Research ArticleDrug Development

Nanoscavenger provides long-term prophylactic protection against nerve agents in rodents

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Science Translational Medicine  02 Jan 2019:
Vol. 11, Issue 473, eaau7091
DOI: 10.1126/scitranslmed.aau7091

A long-lasting poison scavenger

Nerve agents are neurotoxic compounds found in pesticides and chemical weapons. They act by blocking the transmission of nerve impulses to the muscles. After exposure, fatal consequences occur within minutes. Now, Zhang et al. report the development of a nanoparticle-based bioscavenger (nanoscavenger) that breaks down organophosphate nerve agents into innocuous compounds. Prophylactic treatment of rats and guinea pigs with this nanoscavenger revealed its low immunogenicity and good biodistribution. Treated animals were protected from repeated exposure to the nerve agent sarin over 7 days, suggesting that this nanoscavenger might be an effective prophylactic treatment for preventing nerve agent poisoning in subjects at risk.


Nerve agents are a class of organophosphorus compounds (OPs) that blocks communication between nerves and organs. Because of their acute neurotoxicity, it is extremely difficult to rescue the victims after exposure. Numerous efforts have been devoted to search for an effective prophylactic nerve agent bioscavenger to prevent the deleterious effects of these compounds. However, low scavenging efficiency, unfavorable pharmacokinetics, and immunological problems have hampered the development of effective drugs. Here, we report the development and testing of a nanoparticle-based nerve agent bioscavenger (nanoscavenger) that showed long-term protection against OP intoxication in rodents. The nanoscavenger, which catalytically breaks down toxic OP compounds, showed a good pharmacokinetic profile and negligible immune response in a rat model of OP intoxication. In vivo administration of the nanoscavenger before or after OP exposure in animal models demonstrated protective and therapeutic efficacy. In a guinea pig model, a single prophylactic administration of the nanoscavenger effectively prevented lethality after multiple sarin exposures over a 1-week period. Our results suggest that the prophylactic administration of the nanoscavenger might be effective in preventing the toxic effects of OP exposure in humans.


As one of the most dangerous chemical families, organophosphorus (OP) compounds were developed not only as highly effective pesticides but also as chemical warfare agents (1). Such compounds irreversibly inhibit acetylcholinesterase (AChE) in chemical synapses, causing neuromuscular paralysis throughout the entire body and, subsequently, death by asphyxiation (2). The most potent nerve agent VX, for example, has a median lethal dose (LD50) of as low as 12.6 μg/kg in mice (3). These extremely toxic molecules are capable of causing death within minutes when exposed to skin or when inhaled, making it extremely difficult to protect against OP or to treat after OP exposure. OPs appeared in several wars and terrorist attacks. In 2013, thousands of civilians in Syria died due to the usage of sarin in the civil war, and additional tragedies have occurred more recently. Although the most recent anticholinergic antidotes could prevent lethality if administered shortly after exposure, the effects of OPs, including convulsions, incapacitation, performance deficits, and permanent brain damage, would still be devastating (4, 5). OP pesticides, which seem less toxic when compared with other nerve agents, pose an even greater threat to the public health due to their wide applications (6). According to the World Health Organization, 200,000 deaths per year in developing countries were attributed to OP pesticide poisoning (7).

In addition to the post-poisoning treatment, researchers have been seeking a prophylactic strategy to counteract OP threats (8). Protein-based nerve agent bioscavengers are promising candidates because of their ability to scavenge OP compounds in the bloodstream before intoxication. This could avoid the potential side effects and exclude the requirement of rapid administration of the antidotes after exposure (9). In general, bioscavenger proteins function either by stoichiometric binding to OPs or by catalytic hydrolysis of nerve agents into biologically inactive products. The former category includes proteins that bind to nerve agents at a one-to-one ratio, such as cholinesterases and carboxylesterases. Although some candidates of this type seem promising due to their natural occurrence in the human blood, such as human butyrylcholinesterase (10), they have quite low efficiency in OP scavenging due to the stoichiometric binding mechanism. The use of catalytic bioscavengers is considered to be relatively advantageous compared to the use of their stoichiometric counterparts (8). However, because most of these proteins are derived from microorganisms, they suffer from immunologic and pharmacokinetic problems (8). For instance, organophosphorus hydrolase (OPH) is highly effective in hydrolyzing OPs, but its circulation half-life is less than an hour in rodents (1115). For an effective bioscavenger, one dosage should be able to protect human weeks to a month against nerve agents. As a result of these challenges, despite the intensive work over the last 60 years, the current bioscavengers only provide protection within hours after the administration; therefore, there is still an unmet need for effective bioscavengers with long-lasting protection and no side effects. Here, we report the development of a nanoparticulate bioscavenger (nanoscavenger) that offers long-term prophylactic protection against nerve agent poisoning. The nanoscavenger is engineered by coating a zwitterionic nonfouling polymer layer on the surface of an OPH enzyme. When evaluated in rodent models, the nanoscavenger showed long-circulating permanence and minimal immunological responses. One single prophylactic administration of the nanoscavenger protected the animals from multiple nerve agent exposures in a 1-week period.


