Article Database

Nanotherapeutics for combination drug and gene therapy in treating glioblastoma multiforme

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Gliomas represent approximately 80% of all malignant brain tumors, and glioblastoma multiforme (GBM), the most aggressive type, accounts for nearly half of all gliomas. Despite treatment strategies including surgery, radiation, and chemotherapy, the 5-year survival rate for brain cancer is only 35%. New therapeutic strategies are necessary to improve the outcomes of this disease. Chemotherapy with DNA alkylating agents is commonly used in treatment for GBM. Research has shown that better therapeutic response of GBM tumors to alkylating agents and increased survival rate is indicative in patients with epigenetic silencing of O⁶-methylguanine-DNA methyltransferase (MGMT), a gene responsible for DNA repair. Therefore, we propose a treatment strategy combining drug and gene therapy to target and silence MGMT to sensitize cells to treatment with temozolomide (TMZ) or lomustine (CCNU), both DNA alkylating agents. We previously developed cationic, amphiphilic copolymer poly(lactide-co-glycolide)-g-polyethylenimine (PgP) and demonstrated its utility for nucleic acid delivery. Here, we examine the ability of PgP as a drug and siRNA delivery carrier to overcome drug resistance and improve anticancer activity through combination drug and gene therapy for GBM treatment. PgP micelles were designed and synthesized for delivery of hydrophobic drugs in the PLGA core and negatively charged nucleic acids in the positively charged PEI shell through electrostatic interactions. RNA binding and polyplex stability assays were performed using agarose gel electrophoresis. Cytotoxicity of TMZ, CCNU, and/or PgP/siMGMT polyplexes was determined by MTT assay. Silencing of MGMT on the protein and mRNA level was determined using western blotting and qPCR, respectively. Our results demonstrated that PgP effectively forms stable complexes with siRNA and protects siRNAs from serum- and ribonuclease-mediated degradation, confirming the potential of the polyplex for in vivo delivery. We demonstrated that PgP/siMGMT polyplexes mediate knockdown of MGMT protein as well as a significant ~56% and ~68% knockdown of MGMT mRNA in T98G GBM cells compared to cells treated with PgP complexed with non-targeting siRNA (siNT) at a 60:1 and 80:1 nitrogen:phosphate (N:P) ratio, respectively. Further, co-treatment of PgP/siMGMT polyplexes with TMZ or CCNU enhanced anticancer activity in T98G GBM cells compared to treatment with the PgP/siMGMT polyplex, TMZ, or CCNU alone. Future studies will determine efficacy of drug-loaded and siRNA-complexed PgP for combination therapy in vitro as well as using a xenograft GBM model for local delivery. Successful combinatorial drug and gene therapy using PgP may overcome drug resistance and improve therapeutic outcomes for patients with glioblastoma.

Dual peptide-mediated targeted delivery of bioactive siRNAs to oral cancer cells in vivo

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Vectors for glioblastoma gene therapy: viral & non-viral delivery strategies

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Glioblastoma multiforme is the most common and aggressive primary brain tumor. Even with aggressive treatment including surgical resection, radiation, and chemotherapy, patient outcomes remain poor, with five-year survival rates at only 10%. Barriers to treatment include inefficient drug delivery across the blood brain barrier and development of drug resistance. Because gliomas occur due to sequential acquisition of genetic alterations, gene therapy represents a promising alternative to overcome limitations of conventional therapy. Gene or nucleic acid carriers must be used to deliver these therapies successfully into tumor tissue and have been extensively studied. Viral vectors have been evaluated in clinical trials for glioblastoma gene therapy but have not achieved FDA approval due to issues with viral delivery, inefficient tumor penetration, and limited efficacy. Non-viral vectors have been explored for delivery of glioma gene therapy and have shown promise as gene vectors for glioma treatment in preclinical studies and a few non-polymeric vectors have entered clinical trials. In this review, delivery systems including viral, non-polymeric, and polymeric vectors that have been used in glioblastoma multiforme (GBM) gene therapy are discussed. Additionally, advances in glioblastoma gene therapy using viral and non-polymeric vectors in clinical trials and emerging polymeric vectors for glioma gene therapy are discussed.

