Slow-sustained delivery of naloxone reduces typical naloxone-induced precipitated opioid withdrawal effects in male morphine-dependent mice.

Thousands of individuals die each year from opioid-related overdoses. While naloxone (Narcan®) is currently the most widely employed treatment to reverse opioid toxicity, high or repeated doses of this antidote often lead to precipitated opioid withdrawal (POW). We hypothesized that a slow linear release of naloxone from a nanoparticle would induce fewer POW symptoms compared to high-dose free naloxone. First, we measured the acute impact of covalent naloxone nanoparticles (Nal-cNPs) on morphine-induced antinociception in the hotplate test. We found that Nal-cNP treatment blocked the antinociceptive effect of morphine within 15 min of administration. Next, we tested the impact of Nal-cNPs on POW symptoms in male morphine-dependent mice. To induce morphine dependence, mice were treated with 5 mg/kg morphine (or saline) twice-daily for six consecutive days. On day 7 mice received 5 mg/kg morphine (or saline) injections 2 hr prior to receiving treatment of either unmodified free naloxone, a high or low dose of Nal-cNP, empty nanoparticle (cNP-empty), or saline. Behavior was analyzed for 0-6 hr followed by 24 and 48 hr time points after treatment. As expected, free naloxone induced a significant increase in POW behavior in morphine-dependent mice compared to saline-treated mice upon free naloxone administration. In comparison, reduced POW behavior was observed with both doses of Nal-cNP. Side effects of Nal-cNP on locomotion and fecal boli production were measured and no significant side-effects were observed. Overall, our data show that sustained release of naloxone from a covalent nanoparticle does not induce severe POW symptoms in morphine-dependent mice.

Polymer Modification of Lipases, Substrate Interactions, and Potential Inhibition.

An industrially important enzyme, Candida antarctica lipase B (CalB), was modified with a range of functional polymers including hydrophilic, hydrophobic, anionic, and cationic character using a "grafting to" approach. We determined the impact of polymer chain length on CalB activity by synthesizing biohybrids of CalB with each polymer at three different chain lengths, using reversible addition-fragmentation chain transfer (RAFT) polymerization. The activity of CalB in both aqueous and aqueous-organic media mixtures was significantly enhanced for acrylamide (Am) and N,N-dimethyl acrylamide (DMAm) conjugates, with activity remaining approximately constant in 25 and 50% ethanol solvent systems. Interestingly, the activity of N,N-dimethylaminopropyl-acrylamide (DMAPA) conjugates increased gradually with increasing organic solvent content in the system. Contrary to other literature reports, our study showed significantly diminished activity for hydrophobic polymer-protein conjugates. Functional thermal stability assays also displayed a considerable enhancement of retained activity of Am, DMAm, and DMAPA conjugates compared to the native CalB enzyme. Thus, this study provides an insight into possible advances in lipase production, which can lead to new improved lipase bioconjugates with increased activity and stability.

Development and Characterization of Novel Conductive Sensing Fibers for In Vivo Nerve Stimulation.

Advancements in electrode technologies to both stimulate and record the central nervous system's electrical activities are enabling significant improvements in both the understanding and treatment of different neurological diseases. However, the current neural recording and stimulating electrodes are metallic, requiring invasive and damaging methods to interface with neural tissue. These electrodes may also degrade, resulting in additional invasive procedures. Furthermore, metal electrodes may cause nerve damage due to their inherent rigidity. This paper demonstrates that novel electrically conductive organic fibers (ECFs) can be used for direct nerve stimulation. The ECFs were prepared using a standard polyester material as the structural base, with a carbon nanotube ink applied to the surface as the electrical conductor. We report on three experiments: the first one to characterize the conductive properties of the ECFs; the second one to investigate the fiber cytotoxic properties in vitro; and the third one to demonstrate the utility of the ECF for direct nerve stimulation in an in vivo rodent model.

Monoclonal Antibodies Counteract Opioid-Induced Behavioral and Toxic Effects in Mice and Rats.