Nanoscavenger preparation and characterization

When constructing the nanoscavenger, we decided to use OPH enzyme as the core due to its ability to decompose nerve agents into biologically benign products (8). A thin ultrahydrophilic semipermeable poly(carboxybetaine) (PCB) polymer layer was coated on the OPH surface as a protective shell (as shown in fig. S1). The bioscavenger nanoparticle was fabricated by coating PCB polymer hydrogel onto the surface of OPH protein. Dynamic light scattering (DLS) showed an average hydrodynamic size of 32 nm, with polydispersity index of 0.22 ± 0.03 (Fig. 1A). Zeta potential showed a neutral particle surface charge (fig. S2). The dry size of these hydrogel nanoparticles was revealed as about 10 nm under transmission electron microscopy (TEM) (Fig. 1B). Circular dichroism (CD) showed no change in the secondary structure of the OPH enzyme before and after the polymer coating (Fig. 1C). Enzyme kinetics was studied using paraoxon as the substrate (fig. S3). The Michaelis constants (Km) were 0.12 and 0.10 mM for native OPH and nanoscavenger, respectively. The catalytic efficiency (kcat/Km) was determined to be 2.4 × 107 s−1 M−1 for both OPH and encapsulated OPH, indicating that the coated gel layer did not affect the free diffusion of OP molecules into the catalytic center. The storage stability was tested by storing the nanoscavenger in a solution under 4°C for 6 months. More than 90% of its activity was reserved after the long-term storage (Fig. 1D).

Fig. 1 Characterizations of the nanoscavenger.

(A) DLS measurement of the nanoscavenger and native OPH. (B) TEM image of the dried nanoscavenger. (C) CD spectra of the nanoscavenger and native OPH. (D) Storage stability of the samples. The samples were stored for 6 months, and the data are presented as remaining activity after storage.

Pharmacokinetics, immunogenicity, and biodistribution of the nanoscavengers

As a prophylactic countermeasure against nerve agent poisoning, long vascular residence time is a critical requirement for the bioscavenger candidates (16). In addition, post-exposure therapy in scenarios of percutaneous exposure with ongoing agent transfer from skin into the systemic circulation requires long-lasting therapeutic concentrations of bioscavengers to detoxify free agent continuously (17). The pharmacokinetic parameters of unmodified and encapsulated OPH were evaluated by determining the time course of OPH activity in serum after intravenous administration in healthy Sprague-Dawley rats. As shown in Fig. 2A, native OPH was rapidly cleared out from the circulation after administration. By contrast, long circulation behavior was observed in the nanoscavenger group. One compartment model was used to analyze the pharmacokinetic parameters, which are listed in table S1. After the initial injection, unmodified native OPH exhibited a half-life (t1/2) of 0.43 hours, in general agreement with the values reported in the literature (14, 16). The circulation time of nanoscavenger demonstrated a ~60-fold improvement over the native enzyme, with a half-life of 26.2 hours. Moreover, the AUC (area under curve) of the nanoscavenger was 58 times that of the native OPH, indicating higher systemic availability. Pharmacokinetic evaluation of subcutaneous injection also demonstrated a similar trend that the nanoscavenger had significantly longer vascular residence time and higher bioavailability than the native enzyme (fig. S4A). For prophylactic applications, multiple doses of bioscavenger enzymes may be needed to prolong protection against OP poisoning. However, repeated dosing of biological substances may trigger the accelerated blood clearance (ABC) phenomenon (18). To test the performance of nanoscavengers after multiple doses, a second injection was given 2 weeks after the initial administration and pharmacokinetic profiles were evaluated as before (Fig. 2A and fig. S4B). No difference was found between the two doses of nanoscavengers, indicating the likelihood that they could maintain their pharmacokinetic performance after repeated doses.

Fig. 2 Pharmacokinetics, immunogenicity, and biodistribution of the nanoscavenger.

