Characterizing Targeted Therapeutic Delivery and Cellular Dynamics using In Vitro Cancer Disease Models

Cancer is a significant health risk to people living in developed and developing countries, which continues to prove difficult to treat. Common treatment options of cancers include surgical removal, radiation, and chemotherapies, which are often used in combination to improve the likelihood of successful treatment. Such combinatory approaches towards treatment are often taken because each approach is not targeted enough to function perfectly on its own. Being able to delivery therapeutic loads in a more targeted manner to sites of cancer has the capability of improving therapeutic efficiency and improving patient responses. The development of improved therapeutic delivery vehicles and screening systems can help serve the goal of improved targeted therapeutic delivery. The use of microfluidic devices for the study of therapeutic delivery has become popular over the past few decades because of the many benefits that they offer. Specifically, microfluidic devices only require small volumes of therapeutics for testing, which is often ideal because of limited drug supply during screening. Additionally, the high degree of control over channel geometries, ease of fabrication and low cost make microfluidic therapeutic testing devices well suited for higher throughput screening when run together in parallel. The ability to generate shear flow within the microfluidic channels also offers a means of more closely mimicking vascular physiology and conditions that would be experienced during drug delivery in the human body. Lastly, the use of microfluidic in therapeutic testing enables micro-scale data on characteristics such as binding, uptake, cellular permeability and others to be easily collected due to the transparent nature of the devices and ability to facilitate cell cultures. As such, the focus of this dissertation is mainly based around the establishment of microfluidic systems capable of mimicking cancerous environments and testing of various therapeutic vehicles and delivery methods targeted for cancer. In brief, the dissertation demonstrates a few methods of establishing cancerous environments within microfluidic systems of increasing complexity, and how screening of various nanoparticle vehicles and therapeutics is performed.First, a single layer microfluidic device is developed to facilitate the growth of cancer monolayers for the screening of solid lipid nanoparticle drug delivery performance. The device is designed to assist in identifying an optimal ratio of antibody to polymer chains exposed on the surface of the nanoparticles. Improved targeting of nanoparticles to cancer cells is achieved by increasing target specific binding through addition of cancer antibody while reducing non-specific binding through addition of polymer chains on the nanoparticles surface. Conditions for optimal targeting specifically to cancer cells were identified for nanoparticles with 37% of their surface area occupied by polyethelyene glycol (PEG). The cancer cell targeting efficiency for the 37% coated nanoparticles was determined to be a maximum of 81% when a cancer specific antibody was used in conjunction on the nanoparticles surface.Next, to improve the physiological relevance of the microfluidic screening system, a bi-layer setup was fabricated. The nature of the bi-layer device is designed to facilitate the co-culture of cancer and endothelial cells (ECs) in different compartments while still permitting signaling and chemical interactions to occur between the two cell types. The presence of ECs in the device is designed to mimic a blood vessel, as therapeutic delivery within the body relies heavily on the circulatory system from drug transport. As such, understanding the mechanics of therapeutic delivery from mimicked vasculature to cancer is an important consideration. Conditions in the bi-layer system influencing therapeutic transport include endothelial permeability, therapeutic size, system flow rate, and treatment time. Improved therapeutic delivery was achieved using smaller molecules, slower system flow rates, and when the EC monolayer was highly permeabilized. Increased treatment times, resulted in less and less therapeutic transport from the mimicked vessel to the cancer environment as the EC monolayer regained confluency. It was shown that the bi-layer microfluidic system functions to screen therapeutic delivery to a mimicked cancer environment under more physiologically relevant conditions.The next progression with the system was to test nanoparticle delivery and transport from the mimicked vessel to the cancer environment. This was accomplished utilizing the same bi-layer microfluidic setup in conjunction with a range of nanoparticle shapes that were utilized to identify characteristics that facilitate the greatest degree of therapeutic delivery. Specifically, spherical, short rod and long rod/worm-like nanoparticles were tested for their ability to transport therapeutic loads to the cancer environment over the course of 5 day treatments. Optimal nanoparticle shapes for each flow rate varied based on treatment time. Overall, nanoparticle drug delivery should be varied based on the degree of EC permeability which changes with time as the cancer environment is treated.Lastly, to improve the physiological relevance of cancer environments being used, a method for establishing and growing tumor spheroids within the microfluidic devices in an expedited fashion was developed. The ability to perform therapeutic and nanoparticle carrier screening on tumor spheroids as opposed to cancer monolayers provides feedback on efficiency and performance which more closely mimics outcomes observed in animal and clinical testing. In addition, the ability to form tumor spheroids in an expedited manner allows the screening process to be completed in a shorter period of time and with fewer initial cells. The use of convective driven nutrient flow is utilized to achieve such expedited cancer growth in a microfluidic system which also has the potential to facilitate therapeutic screening. The system has been shown to function with adherent and non-adherent cell types where 1.5 to 4.5 times faster growth can be achieved. The ability to cut tumor culturing times from 1 week to 3 days and reducing required cell counts from thousands to tens of cells has the potential to save lives in clinical settings when using patient derived samples