Monoclonal antibodies (mAbs) and vaccines have been proposed as medical countermeasures to treat opioid use disorder (OUD) and prevent opioid overdose. In contrast to current pharmacotherapies (e.g., methadone, buprenorphine, naltrexone, and naloxone) for OUD and overdose, which target brain opioid receptors, mAbs and vaccine-generated polyclonal antibodies sequester the target opioid in the serum and reduce drug distribution to the brain. Furthermore, mAbs offer several potential clinical benefits over approved medications, such as longer serum half-life, higher selectivity, reduced side effects, and no abuse liability. Using magnetic enrichment to isolate opioid-specific B cell lymphocytes prior to fusion with myeloma partners, this study identified a series of murine hybridoma cell lines expressing mAbs with high affinity for opioids of clinical interest, including oxycodone, heroin and its active metabolites, and fentanyl. In mice, passive immunization with lead mAbs against oxycodone, heroin, and fentanyl reduced drug-induced antinociception and the distribution of the target opioid to the brain. In mice and rats, mAb pretreatment reduced fentanyl-induced respiratory depression and bradycardia, two risk factors for opioid-related overdose fatality. Overall, these results support use of mAbs to counteract toxic effects of opioids and other chemical threats. SIGNIFICANCE STATEMENT: The incidence of fatal overdoses due to the widespread access to heroin, prescription opioids, and fentanyl suggests that current Food and Drug Administration-approved countermeasures are not sufficient to mitigate the opioid epidemic. Monoclonal antibodies (mAbs) may provide acute protection from overdose by binding to circulating opioids in serum. Use of mAbs prophylactically, or after exposure in combination with naloxone, may reduce hospitalization and increase survival.

Toward Next-Generation Biohybrid Catalyst Design: Influence of Degree of Polymerization on Enzyme Activity.

Due to their capacity to conduct complex organic transformations, enzymes find extensive use in medical and industrial settings. Unfortunately, enzymes are limited by their poor stability when exposed to harsh non-native conditions. While a host of methods have been developed to stabilize enzymes in non-native conditions, recent research into the synthesis of polymer-enzyme biohybrids using reversible deactivation radical polymerization approaches has demonstrated the potential of increased enzymatic activity in both native and non-native environments. In this manuscript, we utilize the enzyme lipase, as a model system, to explore the impact that modulation of grafted polymer molecular weight has on enzyme activity in both aqueous and organic media. We studied the properties of these hybrids using both solution-phase enzyme activity methods and coarse-grain modeling to assess the impact of polymer grafting density and grafted polymer molecular weight on enzyme activity to gain a deeper insight into this understudied property of the biohybrid system.

In-situ Chemiluminescence-Driven Reversible Addition-Fragmentation Chain-Transfer Photopolymerization.

The power of chemical light generation (chemiluminescence) is used to drive polymerization reactions. A biphasic reaction is developed such that light-generating reactions are confined to the organic phase and photopolymerization occurs in the aqueous phase. Well-defined RAFT-capped polymers are synthesized and the kinetics are shown to be dictated by light generation.

Cationic Hyperbranched Polymers with Biocompatible Shells for siRNA Delivery.

Cationic hyperbranched polymers (HBP) were prepared by self-condensing vinyl polymerization of an atom transfer radical polymerization (ATRP) inimer containing a quaternary ammonium group. Two types of biocompatible shells, poly(oligoethylene glycol) methacrylate (polyOEGMA) and poly(2-(methylsulfinyl) ethyl methacrylate) (polyDMSO), were grafted respectively from HBP core to form core-shell structures with low molecular weight dispersity and high biocompatibility, polyOEGMA-HBP and polyDMSO-HBP. Both of the structures showed low cytotoxicity and good siRNA complexing ability. The efficacy of gene silencing against Runt-related transcription factor 2 ( Runx2) expression and the long-term assessment of mineralized nodule formation in osteoblast cultures were evaluated. The biocompatible core-shell structures were crucial to minimizing undesired cytotoxicity and nonspecific gene suppression. polyDMSO-HBP showed higher efficacy of forming polyplexes than polyOEGMA-HBP due to shell with lower steric hindrance. Overall, the gene silencing efficiency of both core-shell structures was comparable to commercial agent Lipofectamine, indicating long-term potential for gene silencing to treat heterotopic ossification (HO).

Fentanyl Initiated Polymers Prepared by ATRP for Targeted Delivery.

The targeted delivery of polymers to neurons is a challenging yet important goal for polymer based drug delivery. We prepared a fentanyl based atom transfer radical polymerization (ATRP) initiator to target the Mu opioid receptor (MOR) for neuronal targeting. We incorporated our recently discovered rigid acrylate linking group into the initiator to retain a high degree of binding to the MOR and grafted random or block copolymers of poly(oligo(ethylene oxide) methacrylate)-block-(glycidyl methacrylate). Trifluoroethanol promoted amine ring opening of the glycidyl methacrylate was used for post-polymerization modification of the fentanyl initiated polymers to attach a near-infrared fluorescent dye (ADS790WS) or to build a targeted siRNA delivery system via modification with secondary amines. We examined the biocompatibility, cellular internalization, and siRNA binding properties of our polymer library in a green fluorescent protein expressing SY SH5Y neuroblastoma cell-line.