Polymersomes for therapeutic delivery of protein and nucleic acid macromolecules: From design to therapeutic applications

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Macromolecule-based therapeutic agents, particularly proteins, antigens, monoclonal antibodies, transcription factors, nucleic acids, and gene editing enzymes, have the potential to offer cures for previously untreatable diseases. However, they present an enormous delivery challenge due to poor absorption and rapid metabolism in the body. Polymersomes have tremendous potential in delivering these agents to their desired intracellular location due to increased circulation times, decreased macromolecule degradation, and decreased immune responses. In this Review, we highlight the key factors in design, development, and improved performance of these vesicles for macromolecular delivery. The recent progress made toward preclinical application of these vesicles for protein and gene delivery is also covered.

Classifying changes in LN-18 glial cell morphology: a supervised machine learning approach to analyzing cell microscopy data via FIJI and WEKA

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In cell-based research, the process of visually monitoring cells generates large image datasets that need to be evaluated for quantifiable information in order to track the effectiveness of treatments in vitro. With the traditional, end-point assay-based approach being error-prone, and existing computational approaches being complex, we tested existing machine learning frameworks to find methods that are relatively simple, yet powerful enough to accomplish the goal of analyzing cell microscopy data. This paper details the machine learning pipeline for pixel-based classification and object-based classification. Furthermore, it compares the performances of three classifiers. The classifiers evaluated were the fast-random forest (RF), the sequential minimal optimization (SMO), and the Bayesian network (BN). Images were first preprocessed using smoothing and contrast methods found in FIJI. For pixel-based classification, the preprocessed images were fed into the Trainable Waikato Segmentation (TWS). For object-based classification, training and classification were conducted within the Waikato Environment for Knowledge Analysis (WEKA) interface. All classifiers’ performance was evaluated using the WEKA experimental explorer. In terms of performance, the BN had the lowest classification accuracy for both the pixel-based and object-based model. The object-based SMO classifier had the best performance with the lowest mean absolute error of 0.05. The TWS and WEKA interface allows users to easily create and train classifiers for image analysis. However, for analyzing large image datasets, they are not ideal.

Self-assembling peptide hydrogel for delivery of therapeutically active temozolomide in glioblastoma treatment

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Traditional treatment methods for glioblastoma multiforme (GBM) including resection, radiation, and chemotherapy have been largely unsuccessful, with a current 5-year survival rate of 5.6%. In this project we examine the potential of nanosized self-assembling peptide hydrogels to locally deliver and convert temozolomide (TMZ), an FDA-approved pH-sensitive prodrug, for GBM treatment. The peptide hydrogel is designed to load TMZ into the hydrophobic regions of the hydrogels, and during hydrogel degradation in vivo, convert TMZ into its active form. Hydrogel characterization, drug loading and release cellular uptake, and viability are examined to determine the in vitro efficacy of this delivery method. A combination of dynamic light scattering (DLS), scanning electron microscopy (SEM), and circular dichroism (CD) are used to characterize size and structure of the hydrogels. High performance liquid chromatography and microcentrifuge dialysis are used to quantify drug loading and release from the hydrogels. Flow cytometry, fluorescent imaging, and cell viability assays are used to determine uptake and anti-cancer effects of the drug-loaded hydrogels on glioblastoma cells.

Our results show high drug loading efficiency and uptake in drug-resistant T98G and non-resistant LN-18 glioblastoma cell lines using several of our tunable peptide formulations. CD has shown that all peptide formulations form mostly beta-sheet and random structures during self-assembly. SEM and DLS show that certain formulations are fairly consistent in size and structure; we are focused on refining preparation methods to further improve uniform size and structure for these formulations and those that exhibit promising drug loading and uptake. Using a pH-sensitive dye, we have shown that as the peptides degrade, the degradation products cause the local pH to become basic, confirming that more TMZ will convert more quickly as the hydrogels degrade. Preliminary cell viability studies have shown promising results for anti-cancer effects of the drug-loaded hydrogels. Future studies for the project will include further cell viability studies to confirm preliminary results with the most promising peptide formulations once uniform self-assembly has been achieved. Finally, this project will culminate in an in vivo study to confirm the overall anti-cancer effect of the drug-loaded peptide hydrogels in a tumor model of GBM.

