Millicent O. Sullivan

Stimuli-responsive copolymer solution and surface assemblies for biomedical applications

Stimuli-responsive polymeric materials is one of the fastest growing fields of the 21st century, with the annual number of papers published more than quadrupling in the last ten years. The responsiveness of polymer solution assemblies and surfaces to biological stimuli (e.g. pH, reduction-oxidation, enzymes, glucose) and externally applied triggers (e.g. temperature, light, solvent quality) shows particular promise for various biomedical applications including drug delivery, tissue engineering, medical diagnostics, and bioseparations. Furthermore, the integration of copolymer architectures into stimuli-responsive materials design enables exquisite control over the locations of responsive sites within self-assembled nanostructures. The combination of new synthesis techniques and well-defined copolymer self-assembly has facilitated substantial developments in stimuli-responsive materials in recent years. In this tutorial review, we discuss several methods that have been employed to synthesize self-assembling and stimuli-responsive copolymers for biomedical applications, and we identify common themes in the response mechanisms among the targeted stimuli. Additionally, we highlight parallels between the chemistries used for generating solution assemblies and those employed for creating copolymer surfaces.

Polyplexes Traffic through Caveolae to the Golgi and Endoplasmic Reticulum en Route to the Nucleus

The cellular machinery involved in the internalization of nonviral gene carriers and their subsequent trafficking to the nucleus directly impacts their therapeutic efficiency. Hence, identifying key endocytic pathways and organelles that contribute to the successful transfer of polyplexes to the nucleus generates new opportunities for improving carrier design. Previously, we showed that histone H3 tail peptides encoding a sequence known to participate in chromatin activation exhibit synergistic gene delivery activity with poly(ethylenimine) (PEI). Polyplexes containing H3 and PEI exhibited a reduced dependence on endocytic pathways that trafficked to lysosomes, and had enhanced sensitivity to an inhibitor associated with retrograde trafficking through the Golgi apparatus. Thus, we sought to determine whether caveolar uptake and transport through the Golgi and/or endoplasmic reticulum (ER) preceded nuclear delivery. By the use of a panel of chemical endocytic inhibitors, we determined that H3 polyplexes utilized caveolar pathways to a greater degree than PEI polyplexes. Caveolae-mediated endocytosis was found to be a productive route for gene expression by the H3/PEI-pDNA polyplexes, consistent with previous studies of polymer-mediated gene delivery. Additionally, the polyplexes substantially colocalized within the ER after only 5 min of incubation, and utilized retrograde Golgi-to-ER pathways at levels similar to pathogens known to traffic by these routes during infection. The results of this study have expanded our understanding of how caveolar polyplexes are trafficked to cell nuclei, and provide new evidence for the role of Golgi-ER pathways in transfection. These findings suggest new design criteria and opportunities to stragetically target nonviral gene delivery vehicles.

Histone H3 Tail Peptides and Poly(ethylenimine) Have Synergistic Effects for Gene Delivery

This goal of this work was to explore histone H3 tail peptides containing transcriptionally activating modifications for their potential as gene delivery materials. We have found that these H3 tail peptides, in combination with the cationic polymer poly(ethylenimine) (PEI), can effectively bind and protect plasmid DNA. The H3/PEI hybrid polyplexes were found to transfect a substantially larger number of CHO-K1 cells in vitro compared to both polyplexes that were formed with only the H3 peptides and those that were formed with only PEI at the same total charge ratio; however, transfection was similarly high for polyplexes both with and without transcriptionally activating modifications. Transfections with the endolysosomal inhibitors chloroquine and bafilomycin A1 indicated that the H3/PEI hybrid polyplexes exhibited slower uptake and a reduced dependence on endocytic pathways that trafficked to the lysosome, indicating a potentially enhanced reliance on caveolar uptake for efficient gene transfer. In addition, whereas PEI polyplexes typically exhibit a cytotoxic effect, the H3/PEI hybrid polyplexes did not compromise cell viability. In total, the current studies provide new evidence for the potential role for histone-based materials as effective gene transfer agents, and support for the importance of subcellular trafficking for nonviral gene delivery.

Association of diabetic foot ulcer and death in a population-based cohort from the United Kingdom

The presence of diabetic foot ulcers is strongly associated with an increased risk of death. In this study, we investigate whether the effects of diabetes‐associated complications can explain the apparent relationship between diabetic foot ulcers and death.

Size evolution of highly amphiphilic macromolecular solution assemblies via a distinct bimodal pathway

The solution self-assembly of macromolecular amphiphiles offers an efficient, bottom-up strategy for producing well--defined nanocarriers, with applications ranging from drug delivery to nanoreactors. Typically, the generation of uniform nanocarrier architecturesis controlled by processing methods that rely upon cosolvent mixtures. These preparation strategies hinge on the assumption that macromolecular solution nanostructures are kinetically stable following transfer from an organic/aqueous cosolvent into aqueous solution. Herein we demonstrate that unequivocal step-change shifts in micelle populations occur over several weeks following transfer into a highly selective solvent. The unexpected micelle growth evolves through a distinct bimodal distribution separated by multiple fusion events and critically depends on solution agitation. Notably, these results underscore fundamental similarities between assembly processes in amphiphilic polymer, small molecule, and protein systems. Moreover, the non-equilibrium micelle size increase can have a major impact on the assumed stability of solution assemblies, for which performance is dictated by nanocarrier size and structure.