Biocompatible Polymeric Analogues of DMSO Prepared by Atom Transfer Radical Polymerization.

The synthesis of a sulfoxide-based water-soluble polymer, poly(2-(methylsulfinyl)ethyl acrylate) (polyMSEA), a polymeric analogue of DMSO, by atom transfer radical polymerization (ATRP) is reported. Well-defined linear polymers were synthesized using relatively low amounts of copper catalyst (1000 or 100 ppm). Two types of star polymers were synthesized by either an "arm-first" approach or a "core-first" approach using a biodegradable ß-cyclodextrin core. The glass transition temperatures of both the linear polymer (16 °C) and star polymer (32 °C) were determined by differential scanning calorimetry (DSC). The lower critical solution temperature (LCST) of poly(MSEA) was estimated to be ca. 140 °C by extrapolating the LCST of a series of copolymers with NIPAM. Cytotoxicity tests revealed that both the linear and star polymers have low toxicity, even at concentrations up to 3 mg/mL.

Synthesis of Reactive Polymers for Acrolein Capture Using AGET ATRP.

Acrolein is a toxic metabolite of the anticancer agent cyclophosphamide (CP). Current strategies to mitigate acrolein toxicity are insufficient, and in this brief article, we report the synthesis of well-defined low molecular weight block copolymers using activators generated by electron transfer atom transfer radical polymerization (AGET ATRP) capable of reacting with the cytotoxic small molecule acrolein. Acrolein reactivity was introduced into the block copolymers via incorporation of either (a) aminooxy or (b) sulfhydryl groups. The cytoprotective effect of the polymers was compared to sodium 2-sulfanylethanesulfonate (mesna) the current gold standard for protection from CP urotoxicity, and we found that the polymers bearing sulfhydryl moieties demonstrated superior cytoprotective activity.

Synthesis of poly(meth)acrylates with thioether and tertiary sulfonium groups by ARGET ATRP and their use as siRNA delivery agents.

The field of RNA interference depends on the development of safe and efficient carriers for short interfering ribonucleic acid (siRNA) delivery. Conventional cationic monomers for siRNA delivery have utilized the nitrogen heteroatom to produce cationic charges. Here, we polymerized cationic sulfonium (meth)acrylate by activators regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) to form polymers with narrow molecular weight distributions for siRNA delivery. The tertiary sulfonium species was stable toward dealkylation in water but less stable in the polar aprotic solvent dimethyl sulfoxide. Block copolymers poly(ethylene oxide) with poly(meth)acrylate containing sulfonium moieties were prepared as an siRNA delivery platform. Results suggested block copolymers were biocompatible up to 50 µg/mL in vitro and formed polyplexes with siRNA. Additionally, block copolymers protected siRNAs against endonuclease digestion and facilitated knockdown of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) mRNA expression in murine calvarial preosteoblasts. The versatility, biocompatibility, and cationic nature of these tertiary sulfonium groups are expected to find widespread biological applications.

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells.

BACKGROUND: Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation. QUESTIONS/PURPOSES: We hypothesized that (1) nanogel-mediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures. METHODS: Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage™ assay). RESULTS: Gene expression assays demonstrated that nanogel-based RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p < 0.001) and 43.22% ± 18.01% (both p < 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p < 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p < 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that nanogel 1:1, 5:1, and 10:1 RNAi treatments decreased mineralization (ie, HA deposition) from cultures treated only with rhBMP-2 (p < 0.001). However, despite RNAi attack on Runx2 and Osx, HA deposition levels remained greater than non-rhBMP-2-treated cell cultures. CONCLUSIONS: Although mRNA and protein knockdown were confirmed as a result of RNAi treatments against Runx2 and Osx, complete elimination of mineralization processes was not achieved. RNAi targeting mid- and late-stage osteoblast differentiation markers such as ALP, osteocalcin, osteopontin, and bone sialoprotein) may produce the desired RNAi-nanogel nanostructured polymer HO prophylaxis. CLINICAL RELEVANCE: Successful HO prophylaxis should target and silence osteogenic markers critical for heterotopic bone formation processes. The identification of such markers, beyond RUNX2 and OSX, may enhance the effectiveness of RNAi prophylaxes for HO.

Solid-phase incorporation of an ATRP initiator for polymer-DNA biohybrids.

