You-Yeon Won

Missing pieces in understanding the intracellular trafficking of polycation/DNA complexes.

In about 70% of over 1400 gene therapy clinical trials that have been conducted to date worldwide, genetically-modified viruses have been the carrier of choice for delivery of therapeutic genetic material [1]. While the viruses promise both high efficiency of transfer and great protection of the therapeutic genes [2], this approach also carries a risk of causing adverse (inflammatory or immune) reactions [3,4] or even cancer [5]. Non-viral systems, such as cationic lipids and synthetic polymers (in particular, polycations), have attracted the interest of a large number of researchers as safer alternatives [6]. In particular, polycations have become popular components of non-viral gene carriers because of the relative ease with which their chemical and physical properties can be engineered for specific applications. However, the polycation-based approach has been limited in its clinical application in large part due to the poor biological activities of synthetic polymers on both cellular and systemic levels. A major issue is the difficulty associated with target-cell-specific delivery of genetic materials in vivo [6,7]. However, even the basic problem of achieving a sufficient efficiency in the transportation of therapeutic genes across various intracellular barriers also remains one of the leading challenges in the development of superior polycation-based gene delivery systems. In this regard, even the most effective polycation gene carrier (e.g., linear polyethylenimine or PEI for short) remains 10 times less efficient [8] than its viral counterpart [9]. Since the first demonstration of polycation-medicated gene transfection in 1987 [10], many polycation materials (both new and off-the-shelf) have been explored for gene delivery applicationswith themost intensively studied example being the PEI polycation (reviewed in Refs. [11–16]). An obvious reason for the great attention devoted to PEI is that this polycation affords the highest levels of in vitro gene transfection. It is believed that the high gene transfection efficiency observed with PEI is attributable to its unique ability to simultaneously overcome several key barriers to intracellular trafficking of the DNA particles (e.g., escape from endosomes [17,18], protection of DNA from degradation by endonulceases [19], nuclear entry [17,19,20], DNA release and transcription [20]). Currently, however, the exact mechanisms of how PEI orchestrates the sequence of the intracellular processes required for effective expression of the transgene in the host cell, and the particular chemical/molecular attributes of PEI responsible for each event, remain largely unexplained, making it difficult to further improve the performances of the PEI-based carriers in other aspects of the delivery process. One recent example to improve the PEI-based delivery system is the incorporation of intracellularly degradable disulfide bonds in the backbone structure of the PEI molecule [21–24] to reduce the inherent cellular (and systemic) toxicity of the polycation [25–27]. While this modification improves the viability of the transfected cells, thereby enabling the use of the PEI chemistry at high molecular weight without causing cell death [22–24], this improvement accompanies an unwanted decrease in the overall gene transfection efficiency when the performances are compared at an identical PEI molecular weight [24]. Improved understanding of the polycation chemistry vs. performance mechanism relationships will provide useful insights to guide further (chemical and/or physical) modifications of this already useful polycation toward creating multipotent gene carriers that can accommodate all of the sophisticated functional requirements at various stages of the delivery process. In this article, we intend to identify and discuss several key areas which require further improvements in our molecular understanding of the cellular transport processes of polymer/DNA complexes (“polyplexes”).

Rubbery Graft Copolymer Electrolytes for Solid-State, Thin-Film Lithium Batteries

Graft copolymer electrolytes (GCEs) of poly[(oxyethylene)9 methacrylate]-g-poly(dimethyl siloxane) (POEM-g-PDMS) (70:30) have been synthesized by simple free radical polymerization using a macromonomer route. Differentialscanning calorimetry, transmission electron microscopy, and small angle neutron scattering confirmed the material to be microphase-separated with a domain periodicity of ∼25 nm. Over the temperature range 290 200 cycles) at a discharge rate of 2/3 C and could be cycled (charged and discharged) at subambient temperature (0°C).

A Discussion of the pH-Dependent Protonation Behaviors of Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and Poly(ethylenimine-ran-2-ethyl-2-oxazoline) (P(EI-r-EOz))

