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

Antibody Targeting Facilitates Effective Intratumoral SiRNA Nanoparticle Delivery to HER2-Overexpressing Cancer Cells

The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. We investigated the utility of linking an internalizing streptavidinconjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5\u27 RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. Detection of the expected mRNA cleavage product by 5\u27 RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy

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

Fluorescent Labelling of RAFT-Generated Poly(N-Isopropylacrylamide) Via a Facile Maleimide-Thiol Coupling Reaction

We report a facile labeling technique in which the telechelic thiocarbonylthio functionality of well-defined poly-(N-isopropylacrylamide) (PNIPAM) prepared by room temperature RAFT polymerization is first converted to the thiol and subsequently reacted with a maleimido-functional fluorescent dye, N-(1-pyrene)maleimide (PM). Nearly monodisperse PNIPAM (Mn = 39 500 g/mol, Mw/Mn = 1.07) was synthesized using a trithiocarbonate-based CTA, 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (DMP), and a conventional azo-initiator, namely, 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70), as the primary source of radicals. The key to successful conjugation of PM to PNIPAM is the implementation of a two-step reduction process involving (1) the cleavage of the trithiocarbonate with a strong reducing agent, in this case, NaBH4, to form a mixture of polymeric thiols and disulfides and (2) the conjugation of PM to the pure polymeric thiol in the presence of tris(2-carboxyethyl)phosphine·HCI (TCEP). We show that TCEP efficiently eliminates the formation of polymeric disulfides and thus allows for the desired addition of the free polymeric thiol across the maleimide double bond. This concept is demonstrated using SEC-MALLS and UV-vis spectroscopy measurements

PH-Responsive Polymer-Antigen Vaccine Bioconjugates

Protein-based vaccines play an important role in controlling infectious disease but their full clinical impact has been limited by their inability to generate a coordinated cellular CD4+ and CD8+ immune response. Vaccines that better deliver antigens to the cytosolic MHC1 display system could in principle provide a better coordinated response. Here, controlled radical polymerization was employed to prepare a diblock copolymer containing an endosomal releasing segment based on poly(propylacrylic acid) (poly(PAA)) and a hydrophilic segment containing thiol-reactive disulfide moieties for antigen conjugation. Propylacrylic acid (PAA) was polymerized in the presence of a trithiocarbonate based RAFT chain transfer agent (CTA). The resultant poly(PAA) was then employed as a macroCTA in the copolymerization of pyridyl disulfide methacrylamide (PDSMA) (thiol-reactive monomer) and N,N-dimethylacrylamide (DMA) as a hydrophilic comonomer. Copolymer compositions and molecular weights were determined via 1H NMR spectroscopy and size exclusion chromatography. Native polyacrylamide gel electrophoresis (PAGE) showed a complete disappearance of the bands corresponding to free thiolated ovalbumin after conjugation to the polymer at pyridyl disulfide to thiol ratios as low as 2.5. The ability of the poly[(PAA)-b-(DMA)co(PDSMA)]-ovalbumin conjugates to activate CTLs was evaluated in vivo, tumor protection using the EG7 tumor protection model. Tumors were visible in the PBS and free ovalbumin immunized mice by day 7 but were not visible in the polyPAA-ovalbumin conjugate immunized mice until day 18. The mice immunized with the polyPAA-ovalbumin conjugate had a survival rate at day 21 of 100% versus 20% for PBS and 40% for ovalbumin immunized mice

One Pot Synthesis of Thiol-Functional Nanoparticles

Polysorbate 80 (PS80) Was Reacted with 3-Mercaptopropyl Trimethoxysilane (SiSH) Via a Photoinitiated Thiol-Ene Reaction. the Conjugate Was Then Mixed with SiSH and Water to Form Uniform Thiol-Functional Nanoparticles (TFNs) Approximately 22 Nm in Diameter. Aqueous TFN Solutions (20 Wt% Solids) Can Be Used Directly to Conduct Thiol-Ene/thiol-Michael Reactions or Concentrated by Heating at 60 °C to 50 Wt% Solids. the Large Number of Thiol Residues Per Particle Also Provides a Convenient Route for Changing the Physical or Chemical Properties of the Particles. This is Easily Accomplished by Directly Reacting the TFNs with Monofucntional Alkenes under Photochemical Initiation. These Reactions Usually Proceed to Quantitative Conversion within Four to Eight Hours Depending on the Nature of the Alkene and the Photoinitator. Initital Crosslinking Experiments of TFNS with Commercial Alkene Crosslinking Agent PEGDMA (Poly(Ethylene Glycol)dimethacrylate) Showed Improved Cure Rates When Compared to the Crosslinker of PEGDMA by itself. using These Results, TFN Resins Employing Several Commercial Crosslinkers Were Synthesized and Printed on a Commercial DLP Printer. PEGDMA Resins Produced Opaque Prints with Poor Strength While ACMAC (3-(Acryloyloxy)-2-Hydroxypropyl Methacrylate) Produced Several Translucent Prints with Good Strength and Stiffness. the Addition of TFNs to Commercial 3D Printing Resin Resulted in a Significant Rate Enhancement Allowing Digital Light Projection (DLP) Exposure Times of 0.5 Seconds Per Layer. This Simple Scalable One-Pot Process Produces Multifunctional Thiols that Are Soluble in Both Aqueous and Organic Solvents Without the Need for Organic Solvents or Purification

