimuli-responsive mechanisms. This article emphasized the types, features, and various stimuli systems that produce responsive delivery of drugs

Convection-Enhanced Delivery of Macromolecules to the Brain Using Electrokinetic Transport

Electrokinetic transport in brain tissue represents the movement of molecules due to an applied electric field and the interplay between the electrophoretic and electroosmotic velocities that are developed. This dissertation provides a framework for understanding electrokinetic transport and how it may be utilized for short-distance ejections, relevant to capillary iontophoresis, and long-distance infusions, for the clinical management of malignant brain tumors as a novel convection-enhanced drug delivery system.In particular, electrokinetic transport was first analyzed in a series of poly(acrylamide-co-acrylic acid) hydrogels that demonstrated varying electroosmotic velocities. Moreover, a hydrogel was synthesized to mimic the electrokinetic properties of organotypic hippocampal slice cultures (OHSC), as a surrogate for brain tissue. Short- and long-distance capillary infusions of molecules into the hydrogels and OHSC provided a framework to understand the relevant phenomena, such as the effect of varying the capillary tip size, applied electrical current, ζ-potential of the capillary or the outside matrix, infusion time, tortuosity, and properties of the solute (including molecular weight and electrophoretic mobility). Control of the directional transport of molecules was also demonstrated over a distance of several hundred micrometers to millimeters. Finally, electrokinetic infusions were conducted in vivo in the adult rat brain, with results compared to those of pressure-driven infusions.The experiments and results described in this dissertation provide a foundation for further development, by presenting a methodical means to increase the ejection profile and attain clinically relevant penetration distances while minimizing adverse effects to the brain tissue, including from the electric field itself. The rate of electrokinetic transport is greater than the rate of diffusion, and therefore it represents a novel form of convection-enhanced drug delivery system

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells. Using a compartmentalised diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time. The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications

Tumor cell migration in complex microenvironments

Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of applying multiple, simultaneous stimuli and imaging the cellular migratory response in real-time. These next-generation assays will more closely mimic the in vivo microenvironment to provide new insights into tumor progression, inform techniques to control tumor cell migration, and render cancer more treatable.National Science Foundation (U.S.) (Graduate Research Fellowship)Charles Stark Draper Laboratory (Research and Development Program (N.DL-H-550151))National Cancer Institute (U.S.) (R21CA140096

Bacteriophage: from bacteria to targeted gene delivery to mammalian cells

Bacteriophage (phage), bacterial viruses, have been improved as non-human pathogenic viral vectors for the purpose of introducing genetic materials into mammalian cells. Previously, our group generated a novel Adeno-associated virus/Phage (AAVP) hybrid vector as a valuable tool for targeted gene transfer to mammalian cells. However, the efficacy of bacteriophage-based vectors is considered relatively poor, meaning that ways of improving it are of considerable interest. First approach to improve AAVP-mediated gene delivery is through chemical modification. We showed that the transduction efficiency of AAVP was increased by the complexation of phage vectors with cationic molecules and calcium phosphate co-precipitation. Application of the bacteriophage complex carrying a cytotoxic gene resulted in eradication of cultured brain tumour cells. The chemically modified vector showed superior gene delivery over the conventional vector and can thus be regarded as an improved version of phage-based vector that has promise in cancer gene therapy. Next, we demonstrated that Extracellular Matrix (ECM) presents an obstacle for AAVP. Using brain cancer cell lines as a model, AAVP transduction was significantly increased by collagenase and hyaluronidase-mediated degradation of ECM, which can subsequently be translated into tumour cell eradication through AAVP-mediated gene therapy. Our findings prove that combination of AAVP vectors with ECM depletion represents a powerful strategy to advance phage- guided gene transfer. Finally, we engineered the prototype bacteriophage-based multifunctional vector as a proof-of-concept model that can simultaneously display three different peptides and carry a mammalian transgene cassette. Our results show that bacteriophage can be used as a scaffold for constructing multifunctional carriers that integrate multiple functions, which may have great potential for gene delivery applications. Together, the data demonstrate the potential for improved AAVP-based gene transfer to mammalian cells focusing on the use of chemical modification, manipulation of ECM, and the generation of multifunctional phage vectors.Open Acces