Members of the Reineke group are presenting in the Microstructured Polymers Program Review and the Poster Competition at the Industrial Partnership for Research in Interfacial and Materials Engineering (IPrime) Annual Meeting in Keller Hall.
IPrime focuses on creating opportunities for professionals in industry to collaborate with students and researchers at the University of Minnesota. This exchange provides a productive environment for addressing key areas in interfacial and materials science.
Click on the title of each talk below to learn more about it!
[learn_more caption=”Hemocompatibility of Colloidal Glycopolymer-DNA Complexes”]Haley Phillips, Zachary Tolstyka, Bryan Hall, Perry Hackett, Theresa Reineke
Countless gene therapy vectors have been designed from viruses, lipids, and polymers to package, protect, and deliver genetic material into cells. A series of novel cationic polymers, (poly(2-methylacrylamido glucopyranose)-block-poly(2-methylaminoethyl-methacrylate) (pMAG-b-MAEMA), was designed to have superior biocompatibility and circulatory stability compared to other gene delivery vehicles. The purpose of this study was to quantify the biodistribution and hemocompatibility of these glycopolymers when complexed with plasmid DNA, forming polyplexes. To measure blood compatibility, blood was exposed to different polyplex formulations and blood function was observed. A hemolysis assay determined that the polymer vectors did not lyse red blood cells. Coagulation time was also not affected except at higher N/P ratios of 15. Analysis of the DNA cargo and fluorescently tagged polymers both showed widespread distribution in the spleen, heart, liver, lungs, and kidneys following mouse tail intravenous injection, suggesting that the polyplexes are colloidally stable during circulation.[/learn_more]
[learn_more caption=”N-Acetylgalactosamine Block-co-Polycations for Liver-Targeted Plasmid DNA Delivery”]Yogesh Dhande, Bharat Wagh, Bryan Hall, Dustin Sprouse, Perry Hackett, Theresa Reineke
Delivery vehicles targeted to specific tissues are of great interest to the field of nucleic acid therapeutics. The liver is common target for treatment of several diseases such as hemophilia and many lysosomal storage diseases. We synthesized a series of block glycopolycations derived from N-acetylgalactosamine (GNA) to target asialoglycoprotein receptors on liver hepatocytes. The diblock polymers were composed of a GNA-derived block of fixed length and cationic 2-aminoethylmethacrylamide (AEMA) blocks of varying lengths. The polymers were studied for their binding to plasmid DNA (pDNA), polymer-pDNA complex (polyplex) formation and stability, cytotoxicity, and cellular uptake in vitro and in vivo. GNA-derived polymers show cell type-dependent gene expression in vitro, with higher protein expression in HepG2 (human hepatocellular carcinoma) as compared to HeLa (human cervix adenocarcinoma) cells. In vivo studies show higher accumulation of pDNA and GNA-derived polymers in the mouse liver compared with the glucose-derived non-targeted control.[/learn_more]
[learn_more caption=”High-throughput Polymer Screening for Oral Administration: From Discovery to Design”]Jeffrey M Ting, Swapnil R. Tale, Lakmini Widanapathirana, Zachary P. Tolstyka, Seamus D. Jones, Li Guo, Steven J. Guillaudeu, Frank S. Bates, Theresa M. Reineke
Spray-dried dispersions are fascinating polymer–drug mixtures that exploit the amorphous state of a hydrophobic drug to dramatically elevate its aqueous solubility above equilibrium for oral administration. However, these materials have limited fundamental understanding of what polymeric attributes prolong drug supersaturation and combat crystallization in solution. We present an automated protocol to combinatorially explore a vast architectural parameter space for five polymer platforms and elucidate how specific microstructures dictate drug dissolution. Over 60 reversible addition-fragmentation chain transfer (RAFT) polymerizations were conducted in three high-throughput experiments, varying constituent properties such as polymer amphilicity, thermoresponsive phase behavior, and hydrogen bonding capability. The intermolecular interactions of well–defined lead compounds with model drug phenytoin were screened and studied in vitro. This study reports the first results of a polymer–focused screening assay that successfully solubilized an otherwise-intractable drug for oral delivery.[/learn_more]
[learn_more caption=”Isosorbide-based polymethacrylates”]James Gallagher, Marc Hillmyer, Theresa Reineke
The glucose derivative isosorbide has recently become a commercially viable building block for biobased polymers. This work focuses on synthesizing a new monovinyl isosorbide derivative and incorporation thereof into advanced materials such as block copolymers. Acetylated methacrylic isosorbide (AMI) was synthesized in two steps employing scandium (III) triflate as a remarkably efficacious catalyst for the tandem esterification of isosorbide with acetic anhydride and methacrylic anhydride. Poly(AMI) [PAMI] prepared by radical polymerization of a mixture of AMI regioisomers was found to have a high glass transition temperature (Tg ≈ 130 °C) and good thermal stability. Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization of AMI yielded PAMI-CTA samples with controlled molar masses and narrow molar mass distributions. Subsequent chain extension of PAMI-CTA with n-butyl acrylate gave a series of PAMI-b-PnBA block copolymers. All samples of PAMI-b-PnBA exhibited two well-separated Tg values at approximately –45 and 120 °C, indicative of microphase separation.[/learn_more]