(A) Pharmacokinetic profiles of native OPH versus nanoscavenger after repeated dosing in rats. The second dose was injected 2 weeks after the first dose. n = 6. (B) Detection of anti-OPH antibodies by direct enzyme-linked immunosorbent assays (ELISAs). IgG, immunoglobulin G; IgM, immunoglobulin M; I.V., intravenous. (C) Biodistribution of the nanoscavengers. Each value is averaged from three rats. SDs are shown as error bars. Paired Student’s t test was used to compare two small sets of quantitative data. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

We explored the immunogenicity of the OPH-containing nanoparticles in further detail by collecting and testing sera 3 weeks after the second injection. We compared sera from animals treated with the OPH-containing nanoparticles with those from animals treated with OPH alone. As shown in Fig. 2B and fig. S5, high titers of anti-OPH antibodies were found in the groups treated with native OPH via both administration routes (intravenous and subcutaneous). The antibody response was strongly reduced in rats treated with nanoscavengers, suggesting that PCB gel coating mitigated antibody induction. Because anti-polymer antibodies are becoming a growing concern for protein therapeutic formulations such as preexisting and therapy-induced anti–poly(ethylene glycol) (PEG) antibodies (1922), the presence of anti-PCB antibodies was tested in this work. As shown in figs. S6 and S7, no anti-PCB antibodies were observed for either of the administration routes.

To examine the biodistribution of the nanoscavengers, we gathered blood and tissue samples from treated rats 72 hours after injection. The samples were labeled with a fluorescent probe for this test. As shown in Fig. 2C, the nanoscavenger was still present in blood, whereas the native OPH was completely cleared from circulation. Similar to most long-circulating nanoparticles, accumulation of nanoscavengers was mainly found in the liver and spleen. OPH was observed to have less organ accumulation compared with encapsulated enzymes due to different levels of protease stability. Short-term toxicity of the accumulated nanoscavengers was assessed by histological analysis of liver, spleen, and kidney. As shown in fig. S8, no inflammation and cellular damage were observed in these tissues. A hemolysis assay was performed to evaluate the blood compatibility of the nanoscavenger, and no hemolytic activity was observed for both native OPH and the nanoscavenger (fig. S9).

Detoxification and prophylactic efficacy against paraoxon

To explore the detoxification efficacy of the nanoscavenger in vivo, we first conducted a dosage study in a rat model of OP poisoning. Two times of the median lethal dose (2 × LD50, 0.86 mg/kg) (23) of paraoxon, the active metabolite of the insecticide parathion, was injected subcutaneously on the back of each rat, and different dosages of OPH or nanoscavengers were administered intravenously through the tail vein immediately after the OP injection. Once entered into blood vessels, OP molecules irreversibly inhibit butyrylcholinesterase activity; thus, the butyrylcholinesterase activity in blood was measured as the biomarker for OP intoxication (24). As shown in Fig. 3A, the administered OP compounds almost completely inhibited blood butyrylcholinesterase activity in the untreated groups. By contrast, both native OPH and nanoscavenger preserved butyrylcholinesterase from OP inhibition. Five minutes after paraoxon injection, the no treatment and bovine serum albumin (BSA) nanogel control groups exhibited serious signs of OP poisoning, including muscle twitch, salivation, tremors, and respiratory depression. The administration of bioscavengers ameliorated the symptoms induced by paraoxon challenge, and a dosage of 10 to 20 μg/kg completely stopped the onset of intoxication (Fig. 3B). Figure 3C summarizes the animal survival rate measured as the number of death in the first 1.5 hours after OP administration. Both no treatment and BSA nanogel control groups showed rapid death ~10 to 15 min after paraoxon administration due to its acute neurotoxicity. By contrast, the administration of bioscavengers (4 μg/kg) successfully prevented lethality. Considering that the rat blood volume is approximately 64 ml/kg, it can be roughly estimated that the minimum blood concentration of OPH to prevent lethality is 60 ng/ml, and 160 ng/ml is capable of protecting rats from suffering poisoning symptoms. Both native OPH and the nanoscavenger behaved similarly in this set of tests.

Fig. 3 Detoxification efficacy study of the nanoscavenger in vivo.

A dosage of 2 × LD50 paraoxon was injected subcutaneously in rats. Immediately after paraoxon injection, different dosages of native OPH or nanoscavenger were injected intravenously; BSA nanogel control–treated and untreated animals were used for comparison. Blood was drawn 10 min after OPH or nanoscavenger administration. (A) Blood butyrylcholinesterase activity, (B) intoxication signs, and (C) survival rates after paraoxon administration. Each group consisted of six animals. No significant difference was found between the native OPH and nanoscavenger group. One-way analysis of variance (ANOVA) with Bonferroni posttests was used to compare the treated groups with the no-treatment group and to calculate P values. *P < 0.05, **P < 0.01, ***P < 0.001.