In this article, we present results of our experimental and atomistic simulation studies of the pH-dependent protonation behaviors of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and poly(ethylenimine) (PEI). The potentiometric titration profiles of the PDMAEMA polymer and its unpolymerized monomer (i.e., DMAEMA) were measured under identical conditions in order to study the influence of the covalent linkage of the amine groups on their protonation behavior. The titration curves of poly(ethylenimine-ran-2-ethyl-2-oxazoline) (P(EI-r-EOz)) random copolymers with varying monomer composition were measured in order to study the effect of the spacing between the EI monomers on the protonation behavior of the P(EI-r-EOz) copolymer. The results of these two sets of measurements demonstrate that the connectivity and tight spacing between amine groups in a polyamine chain causes the retardation of the protonation of the amine groups relative to the same compounds in their isolated state. The same titration measurements were also performed with added NaCl. The results of these measurements demonstrate that added NaCl weakens the electrostatic repulsion between charged amine groups in a polyamine chain and thus enhances the protonation of the chain, and this effect is quite significant at a physiological NaCl concentration of 150 mM. However, on the quantitative level, the effect of added NaCl was found to be very different between the PDMAEMA and P(EI-r-EOz) cases. In PDMAEMA, since the amine groups are located at the termini of the side chains, the interaction between adjacent charged monomers occurs through the aqueous medium, and therefore at a sufficiently high concentration of added NaCl, the amine groups on the chain behave almost identically to their unpolymerized equivalents. In contrast, the electrostatic interaction between two closely spaced charged EI monomers in a P(EI-r-EOz) chain is significantly less influenced by a change of the ionic strength of the medium, because it is dominated by the local dielectric property of the polymer segment located between the charged monomers. This interpretation is further supported by ab initio electron density functional theory (DFT) calculations on model oligomeric compounds whose structures imitate the repeat unit structures of the polymers. Lastly, in connection with potential applications of the PEI and PDMAEMA polymers in gene delivery technologies, it was also examined how complexation with negatively charged polymers at the physiological NaCl concentration (150 mM) impacts the protonation behaviors of the polyamines. We found that the oppositely charged polyanion greatly stabilizes the protonated form of the amine groups on the polyamine chain. However, the proton buffering capacity of the polyamine in the complexed form under the influence of added 150 mM NaCl for the intracellularly relevant pH change was found to be significantly lower than that of the pure polyamine in the uncomplexed state with no added salt.

Nano carriers that enable co-delivery of chemotherapy and RNAi agents for treatment of drug-resistant cancers.

Tumor cells exhibit drug resistant phenotypes that decrease the efficacy of chemotherapeutic treatments. The drug resistance has a genetic basis that is caused by an abnormal gene expression. There are several types of drug resistance: efflux pumps reducing the cellular concentration of the drug, alterations in membrane lipids that reduce cellular uptake, increased or altered drug targets, metabolic alteration of the drug, inhibition of apoptosis, repair of the damaged DNA, and alteration of the cell cycle checkpoints (Gottesman et al., 2002; Holohan et al., 2013). siRNA is used to silence the drug resistant phenotype and prevent this drug resistance response. Of the listed types of drug resistance, pump-type resistance (e.g., high expression of ATP-binding cassette transporter proteins such as P-glycoproteins (Pgp; also known as multi-drug resistance protein 1 or MDR1, encoded by the ATP-Binding Cassette Sub-Family B Member 1 (ABCB1) gene)) and apoptosis inhibition (e.g., expression of anti-apoptotic proteins such as Bcl-2) are the most frequently targeted for gene silencing. The co-delivery of siRNA and chemotherapeutic drugs has a synergistic effect, but many of the current projects do not control the drug release from the nanocarrier. This means that the drug payload is released before the drug resistance proteins have degraded and the drug resistance phenotype has been silenced. Current research focuses on cross-linking the carriers polymers to prevent premature drug release, but these carriers still rely on environmental cues to release the drug payload, and the drug may be released too early. In this review, we studied the release kinetics of siRNA and chemotherapeutic drugs from a broad range of carriers. We also give examples of carriers used to co-deliver siRNA and drugs to drug-resistant tumor cells, and we examine how modifications to the carrier affect the delivery. Lastly, we give our recommendations for the future directions of the co-delivery of siRNA and chemotherapeutic drug treatments.

Block Copolymer Electrolytes Synthesized by Atom Transfer Radical Polymerization for Solid-State, Thin-Film Lithium Batteries

Block copolymer electrolytes of poly[(oxyethylene) 9 methacrylate]-b-poly(butyl methacrylate) (POEM-b-PBMA) (60:40 by mass) synthesized for the first time by atom transfer radical polymerization (ATRP) exhibited mechanical and electrical properties indistinguishable from those of materials made by the more difficult anionic polymerization method. ATRP offers distinct processing advantages as it is easily scalable and almost solvent-free. Solid-state, thin-film batteries comprised of a metallic lithium anode, a binder-free, additive-free, fully dense vanadium oxide cathode, and an electrolyte of ATRP-synthesized POEM-b-PBMA (60:40) doped with LiCF 3 SO 3 demonstrate resistance to capacity fade during extended cycling at a discharge rate of C/2, and perform comparably to otherwise identical batteries operated with the liquid electrolyte 1 M LiPF 6 in ethylene carbonate:dimethyl carbonate (1:1 by mass).

Targeted Nanotheranostics for Future Personalized Medicine: Recent Progress in Cancer Therapy

Recently, many theranostic nanomaterials have been developed by integrating therapeutic and diagnostic agents in a single regimen. Real-time visualization of nano drug carrier biodistributions, drug release processes and therapeutic responses can provide critical information needed for dynamically optimizing treatment operations in a personalized manner in real time. This review highlights recent progresses in the development of multifunctional nanoparticles possessing both therapeutic and imaging functionalities for cancer therapy. The advantages of using nanoparticle platforms are discussed. Examples demonstrating various combinations of imaging and therapeutic modalities are highlighted.

