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=”Effects of Polycation Structure on DNA-polycation Complexation”]Seyoung Jung, Theresa M. Reineke, Timothy P. Lodge
Deoxyribonucleic acids (DNA), as polyanions, can spontaneously bind with polycations to form polyelectrolyte complexes. When the polycation is a diblock copolymer with one cationic block and one uncharged hydrophilic block, the polyelectrolyte complexes formed with plasmid DNA (pDNA) are often colloidally stable, and show great promise in the field of polymeric gene therapy. While the resulting properties—size, gene delivery efficiency, and toxicity to biological systems—of the complexes have been studied for numerous cationic diblocks, the fundamentals of the pDNA-diblock binding process have not been extensively investigated. Herein, we report how the length of the cationic block influences the pDNA-diblock interactions. Poly(2-deoxy-2-methacrylamido glucopyranose)-b-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA) is used as the model polycation system. Two PMAG-b-PAEMA copolymers with similar PMAG block lengths but distinct PAEMA block lengths, and a PAEMA homopolymer control, are utilized. We show that the enthalpy change from pDNA-diblock interactions is dependent on the cationic diblock composition, and is closely associated with both the binding strength and the pDNA tertiary structure. In addition, the evolution of complex size and dispersity during the titrations of both pDNA and linear DNA with polycations provide insight into the possible structures that DNA-polycation complexes can adopt.[/learn_more]
[learn_more caption=”Glucose-based Block Copolymers as Adhesives and Thermoplastic Elastomers”]Mammad Nasiri, Theresa Reineke
This work proposes a direct modification of glucose, an abundant and inexpensive sugar molecule, to produce new sustainable and functional polymers. Glucose-6-acrylate-1,2,3,4-tetraacetate (GATA) has been synthesized and shown to provide a useful glassy component for developing an innovative family of elastomeric and adhesive materials. A series of diblock and triblock copolymers of GATA and n-butyl acrylate (n-BA) were created via Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. Use of 3,5-Bis(2-dodecylthiocarbonothioylthio-1oxopropoxy)benzoic acid (BTCBA) as the chain transfer agent (CTA) provided a more efficient route to copolymerize GATA and n-BA. Using BTCBA, Poly(GATA)-b-poly(nBA)-b-poly(GATA) triblock copolymers containing 12-25 wt % GATA, with very narrow molar mass distributions (Ɖ ≤ 1.08), were prepared. The synthesized triblock copolymers demonstrated moderate mechanical properties with excellent thermomechanical and adhesion properties. The work herein introduces a new family of glucose-based ABA-type copolymers and demonstrates functionality of a glucose-based feedstock for developing green polymeric materials.[/learn_more]
[learn_more caption=”Building Better Protein Sensors via Surface Functionalization with Glycopolymers”]Victoria Szlag, Matthew Styles, Lindsey Madison, Antonio Campos, Bharat Wagh, Dustin Sprouse, George Shatz, Christy Haynes, Theresa Reineke
Lectins, proteins that bind carbohydrates, can be used as indicators in food safety monitoring due to their ubiquitous biological presence. From bioterror agents to proteins in food allergens, these lectins’ structure and their saccharide binding ability is well characterized but underutilized. To effectively use this monitoring strategy, a method of detection is needed that is sensitive to μg/mL quantities, but is not prohibitively slow or expensive. This work introduces a novel means of lectin detection by pairing synthetic polymers with pendant saccharides, glycopolymers, and surface-enhanced Raman spectroscopy (SERS). SERS is a quick, sensitive technique that can be used to detect the vibrational fingerprint of molecules in low abundance. To employ this approach, however, target molecules must remain within nanometers of the substrate’s surface for the duration of the measurement. In this work, surface functionalization of gold substrates 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 lectins and glycopolymers is 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=”Trehalose-Based Diblock Copolymers as Excipients for Enhancing Solubility of Poorly Water Soluble Drugs”]Anatolii Purchel, Swapnil Tale, Molly Dalsin, Theresa Reineke
Oral administration is the most preferable route of drug delivery, especially during prolonged therapy of chronic diseases. Unfortunately, many effective pharmaceuticals are poorly water-soluble, which leads to decreased bioavailability and shelf life. One of the ways to improve drug solubility and efficacy is to prepare an amorphous solid dispersion (ASD) with a polymer excipient. It is important that the polymer matrix of an ASD will stabilize the drug in the amorphous state and maintain its supersaturated concentration long enough in the dissolution media. Some of the commercial polymeric systems have shown a positive impact on drug dissolution, but most of them are difficult to characterize due to high polydispersity and system complexity. This makes it difficult to understand the structure property relationships and to quantify the effect of drug-polymer specific interactions. Also, most of the available excipients that improve dissolution of poorly water-soluble drugs tend to Hydrogen-bond to the drug while in solution, thus preventing its crystallization. Therefore, a series of block copolymers were synthesized with varied composition of H-bonding monomers including N-isopropylacrylamide, N,N-dimethylacrylamide, and 2-methacrylamidotrehalose using addition-fragmentation chain transfer (RAFT) polymerization. This family of diblock copolymers offers hydrophobic, hydrophilic, or H-bonding functionalities to serve as noncovalent sites for model drug Probucol binding. Role of each binding moiety as well as overall excipient performance was assessed using in-vitro dissolution testing.[/learn_more]