Acknowledgements: This research was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740.

A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro

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Introduction: Ovarian cancer recurrence and survival rates have remained stagnant over time with current therapeutics displaying low efficacy and often rendered ineffective by degradation. This work presents novel fusogenic peptides that will electrostatically complex with siRNAs and cause a pH-responsive insertion into the endosomal membrane, resulting in destabilization and breakdown allowing for release of siRNAs into the cytoplasm of ovarian cancer cells. Delivery of bioactive siRNAs targeting the CSNK2A1 gene in ovarian cancer cells is hypothesized to reduce cell viability, migration, and invasion.

Experimental Procedures: Peptide formulations were complexed with non-targeting siRNAs (siNT) and CSNK2A1 siRNAs (siCSNK2A1) at various N:P ratios for all procedures. Analysis of complex formation was completed through gel-shift assays and dynamic light scattering. After characterization of the complexes, each formulation without siRNA was delivered to OVCAR-3 and CAOV-3 cells for cytotoxicity analysis measured via MTS assay. Observation of cellular uptake and endosomal escape was completed through fluorescent imaging after delivering each peptide formulation complexed with Cy3-labeled siNT (Cy3-siNT) and performing early endosome antigen-1 staining. To confirm bioactivity of targeted siRNAs, complexes with siCSNK2A1 were delivered and gene silencing was analyzed via western blotting.

Results: Complexation of siRNAs with each formulation was achieved at N:P ratios of 40:1 and greater. Each of the three peptide formulations were able to form uniform nanocomplexes, at 195 nm, 111 nm, and 59 nm with 8.25 mV, -14.56 mV, and -18.42 mV surface charges respectively, and did not display vehicle cytotoxicity at an 80:1 ratio. Each peptide also mediated siRNA uptake into ovarian cancer cells, with fluorescent siRNA signal increasing with increasing N:P ratios. Of note, we observed that Cy3-siRNA appeared separate from the endosome, indicative of endosomal escape, which was seen in several peptide formulations with both CAOV-3 and OVCAR-3 cell lines. Confirmation of bioactive delivery of siCSNK2A1 is shown through western blot imaging, demonstrating knockdown of CSNK2A1 in comparison to cells treated with siNT alone or left untreated.

Conclusion: Our novel fusogenic peptides have shown the ability to complex with and deliver siRNAs into ovarian cancer cells. Each peptide mediates endosomal escape activity, with varying degrees of effectiveness, and silencing of CSNK2A1 as measured by western blot. Further study into downstream effects of CSNK2A1 is ongoing. Future work includes optimization of a single fusogenic sequence for optimal delivery of siRNAs and development of a novel targeting peptide to increase cancer targeting and uptake efficiency.

Acknowledgements: This research was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740.

Controlling Media, Controlling Access: The Role of Sport Media on Black Women’s Sport Participation

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The purpose of this paper is to provide a critical argument as to how Black females’ historic and contemporary controlling images in the media contribute to their marginalized participation in sport. According to Collins (2000), controlling images are socially constructed characterizations that emerged out of the slave era to maintain the marginalization of Black women in society based on their race, gender, and social class. As a result, Black sportswomen have faced the most (in)visible journey and are marred by their omission and/or image representation in media. Employing intersectionality theory, this paper explicates how sports media hinders Black females’ access and opportunity in sport at the structural, political, and representational levels.

Critical Race Theory: Black Athletic Sporting Experiences in the United States

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Although elated from the symbolic empowerment that two-term elected president, Barack Obama, provided, substantive changes in improving racial relations and equality lack considerably. Upon his initial election, the expectation was hopeful. Yet at the close of a chapter in US history in 2016 during the sunset of this nation’s first Black president, many Blacks are left wanting. According to late Manning Marable, the celebratory outlook we had during his first election lingered until his second term, till the increased racial tension and violence against the Blacks, specifically in the USA, caused us to question the significance of having a Black face in high place.

Women Coaches of Color: Examining the Effects of Intersectionality

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