Antioxidant Theranostic Copolymer-Mediated Reduction In Oxidative Stress Following Traumatic Brain Injury Improves Outcome In A Mouse Model

Following a traumatic brain injury (TBI), excess reactive oxygen species (ROS) and lipid peroxidation products (LPOx) are generated and lead to secondary injury beyond the primary insult. A major limitation of current treatments is poor target engagement, which has prevented success in clinical trials. Thus, nanoparticle-based treatments have received recent attention because of their ability to increase accumulation and retention in damaged brain. Theranostic neuroprotective copolymers (NPC3) containing thiol functional groups can neutralize ROS and LPOx. Immediate administration of NPC3 following injury in a controlled cortical impact (CCI) mouse model provides a therapeutic window in reducing ROS levels at 2.08–20.83 mg kg−1 in males and 5.52–27.62 mg kg−1 in females. This NPC3-mediated reduction in oxidative stress improves spatial learning and memory in males, while females show minimal improvement. Notably, NPC3-mediated reduction in oxidative stress prevents the bilateral spread of necrosis in male mice, which is not observed in female mice and likely accounts for the sex-based spatial learning and memory differences. Overall, these findings suggest sex-based differences to oxidative stress scavenger nanoparticle treatments, and a possible upper threshold of antioxidant activity that provides therapeutic benefit in injured brain since female mice benefit from NPC3 treatment to a lesser extent than male mice

Theranostic Copolymers Neutralize Reactive Oxygen Species and Lipid Peroxidation Products for the Combined Treatment of Traumatic Brain Injury

Traumatic brain injury (TBI) results in the generation of reactive oxygen species (ROS) and lipid peroxidation product (LPOx), including acrolein and 4-hydroxynonenal (4HNE). The presence of these biochemical derangements results in neurodegeneration during the secondary phase of the injury. The ability to rapidly neutralize multiple species could significantly improve outcomes for TBI patients. However, the difficulty in creating therapies that target multiple biochemical derangements simultaneously has greatly limited therapeutic efficacy. Therefore, our goal was to design a material that could rapidly bind and neutralize both ROS and LPOx following TBI. To do this, a series of thiol-functionalized biocompatible copolymers based on lipoic acid methacrylate and polyethylene glycol monomethyl ether methacrylate (FW ∼950 Da) (O950) were prepared. A polymerizable gadolinium-DOTA methacrylate monomer (Gd-MA) was also synthesized starting from cyclen to facilitate direct magnetic resonance imaging and in vivo tracking of accumulation. These neuroprotective copolymers (NPCs) were shown to rapidly and effectively neutralize both ROS and LPOx. Horseradish peroxidase absorbance assays showed that the NPCs efficiently neutralized H2O2, while R-phycoerythrin protection assays demonstrated their ability to protect the fluorescent protein from oxidative damage. 1H NMR studies indicated that the thiol-functional NPCs rapidly form covalent bonds with acrolein, efficiently removing it from solution. In vitro cell studies with SH-SY5Y-differentiated neurons showed that NPCs provide unique protection against toxic concentrations of both H2O2and acrolein. NPCs rapidly accumulate and are retained in the injured brain in controlled cortical impact mice and reduce post-traumatic oxidative stress. Therefore, these materials show promise for improved target engagement of multiple biochemical derangements in hopes of improving TBI therapeutic outcomes

An influenza virus-inspired polymer system for the timed release of siRNA

Small interfering RNA silences specific genes by interfering with mRNA translation, and acts to modulate or inhibit specific biological pathways; a therapy that holds great promise in the cure of many diseases. However, the naked small interfering RNA is susceptible to degradation by plasma and tissue nucleases and due to its negative charge unable to cross the cell membrane. Here we report a new polymer carrier designed to mimic the influenza virus escape mechanism from the endosome, followed by a timed release of the small interfering RNA in the cytosol through a self-catalyzed polymer degradation process. Our polymer changes to a negatively charged and non-toxic polymer after the release of small interfering RNA, presenting potential for multiple repeat doses and long-term treatment of diseases

Direct, Controlled Synthesis of the Nonimmunogenic Hydrophilic Polymer, Poly(N-(2-Hydroxypropyl)methacrylamide) Via RAFT in Aqueous Media

Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) is a nonimmunogenic, neutral-hydrophilic polymer currently employed in the delivery of anticancer drugs. Herein, we report conditions that facilitate the direct, controlled RAFT polymerization of HPMA in aqueous media. We demonstrate that the use of 4-cyanopentanoic acid dithiobenzoate and 4,4′-azobis(4-cyanopentanoic acid) as the chain transfer agent (CTA) and initiating species, respectively, in the presence of an acetic acid buffer solution at 70 °C is a suitable condition leading to controlled polymerization. The living nature of these polymerizations is demonstrated via chain-extension of an HPMA macroCTA to yield the corresponding poly(HPMA-b-HPMA) homopolymer

PH-Responsive Polymeric Micelle Carriers for SiRNA Drugs

The ability of small interfering RNA (siRNA) to efficiently silence the expression of specific genes provides the basis for exciting new therapies based on RNA interference (RNAi). The efficient intracellular Delivery of siRNA from cell uptake through the endosomal trafficking pathways into the cytoplasm remains a significant challenge. Previously we described the synthesis of a new family of diblock copolymer siRNA carriers using controlled reversible addition-fragmentation chain transfer (RAFT) polymerization. The carriers were composed of a positively charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA binding and a second pHresponsive endosome releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios and butyl methacylate (BMA). Here we describe the Development of a new generation of siRNA Delivery polymers based on this design that exhibit enhanced transfection efficiency and low cytotoxicity. This design incorporates a longer endosomolytic block with increased hydrophobic content to induce micelle formation. These polymers spontaneously form spherical micelles in the size range of 40 nm with CMC (critical micelle concentration) values of approximately 2µg/mL based on dynamic light scattering (DLS), 1H NMR, electron microscopy, and selective partitioning of the small molecule pyrene into the hydrophobic micelle core. The siRNA binding to the cationic shell block did not perturb micelle stability or significantly increase particle size. The self-assembly of the diblock copolymers into particles was shown to provide a significant enhancement in mRNA knockdown at siRNA concentrations as low as 12.5 nM. Under these conditions, the micelle-based systems showed an 89% reduction in GAPDH mRNA levels as compared to only 23% (10 nM siRNA) for the nonmicelle system. The reduction in mRNA levels becomes nearly quantitative as the siRNA concentration is increased to 25 nM and higher. Flow cytometry analysis of fluorescent-labeled siRNA showed uptake in 90% of cells and a 3-fold increase in siRNA per cell compared to a standard lipid transfection agent. These results demonstrate the potential utility of this carrier design for siRNA drug Delivery

Corona-Stabilized Interpolyelectrolyte Complexes of SiRNA with Nonimmunogenic, Hydrophilic/Cationic Block Copolymers Prepared by Aqueous RAFT Polymerization †

ABSTRACT: The complexation of small interfering ribonucleic acid (siRNA) with a series of specifically designed block copolymers consisting of the hydrophilic, nonimmunogenic monomer N-(2-hydroxypropyl)methacrylamide (HPMA) and the cationic monomer N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) has been investigated for potential siRNA stabilization and delivery applications. Specific compositions of poly(HPMAb-DMAPMA) copolymers were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization and characterized using aqueous size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) and 1 H NMR spectroscopy. The degree of soluble complex formation between a model siRNA and the polymers was determined by centrifugal membrane filtration experiments and quantitated by scintillation counting of 32 P ATP-labeled siRNA to determine complex solubility and to estimate the degree of complexation relative to cationic and neutral block lengths. Dynamic and static light scattering methods were employed to determine the hydrodynamic radii, molecular weights, and second virial coefficients of the complexes and to demonstrate their unimodal size distributions. In vitro enzymatic degradation studies of selected siRNA/block copolymer complexes were conducted to demonstrate the enhanced stability of the siRNA/poly(HPMA-b-DMAPMA) complexes. Furthermore, the siRNA/polymer complexes dissociate slowly under gel electrophoresis conditions. Therefore, the siRNA/polymer complexes demonstrate some highly desirable properties for potential applications in therapeutic siRNA stabilization and delivery