[learn_more caption=”Transfection of Primary Fibroblasts with Trehalose-Based Polymers as a Function of Plasmid Size”]William Boyle, Jakub Tolar, Theresa M. Reineke
Primary fibroblasts (PFBs) are an important target for improved gene delivery techniques. They are frequently used in the production of induced pluripotent stem cells (IPSCs). Additionally, gene corrected PFBs represent a potential cell therapy for the inherited skin disease epidermolysis bullosa. However, current gene delivery techniques are lacking in their ability to efficiently transfection PFBs. We have demonstrated the ability of trehalose-based polymers treated with heparin to efficiently transfect PFBs with a relatively small (4.7 kB) GFP reporter gene. However, efficient transfection was not achieved with a clinically relevant 10 kB plasmid in spite of a high rate of polyplex internalization. We hypothesized that this discrepancy was due to suboptimal nuclear delivery with the large plasmid. Increased gene expression with the larger plasmid was achieved by destabilizing the nuclear membrane during transfection using either dexamethasone or cell synchronization. Nuclear localization of polyplexes as a function of plasmid size was also quantified.[/learn_more]
[learn_more caption=”Glycoclusters Formed by RAFT Polymerization Show Enhanced Binding to Receptor Proteins”]Yogesh Dhande, Bharat Wagh, Zhe Tan, Theresa Reineke
[learn_more caption=”Fast, Efficient and Gentle Transfection of Human Adherent Cells in Suspension”]Pranav Agrawal, Nilesh P. Ingle, William S. Boyle, Emily Ward, Jakub Tolar, Kevin D Dorfman, Theresa M Reineke
The current strategy for delivering genetic materials to adherent cells using gene delivery vehicles (GDVs), involves incubating the GDV solution over a cultured cell monolayer, and its performance is limited by slow mass transport of GDVs to the cell surface. We demonstrate a novel suspension-based transfection method that provides efficient delivery of genetic materials to clinically relevant human cell types, such as induced pluripotent stem cells and fibroblasts, using lipid- and polymeric-based commercial transfection reagents (FugeneHD and Glycofect). Our method reduces the time required for transfection by one full day. The reason behind the success of our method is that suspension transfection leads to faster transport of GDVs to the cell surface. This resulted in maximum GDVs attachment to cell surface within 15 minute of transfection time, with atleast 2-fold higher binding efficiency of GDVs as compared to the plating method. Our method is also compatible with common techniques to improve gene delivery, such as cell cycle synchronization (6-fold increase in gene expression compared to control), facilitating easy adaptability in existing setups. Furthermore, to preserve cell physiology during transfection in suspension, we designed a microfluidic version of our method where the transfection takes place in a flow. In this device, the GDV and cell contact time is below one minute, and we achieved high binding of GDVs to cells (>90% GDV-bound live cells) and significant gene expression in several human cell types with minimal effect due to the device flow rates. The fast, efficient and generally applicable transfection methodologies that we will present can be used for rapid screening of different delivery systems and have significant potential for scale-up and high-throughput cell therapy applications.[/learn_more]
[learn_more caption=”Assembly and Structural Evolution of Micelleplexes”]Yaming Jiang, Dustin Sprouse, Jennifer Laaser, Theresa Reineke, Timothy Lodge
Cationic micelles complex with DNA to form micelleplexes, which are attractive vehicles for gene delivery. Compared to linear cationic polymers, cationic micelles hold the potential to co-deliver drug with gene payload and to offer more control on the structure of the complexes. We investigate the formation and structural evolution of micelleplexes in buffered solutions. The micelles are composed of poly((2-dimethylamino)ethyl methacrylate-block-poly(n-butyl methacrylate). The formation of the micelleplexes is monitored via turbidimetric titration and the size and structure of micelleplexes are assessed by dynamic light scattering and cryo-TEM. With DNA oligomers, solutions of the complexes are homogeneous until near the charge neutral point, at which point the complexes precipitate. With plasmid DNA, more than a stoichiometric amount of DNA is needed to reach the inhomogeneous region, which suggests that binding is partially inhibited. This inhibition is not fully relieved when the plasmid DNA is linearized, suggesting that the stiffness of the DNA is the main source of the inhibition. With micelles in excess, the micelleplexes formed at low ionic strength exhibit bimodal size distributions and remain stable in solution. With DNA in excess, soluble micelleplexes aggregate over time and precipitate. We explain the structural evolution of the micelleplexes as an interplay between kinetic trapping and thermodynamic equilibrium, and compare the results for DNA with those for a flexible polyanion.[/learn_more]