Structural changes in block copolymer micelles induced by cosolvent mixtures.

We investigated the influence of tetrahydrofuran (THF) addition on the structure of poly(1,2-butadiene-b-ethylene oxide) [PB-PEO] micelles in aqueous solution. Our studies showed that while the micelles remained starlike, the micelle core-corona interfacial tension and micelle size decreased upon THF addition. The detailed effects of the reduction in interfacial tension were probed using contrast variations in small angle neutron scattering (SANS) experiments. At low THF contents (high interfacial tensions), the SANS data were fit to a micelle form factor that incorporated a radial density distribution of corona chains to account for the starlike micelle profile. However, at higher THF contents (low interfacial tensions), the presence of free chains in solution affected the scattering at high q and required the implementation of a linear combination of micelle and Gaussian coil form factors. These SANS data fits indicated that the reduction in interfacial tension led to broadening of the core-corona interface, which increased the PB chain solvent accessibility at intermediate THF solvent fractions. We also noted that the micelle cores swelled with increasing THF addition, suggesting that previous assumptions of the micelle core solvent content in cosolvent mixtures may not be accurate. Control over the size, corona thickness, and extent of solvent accessible PB in these micelles can be a powerful tool in the development of targeting delivery vehicles.

CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside‐in signaling through integrin αIIbβ3

Summary.  Background: Agonist‐induced inside‐out signaling activates platelet integrin αIIbβ3, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside‐in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside‐in signaling through αIIbβ3 regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating αIIbβ3 function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl3‐induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside‐out signaling appears normal as ADP‐, collagen‐ and PAR4 peptide‐induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside‐in signaling‐dependent tyrosine phosphorylation of the integrin β3 subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.

Light-Mediated Activation of siRNA Release in Diblock Copolymer Assemblies for Controlled Gene Silencing

Controllable release is particularly important for the delivery of small interfering RNA (siRNA), as siRNAs have a high susceptibility to enzymatic degradation if release is premature, yet lack silencing activity if they remain inaccessible within the cytoplasm. To overcome these hurdles, novel and tailorable mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) diblock copolymers containing light-sensitive o-nitrobenzyl moieties and pendant amines are employed to provide both efficient siRNA binding, via electrostatic and hydrophobic interactions, as well as triggered charge reversal and nucleic acid release. In particular, siRNA/mPEG-b-P(APNBMA)23.6 polyplexes show minimal aggregation in physiological salt and serum, and enhanced resistance to polyanion-induced unpackaging compared to polyethylenimine preparations. Cellular delivery of siRNA/mPEG-b-P(APNBMA)23.6 polyplexes reveals greater than 80% cellular transfection, as well as rapid and widespread cytoplasmic distribution. Additionally, UV irradiation indicates ≈70% reduction in targeted gene expression following siRNA/mPEG-b-P(APNBMA)23.6 polyplex treatment, as compared to 0% reduction in polyplex-treated cells without UV irradiation, and only ≈30% reduction for Lipofectamine-treated cells. The results here highlight the potential of these light-sensitive copolymers with a well-defined on/off switch for applications including cellular patterning for guided cell growth and extension, and cellular microarrays for exploring protein and drug interactions that require enhanced spatiotemporal control of gene activation.

Requirements for the nuclear entry of polyplexes and nanoparticles during mitosis.

Nonviral gene delivery has a limited efficacy partly as a result of poor nuclear delivery, yet an understanding of the mechanisms of nuclear entry is limited. The present study aimed to test the common hypothesis that most nonviral vehicles enter the nucleus during cell division.

Structural and functional consequences of poly(ethylene glycol) inclusion on DNA condensation for gene delivery.

Polycationic polymers have been used to condense therapeutic DNA into submicron particles, offering protection from shear‐induced or enzymatic degradation. However, the spontaneous nature of this self‐assembly process gives rise to the formation of multimolecular aggregates, resulting in significant polyplex heterogeneity. Additionally, cytotoxicity issues and serum instability have limited the in vivo efficacy of such systems. One way these issues can be addressed is through the inclusion of poly(ethylene glycol) (PEG). PEG has known steric effects that inhibit polyplex self‐aggregation. A variety of PEGylated gene delivery formulations have been previously pursued in an effort to take advantage of this materials benefits. Because of such interest, our aim was to further explore the consequences of PEG inclusion on the structure and activity of gene delivery vehicle formulations. We explored the complexation of plasmid DNA with varying ratios of a PEGylated trilysine peptide (PEG‐K3) and 25‐kDa polyethylenimine (PEI). Atomic force and scanning electron microscopy were utilized to assess the polyplex size and shape and revealed that a critical threshold of PEG was necessary to promote the formation of homogeneous polyplexes. Flow cytometry and fluorescence microscopy analyses suggested that the presence of PEG inhibited transfection efficiency as a consequence of changes in intracellular trafficking and promoted an increased reliance on energy‐independent mechanisms of cellular uptake. These studies provide new information on the role of PEG in delivery vehicle design and lay the foundation for future work aimed at elucidating the details of the intracellular transport of PEGylated polyplexes. Microsc. Res. Tech. 73:866–877, 2010.