Next, a time-dependent prophylactic efficacy test was performed to evaluate the protection time window provided by the nanoscavengers. The experimental design is shown in Fig. 4A. Nanoscavengers at a dosage of 1 mg/kg were given intravenously at time 0, and 2 × LD50 paraoxon was administered subcutaneously at different time points after nanoscavenger administration. In this test, each animal only received one OP injection. On the basis of the circulatory profiles and the minimum blood OPH concentration required as determined above, it was estimated that the protection time window lies in 24 hours after injection for native OPH and around 1 week for the nanoscavenger. The time points to perform paraoxon challenge were selected on the basis of this estimation. The results are shown in Fig. 4 (B to D). Because of the fast elimination in blood, unmodified OPH provided complete protection (without symptom and death) against 2 × LD50 paraoxon only for 3 hours and a 6-hour time window for the prevention of lethality. By contrast, the long-circulating nanoscavenger protected animals for 6 days without any signs of intoxication and 7 days without lethality (Fig. 4, B to D). Similarly, another set of experiments was carried out to explore the protective efficacy by subcutaneous injection (fig. S10). Because of the slow absorption of large molecules, the subcutaneously injected native OPH exhibited a prolonged vascular residence time compared with intravenous administration, thus demonstrating a longer complete protection time window of 6 hours as well as a lethality protection time window of 15 hours. In contrast, nanoscavenger had a shorter protection time window compared with its own intravenous performance because of the reduced bioavailability. The complete protection was maintained for 3 to 4 days, whereas lethality protection lasted 5 days (fig. S10, B to D).

Fig. 4 Time-dependent prophylactic efficacy test by intravenous administration.

(A to D) The test agents were injected intravenously via the tail veins first (t = 0 hours) at a dosage of 1 mg/kg into rats, and then 2 × LD50 paraoxon was injected at different time points later by subcutaneous administration on the back of each rat. A different group of rats was used for each time point. (A) Schematic showing the experimental design. (B) Survival rates, (C) blood butyrylcholinesterase activity, and (D) intoxication signs after each paraoxon administration. Each group consisted of six animals. (E and F) The bioscavengers were injected intravenously via the tail veins first (t = 0 hours) at a dosage of 1 mg/kg into rats. Three hours later (t = 3 hours), 2 × LD50 paraoxon was injected by subcutaneous administration on the back of each rat. The paraoxon administration was repeated every 24 hours to the survived rats until all animals died (blue arrow represents the bioscavenger administration, and each red arrow represents one paraoxon exposure). (E) Top: Schematic showing the experimental design. Bottom: Survival rates and (F) intoxication signs after each paraoxon administration (n = 3 in saline control group; n = 6 in native OPH and nanoscavenger groups).

To further evaluate the performance of nanoscavenger under different circumstances, as multiple or continuous contacts of OP agents might be encountered in civilian and military settings, we performed a repeated exposure test. In this test, a group of animals received a pretreatment (t = 0 hours) of nanoscavenger via intravenous administration, followed by multiple OP challenges at different time points until death. The experimental design is shown in Fig. 4E. All rats in saline control group did not survive the first challenge of 2 × LD50 paraoxon at the 3-hour time point (Fig. 4E), whereas the other two prophylactically treated groups demonstrated complete protection against the first challenge. The second OP challenge was given to survived rats 24 hours after OPH or nanoscavenger administration, and native OPH failed to protect animals at this time point; 83% of the animals treated with native OPH died after this second challenge (Fig. 4E). Repeated paraoxon injection was then given to the survived rats every 24 hours after the second challenge, and complete protection was observed until the sixth challenge performed 5 days after the nanoscavenger pretreatment (Fig. 4F); at this time point, 50% of the animals treated with the nanoscavenger died.

Prophylactic efficacy against sarin

Last, we tested the protection potency of the nanoscavenger platform against sarin (GB), an OP chemical weapon. Sarin has appeared in multiple wars and terrorist attacks over the last 30 years, which caused thousands of deaths. The wild-type OPH has relatively lower catalytic activity toward sarin substrate; thus, we used the OPH-YT variant with optimized activities (12) against G-type nerve agents to build the sarin-effective nanoscavenger. This evaluation compared the protective efficacy afforded by both OPH-YT and the nanoscavenger-YT after multiple exposures to lethal doses of GB in guinea pigs. Before the test, we first confirmed the long-circulating property of the nanoscavenger in the guinea pig model (fig. S11 and table S2). During this test, the animals first received a pretreatment of nanoscavenger or OPH-YT (5 mg/kg) via intravenous administration (carotid catheter), followed by one sarin injection (2 × LD50, 87 μg/kg, subcutaneously) 20 min after enzyme. Multiple exposures were subsequently administered every 24 hours until death. As shown in Fig. 5, two of five animals in the native enzyme group died after the second exposure of sarin, and no animal survived after the third exposure. By contrast, all animals in the nanoscavenger group were well protected until the eighth exposure, and four of six animals displayed only mild signs of intoxication after the ninth exposure at day 8. Overall, the nanoscavenger provided full protection to guinea pigs receiving a cumulative exposure of 14 × LD50 of GB. These results highlighted the superb protection capacity offered by nanoscavengers against sarin poisoning.