[learn_more caption=”Sugar-derived Hardeners for New Strong Epoxy Resins”]Quanxuan Zhang, Monika Molenda, Theresa M. Reineke
Epoxy resin thermosetting polymers exhibit excellent mechanical strength, thermal and chemical resistance, and strong adhesion to various substrates after cure. Unfortunately, conventional epoxy resins are both unsustainable and frequently contain environmentally/biologically harmful reagents. Carbohydrates have great potential for creating new epoxy polymers due to their sustainability, and the presence of rigid ring structure and multiple reactive sites that imparts a highly crosslinked polymer network. To create sustainably sourced epoxy resins, we synthesized new trehalose and β-cyclodextrin based carboxylic acid hardeners for epoxy resins and examined the thermal, mechanical and adhesive properties of the resulting resins. Trehalose and β-cyclodextrin based hardeners formed homogeneous mixtures with trimethylolpropane triglycidyl ether. The cured resins are thermally stable (Td > 310 °C) and display high Young’s moduli of up to 1.4 and 1.8 GPa with mechanical strength of 45 and 64 MPa for trehalose and β-cyclodextrin based epoxy resins, respectively. The cured epoxy resins degraded in both acid and basic solutions, and exhibit excellent adhesion shear strength of 3600 and 2100 psi, respectively. These results imply that this class of novel hardeners could hold great promise for sustainable high performance epoxy resins.[/learn_more]
[learn_more caption=”Hemocompatibility and In Vivo Biodistribution of Colloidally Stable Glycopolymer-DNA Complexes”]Haley R. Phillips, Zachary P. Tolstyka, Bryan C. Hall, Perry B. Hackett, Theresa M. Reineke
Gene therapy holds great promise for the cure of monogenic diseases, however, off-target effects including random gene insertion as well as tissue non-specificity limit the application of genetic therapies. Nonviral transfection of plasmids can aid in avoiding random gene insertion, however it is not without its own limitations. Strategic polymer design can avoid nonviral delivery vehicle aggregation and contribute other desirable properties such as stable DNA loading and protection, low cytotoxicity, and high cellular uptake and gene expression. Gene delivery vehicles made of poly(methacrylamido glucopyranose-block-2-methylaminoethylmethacrylate) (P(MAG-b-MAEMT)) were previously shown to stabilize polyplexes in serum solutions and promoted significant gene expression in human cancer cell lines. We hypothesized that the hydrophilic P(MAG) block would also prevent interactions with blood protein and cells and avoid passive trapping in mononuclear phagocytic organs such as the liver, lungs, and spleen. A combination of hemocompatibility and biodistribution studies demonstrated the ability of P(MAG-b-MAEMT) to prevent undesired interactions with human blood and passive trapping in mouse organs.
Blood compatibility was assessed ex vivo by hemolysis, red blood cell morphology, complement activation, and coagulation analyses. In addition to maintaining a stable diameter in serum solution over time, P(MAG-b-MAEMT) polyplexes prevented blood cell lysis more effectively than PEG-b-MAEMT polyplexes. Qualitative cell morphology observations suggest that PEG-b-MAEMT polyplexes at both higher and lower N/P disrupt cell membrane integrity, whereas all P(MAG-b-MAEMT) polyplex formulations are safe to mix with human red blood cells. However, care must be taken when determining dose for in vivo work as all polyplex formulations, regardless of polymer type, activated the complement system via the alternative pathway. Higher polyplex N/P formulations for all polymers also delayed coagulation compared to normal human plasma. These ex vivo studies provided valuable information about polyplex-blood component interactions, and in vivo biodistribution studies demonstrated the ability of polyplexes to circulate throughout the body. Polymer and pDNA distribution were analyzed separately using fluorescent imaging and qPCR. Results suggest polyplexes remain intact during circulation and tissue uptake. P(MAG-b-MAEMT)-2 at N/P=15 polyplexes avoided accumulation in six major organs (shown by low, even levels of organ uptake), while P(MAG-b-MAEMT) N/P=5 and PEG-b-MAEMT N/P=5 polyplexes were captured by the liver and other mononuclear phagocytic organs. The biodistribution information of the polymer vehicles and the pDNA cargo, combined with the stability shown in serum solutions and low levels of blood interaction support P(MAG-b-MAEMT)-2 N/P=15 as a hemocompatible, stable, and stealthy gene delivery vehicle.[/learn_more]