Selecting the appropriate hurdles and endpoints for pentilludin, a novel antiaddiction pharmacotherapeutic targeting the receptor type protein tyrosine phosphatase D

Substance use disorders provide challenges for development of effective medications. Use of abused substances is likely initiated, sustained and “quit” by complex brain and pharmacological mechanisms that have both genetic and environmental determinants. Medical utilities of prescribed stimulants and opioids provide complex challenges for prevention: how can we minimize their contribution to substance use disorders while retaining medical benefits for pain, restless leg syndrome, attention deficit hyperactivity disorder, narcolepsy and other indications. Data required to support assessments of reduced abuse liability and resulting regulatory scheduling differs from information required to support licensing of novel prophylactic or therapeutic anti-addiction medications, adding further complexity and challenges. I describe some of these challenges in the context of our current efforts to develop pentilludin as a novel anti-addiction therapeutic for a target that is strongly supported by human and mouse genetic and pharmacologic studies, the receptor type protein tyrosine phosphatase D (PTPRD)

Self-Management and Team-Making in Cross-Functional Work Teams: Discovering the Keys to Becoming an Integrated Team

Project teams are rapidly becoming the primary mechanisms for innovation and change in modern organizations. As such, they are designed to capitalize on leadership and integrated cross-functional teamwork and to negate subordination and individual gamesmanship. Unfortunately, research on cross-functional project teams is scarce and largely atheoretical. The increasing use of these project teams by modern organizations, however, calls for theory development in this area. In the present paper, self-management and team-making models are applied to cross-functional project designs to develop a theoretical framework for the investigation of teamwork effectiveness for integrated cross-functional project teams. Future issues for theory development and research methodology are presented

Quantitative pharmacologic MRI: Mapping the cerebral blood volume response to cocaine in dopamine transporter knockout mice

The use of pharmacologic MRI (phMRI) in mouse models of brain disorders allows noninvasive in vivo assessment of drug-modulated local cerebral blood volume changes (ΔCBV) as one correlate of neuronal and neurovascular activities. In this report, we employed CBV-weighted phMRI to compare cocaine-modulated neuronal activity in dopamine transporter (DAT) knockout (KO) and wild-typemice. Cocaine acts to block the dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT) that clear their respective neurotransmitters from the synapses, helping to terminate cognate neurotransmission. Cocaine consistently reduced CBV, with a similar pattern of regional ΔCBV in brain structures involved inmediating reward in both DAT genotypes. The largest effects (−20% to −30% ΔCBV) were seen in the nucleus accumbens and several cortical regions. Decreasing response amplitudes to cocaine were noted in more posterior components of the cortico-mesolimbic circuit. DAT KO mice had significantly attenuated ΔCBV amplitudes, shortened times to peak response, and reduced response duration in most regions. This study demonstrates that DAT knockout does not abolish the phMRI responses to cocaine, suggesting that adaptations to loss of DAT and/or retained cocaine activity in other monoamine neurotransmitter systems underlie these responses in DAT KO mice

Hemicraniectomy for Dominant vs Nondominant Middle Cerebral Artery Infarction : A Systematic Review and Meta- Analysis