Fig. 5 Survival rates of the guinea pigs after repeated sarin exposure.

The bioscavengers were injected intravenously via a catheter (day 0, blue arrow) into guinea pigs at a dosage of 5 mg/kg. Sarin (2 × LD50) was then administered at 20 min and then repeatedly every 24 hours to the survived animals (red arrows). *Four animals did not show intoxication signs 1 hour after the ninth exposure at day 8 (n = 5 in native OPH-YT group; n = 6 in nanoscavenger-YT group).


OP poisoning remains as a major medical issue for public health, and nerve agents intimidate both soldiers and civilians in war zones. The acute poisoning nature demands effective therapeutic and prophylactic interventions. The concept of a prophylactic bioscavenger that eliminates toxic molecules in blood before intoxication was proposed decades ago (8). Since then, numerous efforts have been devoted to searching for a qualified candidate, including stoichiometric and catalytic bioscavengers. Stoichiometric bioscavengers have been the subject of intensive research but have limitation due to the extremely low scavenging efficiency (8). On the other hand, although the later approach based on catalytic bioscavengers presents great advantages over the former approach in terms of scavenging efficiency, in vivo applications are hindered due to inadequate pharmacokinetics and strong protein immunogenicity (8). Because of these challenges, there is still no approved bioscavenger to date that can provide long-term prophylactic protections for practical applications.

In this study, a nanoscavenger capable of hydrolyzing OPs was developed by chemical modification of OPH enzyme with zwitterionic materials. The two key problems faced by catalytic bioscavengers were addressed by coating the enzyme with a zwitterionic PCB gel network, forming a nanocapsule that confers excellent “stealth” properties upon the protein core. Zwitterionic polymers have been extensively studied as nonfouling materials for various biomedical applications due to their high hydration property and low protein adsorption (25). Surfaces modified with PCB have exhibited protein adsorption below 0.3 ng/cm2 from 100% blood plasma or serum (26). When coated on a nanoparticle surface, the PCB layer resists opsonization, inhibits cellular uptake, and extends circulation half-life (27). Previously, conjugation of PCB to model proteins has been shown to stabilize them without sacrificing their affinity to substrates (28) and increase circulation half-life with minimal immunogenicity (29). The gel encapsulation strategy further promoted these merits by providing complete protein surface coverage (30). When the OPH enzyme is encapsulated in a hydrated zwitterionic gel nanocapsule, the prolonged blood residence time makes long-term protection feasible and the porous gel network still allows free diffusion of small OP molecules into the catalytic site. In a rat model, the catalytic nanoscavenger demonstrated long vascular residence time with minimal immune responses. As a microorganism-derived protein, the fast clearance remains one of the major obstacles that hinder the translation of OPH into clinical use (8). OPH has a dimeric structure with a molecular weight of ∼72 kDa, slightly larger than the kidney clearance threshold of protein (∼60 kDa) (31). The irreversible uptake by reticuloendothelial system might be a major culprit for the rapid clearance of native OPH. Coating the native protein with a highly hydrated zwitterionic polymer gel not only markedly increased its hydrodynamic size but also hid the enzyme core from immune system, ensuring its long vascular residence. Because rodents have much smaller size and faster metabolism when compared to human, we would expect a much longer circulation half-life in human. Consulting the agents that have comparable circulatory behavior in rodents, it is reasonable to expect a human half-life of more than 100 hours (32). In addition, no ABC was observed for the nanoscavenger in the repeated administration tests. ABC phenomenon is a direct consequence of humoral immune responses elicited by multiple administrations of the same exogenous biologic. Reduced vascular residence time of bioscavengers will limit their performance when multiple administrations are on demand. With no ABC phenomenon, it is likely for the nanoscavenger to maintain its performance after repeated administrations.

Benefiting from its long blood residence time and high catalytic efficiency, the nanoscavenger demonstrated desired protection efficacy against OP. In addition to the rescue of animals after lethal OP challenge, a single prophylactic intravenous injection of the nanoscavenger has successfully protected rats from paraoxon poisoning for over a week. In a guinea pig model, the long-circulating property of the nanoscavenger was confirmed, and a nearly 1-week prophylactic protection time window was achieved against the real nerve agent sarin. By contrast, most of the bioscavengers on record were tested prophylactically only minutes to hours after pretreatment (4, 9, 15, 3335). In addition to the intravenous route, the successful protection provided by subcutaneous administration demonstrated its flexibility in administration routes, which is critical under circumstances when intravenous administration is not applicable. Because the vascular residence time of modified proteins in human can be several-fold longer than in rodents (32), we can reasonably expect a protection time window of weeks to a month in human prophylactic applications, which should be long enough to fulfill the mission of bioscavengers.