Multifunctional Triblock Copolymers for Intracellular Messenger RNA Delivery

Messenger RNA (mRNA) is a promising alternative to plasmid DNA (pDNA) for gene vaccination applications, but safe and effective delivery systems are rare. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize a series of triblock copolymers designed to enhance the intracellular delivery of mRNA. These materials are composed of a cationic dimethylaminoethyl methacrylate (DMAEMA) segment to mediate mRNA condensation, a hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) segment to enhance stability and biocompatibility, and a pH-responsive endosomolytic copolymer of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) designed to facilitate cytosolic entry. The blocking order and PEGMA segment length were systematically varied to investigate the effect of different polymer architectures on mRNA delivery efficacy. These polymers were monodisperse, exhibited pH-dependent hemolytic activity, and condensed mRNA into 86-216 nm particles. mRNA polyplexes formed from polymers with the PEGMA segment in the center of the polymer chain displayed the greatest stability to heparin displacement and were associated with the highest transfection efficiencies in two immune cell lines, RAW 264.7 macrophages (77%) and DC2.4 dendritic cells (50%). Transfected DC2.4 cells were shown to be capable of subsequently activating antigen-specific T cells, demonstrating the potential of these multifunctional triblock copolymers for mRNA-based vaccination strategies

3D Printing Soluble Solids Via PISA

Polymer-Induced Self Assembly (PISA) is a technique that often involves the use of a macro chain transfer agent (macro-CTA) polymerized through reversible addition-fragmentation chain transfer (RAFT), which is then chain extended with a monomer to induce phase separation during block copolymer formation. The process results in the in situ generation of nanostructures with various morphologies. Prior studies have indicated that increasing the functionality of RAFT macro-CTAs can cause the formation of flower or loop-like coronas during polymer self-assembly, enabling physical crosslinks between particles and gel formation. This study focuses on developing a PISA resin for 3D printing using a difunctional macro-CTA, with the expectation that physical crosslinks between difunctional macro-CTAs would allow printed parts to maintain their shape without the need for a crosslinker. In this research, a difunctional poly(ethylene glycol) (PEG) macro-CTA was chain extended with diacetone acrylamide (DAAm) in water and 3D printed via digital light projection (DLP) using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator and phenol red as a photoabsorber. Resins were printed with or without the addition of 2.5 wt% N′N-methylene bisacrylamide (MBAc) crosslinker. The DAAm PISA frog without crosslinker, being water-insoluble, completely dissolved in N,N-dimethylformamide (DMF) while the one with crosslinker did not. Controlled dissolution of the part architecture was achieved by printing components containing both crosslinked and uncrosslinked resins. Atomic Force Microscopy (AFM) images displayed interlocked particle (worm-like) morphologies and/or phase-separated domains, indicating self-assembly during the printing process. Scanning Electron Microscopy (SEM) imaging of these physically crosslinked, 3D printed PISA polymers revealed a vasculature-like network, which holds potential for use an scaffolds in tissue engineering applications

Neutral Polymeric Micelles for RNA Delivery

RNA interference (RNAi) drugs have significant therapeutic potential, but delivery systems with appropriate efficacy and toxicity profiles are still needed. Here, we describe a neutral, ampholytic polymeric delivery system based on conjugatable diblock polymer micelles. The diblock copolymer contains a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide-co-N-(2-(pyridin-2- yldisulfanyl)ethyl)methacrylamide) (poly[HPMA-co-PDSMA]) segment to promote aqueous stability and facilitate thiol-disulfide exchange reactions and a second ampholytic block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The poly[(HPMA-co-PDSMA)-b- (PAA-co-DMAEMA-co-BMA)] was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization with an overall molecular weight of 22 000 g/mol and a PDI of 1.88. Dynamic light scattering and fluorescence measurements indicated that the diblock copolymers self-assemble under aqueous conditions to form polymeric micelles with a hydrodynamic radius and critical micelle concentration of 25 nm and 25 μg/mL, respectively. Red blood cell hemolysis experiments show that the neutral hydrophilic micelles have potent membrane destabilizing activity at endosomal pH values. Thiolated siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was directly conjugated to the polymeric micelles via thiol exchange reactions with the pyridal disulfide groups present in the micelle corona. Maximum silencing activity in HeLa cells was observed at a 1:10 molar ratio of siRNA to polymer following a 48 h incubation period. Under these conditions 90% mRNA knockdown and 65% protein knockdown at 48 h was achieved with negligible toxicity. In contrast the polymeric micelles lacking a pH-responsive endosomalytic segment demonstrated negligible mRNA and protein knockdown under these conditions. The potent mRNA knockdown and excellent biocompatibility of the neutral siRNA conjugates demonstrate the potential utility of this carrier design for delivering therapeutic siRNA drugs