The combination of polymers with nucleic acids leads to materials with significantly advanced properties. To obviate the necessity and complexity of conjugating two macromolecules, a polymer initiator is described that can be directly covalently linked to DNA during solid-phase synthesis. Polymer can then be grown from the DNA bound initiator, both in solution after the DNA-initiator is released from the solid support as well as directly on the solid support, simplifying purification. The resulting polymer-DNA hybrids were examined by chromatography and fluorescence methods that attested to the integrity of hybrids and the DNA. The ability to use DNA-based supports expands the range of readily available molecules that can be used with the initiator, as exemplified by direct synthesis of a biotin polymer hybrid on solid-support. This method expands the accessibility and range of advanced polymer biohybrid materials.

Cooperative, reversible self-assembly of covalently pre-linked proteins into giant fibrous structures.

We demonstrate a simple bioconjugate polymer system that undergoes reversible self-assembling into extended fibrous structures, reminiscent of those observed in living systems. It is comprised of green fluorescent protein (GFP) molecules linked into linear oligomeric strands through click step growth polymerization with dialkyne poly(ethylene oxide) (PEO). Confocal microscopy, atomic force microscopy, and dynamic light scattering revealed that such strands form high persistence length fibers, with lengths reaching tens of micrometers, and uniform, sub-100 nm widths. We ascribe this remarkable and robust form of self-assembly to the cooperativity arising from the known tendency of GFP molecules to dimerize through localized hydrophobic patches and from their covalent pre-linking with flexible PEO. Dissipative particle dynamics simulations of a coarse-grained model of the system revealed its tendency to form elongated fibrous aggregates, suggesting the general nature of this mode of self-assembly.

Are carfentanil and acrylfentanyl naloxone resistant?

The rapid rise in deaths since 2012 due to opioid poisoning is correlated with the proliferation of potent synthetic opioid agonists such as fentanyl, acrylfentanyl, and carfentanil. The efficacy of frontline antidotes such as naloxone in reversing such poisoning events has been questioned, and the possibility of naloxone-resistant synthetic opioids has been raised. In this manuscript, we applied in vitro techniques to establish the median effective inhibitory concentrations for fentanyl, acrylfentanyl, and carfentanil and subsequently evaluate naloxone's ability to reverse agonist-receptor interactions.

Evaluating the rate of reversal of fentanyl-induced respiratory depression using a novel long-acting naloxone nanoparticle, cNLX-NP.

Introduction: Fentanyl and fentanyl analogs (F/FA) have become increasingly common adulterants in counterfeit prescription pills and illicit street drug mixtures due to their ease of synthesis and exceedingly high potency. The ongoing epidemic of fatal overdoses fueled by F/FA continues to highlight the need for longer-acting therapies than naloxone (NLX), the current gold-standard for reversing opioid overdoses, which shows limited efficacy to prevent renarcotization associated with F/FA toxicity. A novel opioid reversal agent based on covalent naloxone nanoparticles (cNLX-NP) has been shown to blunt fentanyl-induced respiratory depression out to 48 hr, demonstrating its potential therapeutic utility. The purpose of this study was to characterize how rapidly cNLX-NP reverses fentanyl-induced respiratory effects as well as the duration of its protective effects. Methods: Sprague Dawley male rats (n=6/group) were tested on an oximeter for baseline percent arterial oxygen saturation (%SaO2) challenged with 0.1 mg/kg SC fentanyl and 15 min later given 10 mg/kg IM doses of NLX, nalmefene (NLMF), or cNLX-NP and continuously monitored via oximetry for 10 minutes. One week later the experiment was repeated using a 1:1 mixture of NLX:cNLX-NP as the reversal agent in the rats that previously received NLX alone. Results: While both NLX and NLMF rapidly reversed %SaO2 to baseline within 1 min, rats that received cNLX-NP did not return to >90% SaO2 levels until 9 min after administration. Similarly, heart and breath rates returned to baseline within 1 min of treatment with NLX and NLMF but did not return to baseline until 10 minutes after cNLX-NP administration. In contrast, NLX:cNLX-NP reversed all fentanyl-induced respiratory depressive effects within one minute. Discussion: While cNLX-NP alone may not sufficiently reverse F/FA overdose in a timely manner, mixing free NLX with cNLX-NP can provide a mechanism to both rapidly reverse fentanyl-related effects and maintain extended protection against synthetic opioid toxicity. These data support further development of cNLX-NP as a fast-acting and long-lasting antidote to treat F/FA-induced respiratory depression and overdose, and potentially prevent renarcotization in humans.