Under multiple OP exposure challenges, a “leakage” phenomenon might shorten the protection time window compared to the single-exposure test. In brief, when each time an animal is exposed to nerve agents, the agents will distribute through the bloodstream and out into the rest of the body. Although the endogenously administered nanoscavenger will hydrolyze most of the nerve agent molecules, a small portion will inevitably reach and irreversibly bind to their physiological target AChE without signs of intoxication. This leakage will accumulate after multiple OP exposures, and during this process, the victim becomes more and more vulnerable to OP intoxication until death occurs. The catalytic mechanism has intrinsic advantages over stoichiometric counterparts, particularly in dealing with multiple contact situations, because the nanoscavengers will not be consumed in the detoxification process. These results proved that a long-term protection can also be achieved with pretreatment of nanoscavengers under rigorous multiple OP exposure situations.

Evaluation in a guinea pig model confirmed that the nanoscavenger made from OPH-YT provides effective protection against GB. Because the YT variant has similar catalytic activities across G-type OPs (GB, GD, and GF) (12), we can reasonably conclude the effectiveness of nanoscavenger as a prophylactic countermeasure against the threats from this major type of nerve agents. The field is constantly seeking for a bioscavenger system that counteracts a broad range of nerve agents across both G-type and V-type OPs. As a major limitation, the current catalytic nanoscavenger only demonstrated sufficient activity against G-type agents. Continuous efforts need to be devoted in developing enzymes with high activity in hydrolyzing V-type nerve agents. Analogs and mutation strategies have been shown to greatly facilitate the optimization of OPH for the hydrolysis of VX by more than two orders of magnitude (13). By changing the enzyme core into different variants, nanoscavenger against different nerve agents might be easily fabricated. A nanoscavenger cocktail against a broad spectrum of OP compounds might also be feasible with the rational design of OPH variants.

Collectively, the nanoscavenger demonstrated the potential to be translated to counteract OP threatening in both clinical and military settings. However, the development is still in an early phase, and several issues need to be resolved in the preclinical tests. First, a large-scale fabrication process of the nanoscavengers needs to be developed, and a quality control system should be established to ensure batch-to-batch consistency. Second, the formula of the nanoscavenger consists of nonbiodegradable PCB polymers. Although our data demonstrated its safety in short term, its metabolism and long-term safety require further investigation. Last, the protection efficacy needs to be confirmed in a large animal model, preferably nonhuman primates.


Study design

This study was designed to examine the prophylactic protection efficacy of the constructed nanoscavengers against nerve agent poisoning. A Sprague-Dawley rat model was first used to determine the pharmacokinetic parameters, immune responses, and protection efficacy against paraoxon. All experiments using rats as the animal model were conducted at the University of Washington, adhered to federal guidelines, and were approved by the University of Washington Institutional Animal Care and Use Committee. The test groups were randomly assigned, data were analyzed by blinded observers, and biological replicate numbers were stated with each result. Hartley guinea pig was used as the animal model to repeat the pharmacokinetic test and to perform protection efficacy tests against sarin. Animal experiments using guinea pigs as the animal model were conducted at the U.S. Army Medical Research Institute of Chemical Defense (USAMRICD). The experimental protocol used for this work was approved by the Animal Care and Use Committee at the USAMRICD, and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and the Animal Welfare Act of 1966 (P.L. 89-544), as amended.

Nanoscavenger preparation

To synthesize the nanoscavenger, OPH or OPH-YT was first modified to introduce acryloyl groups onto its surface. Initially, acryloyl groups were introduced onto the protein by reacting surface lysine residues with N-acryloxysuccinimide. However, serious protein aggregation and precipitation were observed during reaction because of the increased hydrophobicity of the proteins (fig. S12). Inspired by the fact that glycine betaine, which is the main component of the carboxybetaine acrylamide (CBAA) monomer, is a natural osmolyte and is used as a protein stabilizer, we changed the feeding order of this reaction—CBAA monomers and cross-linkers were added before the first acryloylation step. In the presence of CBAA monomers, OPH protein remained stable throughout the whole reaction process, and the nanoscavengers were successfully fabricated by free radical polymerization. The reaction was performed by dissolving 10 mg of OPH, 100 mg of CBAA monomer, and 20 mg of CBAAX cross-linker into 5 ml of 50 mM Hepes buffer (pH 8.5), followed by adding 50 μl of N-acryloxysuccinimide dimethyl sulfoxide (DMSO) solution (20 mg/ml) dropwise. The reaction was stirred at 4°C for 2 hours. The polymer encapsulation was done via in situ radical polymerization by adding 4 mg of ammonium persulfate and 16 μl of tetramethylethylenediamine into the former reaction solution. After stirring for another 2 hours, the reaction mixture was concentrated and washed extensively by phosphate-buffered saline (PBS) (pH 7.4) using 150-kDa molecular weight cutoff centrifugal filters. The nanoscavengers were then passed through a hydrophobic interaction column (Phenyl-Sepharose CL-4B, GE Healthcare Life Sciences) to remove any unencapsulated proteins using PBS (pH 7.4) (50 mM and 0.1 mM CoCl2) as elution buffer. The final products were then concentrated and stored at 4°C for further studies. The nanogel control sample was prepared following the same procedure except changing OPH to BSA.