Objectives: Decompressive hemicraniectomy decreases mortality and severe disabil-ity from space-occupying middle cerebral artery infarction in selected patients. However, attitudes towards hemicraniectomy for dominant-hemispheric stroke have been hesitant. This systematic review and meta-analysis examines the associa-tion of stroke laterality with outcome after hemicraniectomy. Materials and methods: We performed a systematic literature search up to 6th February 2020 to retrieve original articles about hemicraniectomy for space-occupying middle cere-bral artery infarction that reported outcome in relation to laterality. The primary outcome was severe disability (modified Rankin Scale 4-6 or 5-6 or Glasgow Out -come Scale 1-3) or death. A two-stage combined individual patient and aggregate data meta-analysis evaluated the association between dominant-lateralized stroke and (a) short-term ( 3 months) outcome. We per -formed sensitivity analyses excluding studies with sheer mortality outcome, sec -ond-look strokectomy, low quality, or small sample size, and comparing populations from North America/Europe vs Asia/South America. Results: The analysis included 51 studies (46 observational studies, one nonrandomized trial, and four randomized controlled trials) comprising 2361 patients. We found no asso-ciation between dominant laterality and unfavorable short-term (OR 1.00, 95% CI 0.69-1.45) or long-term (OR 1.01, 95% CI 0.76-1.33) outcome. The results were unchanged in all sensitivity analyses. The grade of evidence was very low for short -term and low for long-term outcome. Conclusions: This meta-analysis suggests that patients with dominant-hemispheric stroke have equal outcome after hemicraniec-tomy compared to patients with nondominant stroke. Despite the shortcomings of the available evidence, our results do not support withholding hemicraniectomy based on stroke laterality.Peer reviewe

Genome Wide Association for Addiction: Replicated Results and Comparisons of Two Analytic Approaches

BACKGROUND: Vulnerabilities to dependence on addictive substances are substantially heritable complex disorders whose underlying genetic architecture is likely to be polygenic, with modest contributions from variants in many individual genes. "Nontemplate" genome wide association (GWA) approaches can identity groups of chromosomal regions and genes that, taken together, are much more likely to contain allelic variants that alter vulnerability to substance dependence than expected by chance. METHODOLOGY/PRINCIPAL FINDINGS: We report pooled "nontemplate" genome-wide association studies of two independent samples of substance dependent vs control research volunteers (n = 1620), one European-American and the other African-American using 1 million SNP (single nucleotide polymorphism) Affymetrix genotyping arrays. We assess convergence between results from these two samples using two related methods that seek clustering of nominally-positive results and assess significance levels with Monte Carlo and permutation approaches. Both "converge then cluster" and "cluster then converge" analyses document convergence between the results obtained from these two independent datasets in ways that are virtually never found by chance. The genes identified in this fashion are also identified by individually-genotyped dbGAP data that compare allele frequencies in cocaine dependent vs control individuals. CONCLUSIONS/SIGNIFICANCE: These overlapping results identify small chromosomal regions that are also identified by genome wide data from studies of other relevant samples to extents much greater than chance. These chromosomal regions contain more genes related to "cell adhesion" processes than expected by chance. They also contain a number of genes that encode potential targets for anti-addiction pharmacotherapeutics. "Nontemplate" GWA approaches that seek chromosomal regions in which nominally-positive associations are found in multiple independent samples are likely to complement classical, "template" GWA approaches in which "genome wide" levels of significance are sought for SNP data from single case vs control comparisons

MPP+ toxicity and plasma membrane dopamine transporter: study using cell lines expressing the wild-type and mutant rat dopamine transporters

AbstractThe Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) causes specific cell death in dopaminergic neurons after accumulation by the dopamine transporter (DAT). COS cells, a non-neuronal cell line insensitive to high doses of MPP+, becomes sensitive to MPP+ when transfected with the rat DAT cDNA. We analyzed the bi-directional transport of MPP+ and its toxicity in several cell lines expressing wild or mutant DATs. Cell death in COS cells expressing wild DAT by exposure to MPP+ was concentration-dependent and cocaine-reversible. Increased wild DAT expression caused higher sensitivities to the toxin in HeLa cells. Although several mutant DATs demonstrated greater transport activity than the wild-type, they displayed similar or lower sensitivity to MPP+ toxicity. Reverse transport of preloaded [3H]MPP+ through DAT was facilitated in COS cells expressing certain mutant DATs, which consistently displayed less sensitivity to MPP+ toxicity. These results suggest that re-distribution of MPP+ due to influx/efflux turnover through the transporter is a key factor in MPP+ toxicity