[learn_more caption=”Carbohydrate-Based Polycations as Gene Delivery Vectors in Hard to Transfect Cells “]William Boyle, Theresa M. Reineke
Gene and cell therapies are promising methods for treatment of many acquired and inherited diseases. However, the efficient treatment of a variety of diseases is limited by the ability to efficiently deliver DNA or RNA to the desired cells or tissue. Cationic polymers can form complexes, termed polyplexes, with negatively charged nucleic acids, and help decrease the hydrodynamic radius of the cargo, protect the cargo from degredation, and increase cellular uptake and nuclear delivery leading to exogenous gene expression. This process is known as transfection. Dystrophic Epidermolysis Bullosa (DEB) is a rare inherited skin condition caused by a defect in COL7A1 gene, which codes for type VII collagen. It is characterized by severe, widespread blistering, and can frequently lead to squamous cell carcinoma, and many patients do not reach adulthood. With the exception of hematopoietic cell transplantation, no efficient treatment exists, and physicians are largely limited to palliative care. Treatment of DEB with gene therapy will require efficient gene delivery to primary human cells, such as keratinocytes or fibroblasts. However, primary skin fibroblasts are known to be difficult to transfect compared to other more commonly studied immortalized cell lines. In this work, trehalose-oligoethyleneamine click polymers were used as to successfully transfect primary human fibroblasts with plasmid DNA In Vitro compared to controls jetPEITM and GlycofectTM. The presence of heparin in the transfection medium proved to be vital for optimal transfection. Heparin is a highly sulfated anionic polysaccharide chiefly used in biomedical applications as a blood thinner. Due to its highly negative charge at physiological pH, it usually inhibits transfection by decomplexing the negatively charged DNA from the cationic polymer. However, polymers developed in the Reineke group exhibit increased transfection efficiency in a variety of cell lines when treated with low concentrations of heparin. This heparin-induced increase in transfection efficiency is driven partially by increased uptake, however, the presence of heparin may also play a role in nuclear localization as well. Further experiments were performed to examine how plasmid size affects the transfection of primary human fibroblasts. Polyplexes were formed with 3 different sized GFP-expressing plasmids, and characterized by DLS and zeta potential. DNA binding was also studied by gel electrophoresis a Picogreen dsDNA quantitation assay. Polyplex uptake and transfection efficiency were examined by delivery of Cy-5-labled pDNA and a GFP-expressing plasmid respectively, and quantified by FACS. Toxicity was measured by MTT assay.[/learn_more]
[learn_more caption=”Pairing Glycopolymers and Surface-Enhanced Raman Spectroscopy (SERS) for the Detection of Toxic Lectins”]Victoria Szlag, Matthew Styles, Antonio Campos, Dustin Sprouse, Bharat Wagh, Theresa Reineke, Christy Haynes
This work explores a novel means to detect toxic carbohydrate-binding proteins (lectins) by pairing glycopolymers, synthetic polymers with pendant saccharides, and surface-enhanced Raman spectroscopy (SERS). The goal is to create a method of detection that is sensitive to μg/mL quantities, but is not prohibitively slow or expensive. SERS on gold-film-over-nanospheres (AuFONS) is a quick, sensitive technique that can be used to study molecules in low abundance. However, to employ this approach, target molecules must remain within nanometers of the substrate’s surface for the duration of the measurement. In this work, surface functionalization of AuFONS with glycopolymers provides a simple, tunable means to capture target lectins. Glycopolymers provide saccharide multivalency for enhanced lectin binding and, if synthesized by a controlled technique, access to short chain lengths for near-surface lectin capture. In the development of this novel detection platform, the affinity between ricin B chain, the binding portion of a potent ribosome-inactivating protein, and glycopolymers was explored. Ongoing research focuses on how this affinity impacts different aspects of the platform, such as its sensitivity, selectivity, reversibility, and practical applicability.[/learn_more]
[learn_more caption=”Microfluidics for controlled delivery of nucleic acids to cells”]Pranav Agrawal, Nilesh Ingle, Kevin Dorfman, Theresa M. Reineke
The study focuses on a high throughput and controlled delivery of nucleic acids to a single cells by means of a microfluidic device. The device is fabricated in polydimethylsiloxane (PDMS) using photolithography techniques, and is employed to control the interaction of cells and nucleic acid-containing nanoparticles (polyplex and lipoplex). Here, we present our efforts to further optimize the design and key working parameters of the device: (a) effect of flow rate, (b) effect of off-device incubation time, (c) effect of cell type, and (d) effect of nanoparticle type. Nanoparticles were formulated by complexing Glycofect polymer (20 N/P) with plasmid DNA (0.02 μg/μL) and FugeneHD with plasmid DNA (3:1 ratio). HeLa and HDFn cells in suspension are treated with nanoparticles to observe: (1) cellular uptake and (2) GFP gene expression. We show that a transient transfection time of microseconds leads to >90% cell surface attachment of nanoparticles, using a Cy5 assay. Subsequently, these nanoparticles are internalized via endocytosis and the particles deliver nucleic acids to the nucleus. Overall success of all transport steps has been quantified using a GFP assay.[/learn_more]