Pharmacokinetic study in rats

The pharmacokinetics of native and modified OPH was studied using Sprague-Dawley rats (female; body weight, 74 to 100 g) as animal model. Each sample group has six animals. For pharmacokinetic studies, each protein sample was administered intravenously via tail vein or subcutaneously on the back at a dosage of 1 mg/kg body weight. The dosage of nanoscavenger refers to the amount of enzyme within the nanoparticles. Blood samples were collected from the tail vein at different time points after the injection. The blood samples were put in heparinized vials and centrifuged, and the enzyme content in plasma was estimated by activity assay using paraoxon as the substrate at a concentration of 2 mM. The enzyme activity was converted into the concentration using a standard curve, as shown in fig. S13. The injections and bleeding procedure were repeated twice with a 2-week time interval between each injection. Five weeks after the first injection, the animals were euthanized by overdose of isoflurane followed by confirmatory thoracotomy, and blood was taken for antibody measurements. The concentration-time profiles after administration were analyzed using PKSolver following instructions (36). One-compartment model was used to calculate pharmacokinetic parameters.

Biodistribution study in rats

Samples used for biodistribution test were labeled by fluorescein isothiocyanate (FITC). OPH or nanoscavengers were dissolved in 0.1 M sodium carbonate buffer (pH 9) at a protein concentration of 2 mg/ml. Then, 50 μl of FITC DMSO solution (1 mg/ml) was added slowly to each milliliter of the sample solution. The reaction was kept at 4°C for 8 hours in the dark. After reaction, the labeled OPH and nanoscavengers were concentrated and washed extensively with PBS (pH 7.4) using 50- or 100-kDa molecular weight cutoff centrifugal filters.

For biodistribution study, rats (n = 3) were injected with the FITC-labeled samples through the tail vein. At 72 hours after injection, all rats were euthanized by overdose of isoflurane followed by confirmatory thoracotomy, and the heart, liver, spleen, lung, kidney, and blood were collected for further analysis. The collected organ tissues were homogenized using a tissue ruptor, followed by centrifugation at 3200g for 30 min at room temperature. The fluorescence of particles in the tissues was measured using a microplate reader.

Dosage-dependent efficacy study in rats

A dosage of 2 × LD50 paraoxon (0.86 mg/kg) dissolved in pharmaceutical-grade saline was injected subcutaneously into each group of Sprague-Dawley rats (n = 6) under isoflurane anesthesia. Before the paraoxon administration, 50 μl of blood was drawn from the tail vein to measure the blood butyrylcholinesterase activity. Immediately after paraoxon injection, different dosages of OPH or nanoscavenger were injected intravenously into each group. The dosages of OPH include 1, 2, 4, 10, and 20 μg/kg. BSA nanogel control was injected at a dosage of 100 μg/kg, and saline control was injected at the same volume. Ten minutes after OPH injection, another 50 μl of blood was drawn from the tail vein to measure the blood butyrylcholinesterase activity. Butyrylcholinesterase activity was measured using a commercially available activity kit (Arbor Assays) following the manufacturer’s protocol. Symptoms (such as tremors, salivation, and respiratory depression) were observed and scored within 90 min after injection. The clinical signs were scored on the basis of a 0 to 4 scale. The score definitions are listed in table S3. Survival rates for 90 min after OP injection were recorded. Any rats that survived a maximum of 90 min after challenge were euthanized immediately by overdose of isoflurane followed by confirmatory thoracotomy.

Prophylactic protection efficacy study in rats

The test bioscavengers were injected by intravenous or subcutaneous administration first (t = 0 hours) at a dosage of 1 mg/kg into rats (n = 6) under isoflurane anesthesia, and then 2 × LD50 paraoxon was injected at different time points later by subcutaneous administration on the back. Blood (50 μl) was drawn from the tail vein before and 10 min after paraoxon injection to measure the blood butyrylcholinesterase activity change. Symptoms and death rates were observed and recorded within 90 min after injection. In this set of tests, each animal received one injection of paraoxon.