Genomic Regions Identified by Overlapping Clusters of Nominally-Positive SNPs from Genome-Wide Studies of Alcohol and Illegal Substance Dependence

Declaring “replication” from results of genome wide association (GWA) studies is straightforward when major gene effects provide genome-wide significance for association of the same allele of the same SNP in each of multiple independent samples. However, such unambiguous replication is unlikely when phenotypes display polygenic genetic architecture, allelic heterogeneity, locus heterogeneity and when different samples display linkage disequilibria with different fine structures. We seek chromosomal regions that are tagged by clustered SNPs that display nominally-significant association in each of several independent samples. This approach provides one “nontemplate” approach to identifying overall replication of groups of GWA results in the face of difficult genetic architectures. We apply this strategy to 1 M SNP GWA results for dependence on: a) alcohol (including many individuals with dependence on other addictive substances) and b) at least one illegal substance (including many individuals dependent on alcohol). This approach provides high confidence in rejecting the null hypothesis that chance alone accounts for the extent to which clustered, nominally-significant SNPs from samples of the same racial/ethnic background identify the same sets of chromosomal regions. It identifies several genes that are also reported in other independent alcohol-dependence GWA datasets. There is more modest confidence in: a) identification of individual chromosomal regions and genes that are not also identified by data from other independent samples, b) the more modest overlap between results from samples of different racial/ethnic backgrounds and c) the extent to which any gene not identified herein is excluded, since the power of each of these individual samples is modest. Nevertheless, the strong overlap identified among the samples with similar racial/ethnic backgrounds supports contributions to individual differences in vulnerability to addictions that come from newer allelic variants that are common in subsets of current humans

Quantitative Detection of µ Opioid Receptor: Western Blot Analyses Using µ Opioid Receptor Knockout Mice

Increasing evidence suggests that µ opioid receptor (MOP) expression is altered during the development of and withdrawal from substance dependence. Although anti-MOP antibodies have been hypothesized to be useful for estimating MOP expression levels, inconsistent MOP molecular weights (MWs) have been reported in studies using anti-MOP antibodies. In the present study, we generated a new anti-MOP antibody (N38) against the 1-38 amino acid sequence of the mouse MOP N-terminus and conducted Western blot analysis with wildtype and MOP knockout brain lysates to determine the MWs of intrinsic MOP. The N38 antibody detected migrating bands with relative MWs of 60-67 kDa in the plasma membrane fraction isolated from wildtype brain, but not from the MOP knockout brain. These migrating bands exhibited semi-linear density in the range of 3-30 µg membrane proteins/lane. The N38 antibody may be useful for quantitatively detecting MOP

MPP+ toxicity and plasma membrane dopamine transporter: study using cell lines expressing the wild-type and mutant rat dopamine transporters

AbstractThe Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) causes specific cell death in dopaminergic neurons after accumulation by the dopamine transporter (DAT). COS cells, a non-neuronal cell line insensitive to high doses of MPP+, becomes sensitive to MPP+ when transfected with the rat DAT cDNA. We analyzed the bi-directional transport of MPP+ and its toxicity in several cell lines expressing wild or mutant DATs. Cell death in COS cells expressing wild DAT by exposure to MPP+ was concentration-dependent and cocaine-reversible. Increased wild DAT expression caused higher sensitivities to the toxin in HeLa cells. Although several mutant DATs demonstrated greater transport activity than the wild-type, they displayed similar or lower sensitivity to MPP+ toxicity. Reverse transport of preloaded [3H]MPP+ through DAT was facilitated in COS cells expressing certain mutant DATs, which consistently displayed less sensitivity to MPP+ toxicity. These results suggest that re-distribution of MPP+ due to influx/efflux turnover through the transporter is a key factor in MPP+ toxicity