Self-consistent field theory study of the effect of grafting density on the height of a weak polyelectrolyte brush.

The height of weakly basic polyelectrolyte brushes in the osmotic brush regime is studied as a function of the grafting density using a numerical self-consistent field theory derived from the (semi)grand canonical partition function. The theory is shown to properly account for the local nature of the charge equilibrium and to capture the basic behaviors of polyelectrolyte brushes. On one hand, we find, in agreement with recent experiments, that the scaling of brush height with grafting density can be qualitatively different at intermediate chain lengths than that predicted by basic scaling arguments. This difference is attributed to the relative strength of electrostatic type interactions compared to finite segment size packing constraints. On the other hand, the trend of decreasing brush height with increasing grafting density predicted by the classic scaling analysis is recovered for large molecular weight polymers immersed in a solution of very weak ionic strength.

Fabrication of high-quality non-close-packed 2D colloid crystals by template-guided Langmuir–Blodgett particle deposition

We present a new method of fabricating highly-ordered two-dimensional (2D) colloid crystals with non-closed-packed (NCP) symmetries. In this method, using the Langmuir-Blodgett (LB) monolayer deposition technique, we transfer a Langmuir monolayer of colloidal particles constructed at the air-water interface onto a substrate that contains micro-fabricated topological patterns. We demonstrate that by using this template-guided LB deposition method, near perfect single 2D colloid crystal domains of the order of a hundred micrometres can be easily fabricated under typical LB processing conditions. We investigate the effects of various control parameters (such as the initial particle density at the air-water interface and the substrate lifting speed during the LB particle deposition process) on the density of the deposited particles in the resultant LB monolayer; the final density of the particles deposited on the patterned surface is found to be systematically lower than the particle packing density of the initial Langmuir monolayer, and this dilation is an increasing function of the LB deposition speed. On the length scales larger than approximately a hundred micrometres, we typically observe the formation of stripe patterns in the template-guided LB particle monolayer film, which (we believe) indicates that the contact line of the water meniscus is not stationary. Rather, its position undulates periodically due to the water evaporation during the LB deposition, contrary to what has been commonly assumed in many previous models describing the LB (dip-coating) processes. We present a simple theoretical model, which by taking into account the effects of the evaporation-induced subphase water flow and the particle concentration gradient around the meniscus contact line, can explain all the above-stated experimental observations. Finally, we provide experimental evidence that under high-humidity conditions in which water evaporation is suppressed, the pattern-guided LB deposition technique can indeed produce a high-quality 2D colloid crystal structure that is homogeneous throughout the entire area of the micro-patterned region of the substrate.

Imaging Nanostructured Fluids Using Cryo-TEM

Cryogenic transmission electron microscopy (cryo-TEM) has gained increasing popularity among researchers in the field of amphiphilic self-assembly as an experimental method of choice because of its unique ability to visualize nanostructures in complex fluids. Due to many recent technical improvements in the instrumentation, cryo-TEM experiments are now common. However, some limitations and possible artifacts are still a problem, and awareness of them remains a prerequisite for reliable operations and interpretation. The goal of this paper is to provide a review of the basic methods and procedures by which the cryo-TEM experiments are typically practiced. As examples, this paper also presents recent results derived from our cryo-TEM studies of micellization of block copolymer blends which clearly illustrate the unique usefulness of the technique for exploring the unexpected, complex, and/or heterogeneous nanostructures in amphiphilic solutions.

Study of the Air−Water Interfacial Properties of Biodegradable Polyesters and Their Block Copolymers with Poly(ethylene glycol)

It has been reported that the surface pressure-area isotherm of poly(D,L-lactic acid-ran-glycolic acid) (PLGA) at the air-water interface exhibits several interesting features: (1) a plateau at intermediate compression levels, (2) a sharp rise in surface pressure upon further compression, and (3) marked surface pressure-area hysteresis during compression-expansion cycles. To investigate the molecular origin of this behavior, we conducted an extensive set of surface pressure and AFM imaging measurements with PLGA materials having several different molecular weights and also a poly(D,L-lactic acid-ran-glycolic acid-ran-caprolactone) (PLGACL) material in which the caprolactone monomers were incorporated as a plasticizing component. The results suggest that (i) the plateau in the surface pressure-area isotherm of PLGA (or PLGACL) occurs because of the formation (and collapse) of a continuous monolayer of the polymer under continuous compression; (ii) the PLGA monolayer becomes significantly resistant to compression at high compression because under that condition the collapsed domains become large enough to become glassy (such behavior was not observed in the nonglassy PLGACL sample); and (iii) the isotherm hysteresis is due to a coarsening of the collapsed domains that occurs under high-compression conditions. We also investigated the monolayer properties of PEG-PLGA and PEG-PLGACL diblock copolymers. The results demonstrate that the tendency of PLGA (or PLGACL) to spread on water allows the polymer to be used as an anchoring block to form a smooth biodegradable monolayer of block copolymers at the air-water interface. These diblock copolymer monolayers exhibit protein resistance.