Multivalent Vaccination Strategies Protect against Exposure to Polydrug Opioid and Stimulant Mixtures in Mice and Rats.

Illicit drug mixtures containing opioids and stimulants have been responsible for the majority of fatal drug overdoses among occasional users, and those with either opioid use disorder (OUD) or substance use disorder (SUD). As a complementary strategy to current pharmacotherapies, active immunization with conjugate vaccines has been proposed as a viable intervention to treat OUD as well as other SUD for which there are either limited or no treatment options. Vaccination against opioids and stimulants could help address the limitations of current medications (e.g., patient access, compliance, misuse liability, and safety) by providing an additional tool to prevent drug misuse and/or overdoses. However, more research is needed to fully understand the potential benefits and limitations of using vaccines to treat SUD and overdose and to inform us on how to deploy this strategy in the field. Previous reports have shown promise by combining two vaccines into bivalent vaccine formulations to concurrently target multiple drugs. Here, multiple individual candidate monovalent vaccines were incrementally combined in multivalent vaccine formulations to simultaneously target fentanyl, carfentanil, oxycodone, heroin, methamphetamine, and their analogs or metabolites. Bi-, tri-, and quadrivalent vaccine formulations induced the formation of independent serum antibody responses against their respective opioid targets and selectively attenuated the distribution of each individual drug to the brain in mice and rats. Results indicate that a single injection of an admixed multivalent vaccine formulation may be more effective than coinjecting multiple monovalent vaccines at multiple sites. Finally, adding a methamphetamine conjugate vaccine to an quadrivalent opioid vaccine in a pentavalent formulation did not interfere with the production of effective antiopioid IgG antibodies. Multivalent vaccines could provide multifaceted, yet selective, protection against polydrug use and exposure.

Autotransfecting short interfering RNA through facile covalent polymer escorts.

Short interfering ribonucleic acids (siRNAs) are important agents for RNA interference (RNAi) that have proven useful in gene function studies and therapeutic applications. However, the efficacy of exogenous siRNAs for gene knockdown remains hampered by their susceptibility to cellular nucleases and impermeability to cell membranes. We report here new covalent polymer-escort siRNA constructs that address both of these constraints simultaneously. By simple postsynthetic click conjugation of polymers to the passenger strand of an siRNA duplex followed by annealing with the complementary guide strand, we obtained siRNA in which one strand includes terminal polymer escorts. The polymer escorts both confer protection against nucleases and facilitate cellular internalization of the siRNA. These autotransfecting polymer-escort siRNAs are viable in RNAi and effective in knocking down reporter and endogenous genes.

Star polymers with a cationic core prepared by ATRP for cellular nucleic acids delivery.

Poly(ethylene glycol) (PEG)-based star polymers with a cationic core were prepared by atom transfer radical polymerization (ATRP) for in vitro nucleic acid (NA) delivery. The star polymers were synthesized by ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and ethylene glycol dimethacrylate (EGDMA). Star polymers were characterized by gel permeation chromatography, zeta potential, and dynamic light scattering. These star polymers were combined with either plasmid DNA (pDNA) or short interfering RNA (siRNA) duplexes to form polyplexes for intracellular delivery. These polyplexes with either siRNA or pDNA were highly effective in NA delivery, particularly at relatively low star polymer weight or molar ratios, highlighting the importance of NA release in efficient delivery systems.

Anti-Strychnine Immunoconjugate Reduces the Effects of Strychnine-Induced Toxicity in Mice.

Strychnine poisoning induces seizures that result in loss of control of airway muscles, leading to asphyxiation and subsequent death. Current treatment options are limited, requiring hands-on medical care and isolation to low-stimulus environments. Anticonvulsants and muscle relaxants have shown limited success in cases of severe toxicity. Furthermore, nonfatal strychnine poisoning is likely to result in long-term muscular and cognitive damage. Due to its potency, accessibility, and lack of effective antidotes, strychnine poses a unique threat for mass casualty incidents. As a first step toward developing an anti-strychnine immunotherapy to reduce or prevent strychnine-induced seizures, a strychnine vaccine was synthesized using subunit keyhole limpet hemocyanin. Mice were vaccinated with the strychnine immunoconjugate and then given a 0.75 mg/kg IP challenge of strychnine and observed for seizures for 30 min. Vaccination reduced strychnine-induced events, and serum strychnine levels were increased while brain strychnine levels were decreased in vaccinated animals compared to the control. These data demonstrate that strychnine-specific antibodies can block the seizure-inducing effects of strychnine and could be used to develop a therapeutic for strychnine poisoning.