Repeated paraoxon exposure test in rats

The test bioscavengers were injected intravenously via tail vein first (t = 0 hours) at a dosage of 1 mg/kg into rats (n = 3 in saline control group; n = 6 in native OPH and nanoscavenger groups). Three hours later (t = 3 hours), 2 × LD50 paraoxon was injected by subcutaneous administration on the back. Symptoms and death rates were observed and recorded in the same way as previous tests. At t = 24 hours, another injection of 2 × LD50 paraoxon was given to the survived animals, and symptoms and death rates were recorded as previously described. The paraoxon administration was repeated every 24 hours to the survived rats until all animals died.

Pharmacokinetics in guinea pigs

Native OPH and nanoscavenger were administered to male Hartley guinea pigs (n = 3 per formulation; weights, 335 ± 23 g; means ± SD) via a carotid catheter (5 mg/kg). Blood samples were collected (via toe nail clip) from the animals into heparinized tubes at multiple time points after administration. The samples were centrifuged to separate the plasma, which was subsequently frozen and stored at −80°C until assay. The activity of OPH was determined in these samples by colorimetric assay; in brief, using MOPS buffer (50 mM at pH 7.0 and 20°C), diluted plasma samples (200 μl) were incubated with 5 mM paraoxon and change in absorbance at 405 nm was measured over 5 min. To determine concentration, standard curves using the stock formulations were constructed, and activities were used to interpolate the concentration. All standard curves and samples were assayed in triplicate.

Repeated sarin exposure test in guinea pigs

Male Hartley guinea pigs (n = 11; weights, 332 ± 19 g; means ± SD) were administered OPH-YT (5 mg/kg) (n = 5) or nanoscavenger-YT (5 mg/kg) (n = 6) intravenously via a carotid catheter. Animals were exposed to 2 × LD50 of GB (87 μg/kg) in a saline vehicle via subcutaneous injection on the caudal lateral flank area 20 min after enzyme administration followed by repeated exposures every 24 hours for 8 days. Survival was assessed every 24 hours with immediate exposure to surviving animals. Negative control animals died due to intoxication within 20 min after exposure.


Paired Student’s t test was used to compare two small sets of quantitative data from pharmacokinetics, biodistribution studies, and ELISA experiments, with P < 0.05 being considered as statistically significant. One-way ANOVA with Bonferroni posttests were completed on data from paraoxon detoxification tests to determine significance and calculate P values. Differences between groups were considered statistically significant when P < 0.05.



Fig. S1. Protection mechanism of the nanoscavenger.

Fig. S2. Zeta potential of the native OPH enzyme and nanoscavenger.

Fig. S3. Enzyme kinetic measurements of the native OPH and nanoscavenger.

Fig. S4. Pharmacokinetic profiles of the native OPH versus nanoscavenger after subcutaneous injections.

Fig. S5. Serum anti-OPH antibody titers after subcutaneous administration.

Fig. S6. Detection of anti-PCB antibodies in nanoscavenger groups by direct ELISAs.

Fig. S7. Detection of anti-PCB antibodies in nanoscavenger groups by SPR sensors.

Fig. S8. Histologic examinations of rat organs.

Fig. S9. Hemolytic activity tests of the nanoscavengers.

Fig. S10. Time-dependent prophylactic efficacy test by subcutaneous administration.

Fig. S11. Pharmacokinetic profiles of the native OPH versus nanoscavenger in guinea pigs after intravenous administrations.

Fig. S12. DLS of the OPH protein before and after acryloylation.

Fig. S13. Standard curves used to convert enzyme activity into concentration.

Table S1. Pharmacokinetic parameters after intravenous and subcutaneous injections in rats.

Table S2. Pharmacokinetic parameters after intravenous administration in guinea pigs.

Table S3. Intoxication score definitions.


Funding: This work was supported by the Defense Threat Reduction Agency (HDTRA1-13-1-0044) and the University of Washington. This work was partially supported by the Defense Threat Reduction Agency–Joint Science and Technology Office, Medical S&T Division (CB3945). This research was also supported, in part, by an appointment to the Postgraduate Research Participation Program at the USAMRICD administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAMRMC. The views expressed in this manuscript are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. Author contributions: P.Z. and S.J. conceived and designed the project. E.J.L., J.M., A.N.B., and F.M.R. produced OPH and OPH-YT enzymes. P.J. synthesized the zwitterionic monomer and cross-linkers. P.Z., Y.C., and W.L. performed the nanoscavenger preparation and characterizations. P.Z. and C.T. performed in vivo experiments in rats and analyzed the samples. F.S., H.-C.H., and Z.Y. helped to analyze the blood samples. S.A.K. and D.M.C. designed the in vivo experiments in guinea pigs. M.V.B., T.L.D., S.J.D., C.L.C., C.A.B., and T.C.O. performed the in vivo experiments in guinea pigs. P.Z. and S.J. wrote the paper. All authors discussed the results and commented on the manuscript. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: All data are present in the paper and/or the Supplementary Materials.
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