Advancements in bioscavenger mediated detoxification of organophosphorus poisoning

Abstract

Background

Organophosphorus compounds, widely used in agriculture and industry, pose a serious threat to human health due to their acute neurotoxicity. Although traditional interventions for organophosphate poisoning are effective, they often come with significant side effects.

Objective

This paper aims to evaluate the potential of enzymes within biological organisms as organophosphorus bioclearing agents. It analyses the technical challenges in current enzyme research, such as substrate specificity, stereoselectivity, and immunogenicity, while exploring recent advancements in the field.

Methods

A comprehensive review of literature related to detoxifying enzymes or proteins was conducted. Existing studies on organophosphorus bioclearing agents were summarised, elucidating the biological detoxification mechanisms, with a particular focus on advancements in protein engineering and novel delivery methods.

Results

Current bioclearing agents can be categorised into stoichiometric and catalytic bioclearing agents, both of which have shown some success in preventing organophosphate poisoning. Technological advancements have significantly improved various properties of bioclearing agents, yet challenges remain, particularly in substrate specificity, stereoselectivity, and immunogenicity. Future research will focus on expanding the substrate spectrum, enhancing catalytic efficiency, prolonging in vivo half-life, and developing convenient administration methods.

Conclusion

With the progression of clinical trials, bioclearing agents are expected to become widely used as a new generation of therapeutic organophosphate detoxifiers.

Introduction

Organophosphorus compounds (OPs) are widely used in agriculture and industry, posing a significant threat to human health due to their potent neurotoxicity. These compounds irreversibly inhibit the enzyme acetylcholinesterase (AChE) by covalently binding to a critical serine residue in its active site. This binding prevents AChE from breaking down acetylcholine (ACh), a crucial neurotransmitter. The resulting accumulation of ACh at neural synapses disrupts signal transmission, leading to a cascade of severe neurological symptoms and systemic damage. Further complicating treatment, some phosphorylated AChE can undergo a process called “aging,” where a side chain within the phosphorylated enzyme-phosphate complex is cleaved. This “aged” AChE is less susceptible to reactivation by conventional treatments, making it even harder to restore normal nerve function. These processes culminate in the accumulation of acetylcholine at the neural synapses, thereby blocking neural signal transmission and triggering a series of severe neurological symptoms and systemic damage.

It can also inhibit butyrylcholinesterase (BChE) and penetrate the blood–brain barrier (BBB), causing inactivation of AChE at synaptic junctions in the central nervous system.¹ The accumulation of acetylcholine at synapses can lead to asphyxiation and death. Even if patients survive, residual OPs released from storage sites can result in subsequent brain damage and cholinergic syndrome.² Current treatments for organophosphorus nerve poisoning include reactivators, such as 2-pyridine aldoxime methyl chloride (2-PAM), obidoxime, and HI-6 dimethanesulfonate, as well as atropine and anticonvulsants. Additionally, supportive measures such as airway management, hemodialysis, and cardiopulmonary resuscitation can improve survival rates and alleviate poisoning symptoms.³ However, these drugs cannot mitigate the irreversible brain damage caused by OP poisoning and may also affect normal bodily functions, such as central nervous system damage, increased blood pressure, and accelerated heart rate.⁴

In recent years, there has been a surge in the development of enzymes, antibodies, and other proteins with organophosphate hydrolysis activity, serving as biological scavengers against OPs. These substances can be administered intravenously or absorbed via the alveoli, enabling them to effectively neutralize the toxicity of OPs before they reach their targets. Furthermore, they exhibit sustained activity within the body, preventing the prolonged toxicity of OPs. Animal studies have unequivocally confirmed the efficacy and safety of these biological scavengers in both preventing OP poisoning and neutralizing post-exposure toxicity.^5,6

Classification of biological scavengers

Biological scavengers are enzymes and proteins with OP hydrolytic activity found in bacteria or mammals. As shown in Fig. 1, biological scavengers and their nanoparticles can be delivered to the body through intravenous injection, where they neutralize and degrade OP in the blood or at synapses to achieve detoxification. Biological scavengers currently used for the detoxification of OPs are classified into three categories¹: stoichiometric bioscavengers. These include cholinesterase (ChE), carboxylesterase (CarE), and other β-esterases. They irreversibly bind to OP in a 1:1 ratio, inactivating both themselves and the OP.² catalytic bioscavenger. These are enzymes that catalyze the hydrolysis of OPs, producing non-toxic products. They have significant advantages over stoichiometric bioscavengers. A small amount of catalytic bioscavenger can detoxify a lethal dose of organophosphorus nerve agent (OPNA). They provide sustained protection without being consumed.³ pseudocatalytic bioscavenger. These are a type of dual-component catalytic system, consisting of a stoichiometric bioscavenger and a reactivator.⁷ One example of this category is the mixture of ChE and oxime drugs. The function of this dual-component system depends on the rate at which the phosphorylated group is displaced to reactivate the enzyme. To achieve a detoxification effect, this rate must be faster than the deactivation rate caused by OP.⁸

As essential life-saving agents, biological scavengers must possess attributes that make them efficient detoxifiers. High catalytic efficiency is a crucial attribute of biological scavengers. The detoxification reaction must occur quickly to prevent the organism from being poisoned. Even in severe cases of poisoning, the concentration of OP in the blood remains low. Stoichiometric and catalytic bioscavengers should exhibit a strong binding affinity for OPs and possess high catalytic efficiency, typically characterized by the K_cat/K_m ratio. Additionally, pseudocatalysts, besides requiring a regeneration rate surpassing the deactivation rate, should also demonstrate high catalytic efficiency. Another desired property of biological scavengers is in vivo stability. Being foreign proteins to the human body, they can elicit an immune response, leading to their elimination from circulation. Therefore, these scavengers should have low immunogenicity to maintain their activity in the bloodstream.⁹ Broad-spectrum substrate specificity is another crucial property for effective biological scavengers. Ideally, such scavengers should exhibit rapid and efficient detoxification of a wide range of highly toxic OPs. Additionally, overcoming delivery challenges, particularly in crossing BBB, is essential. They must be easily deliverable and capable of penetrating BBB to detoxify OPs present in the central nervous system.

Stoichiometric bioscavengers

AChE, BChE, and carboxylesterase (CarE) are some exaples of stoichiometric scavengers. Among these, AChE poses a unique challenge. Due to its crucial role in regulating the cholinergic nervous system, freely circulating AChE can disrupt neural transmission and lead to severe side effects. This renders it unsuitable for direct use as a biological scavengers.¹⁰ Researchers have addressed this issue by immobilizing AChE onto red blood cell (RBC) membranes to form nanoparticles (RBC-NPs), which retain AChE activity and effectively degrade OPs in the blood.¹¹

BChE is the most extensively studied cholinesterase and exhibits better neutralization activity against OPs compared to that of AChE. Thus, it can effectively treat human patients suffering from OP poisoning. Theoretical calculations suggest that an intravenous injection of BChE at a dose of 200-300 milligrams into the human body can increase the erythrocyte AChE activity by 30% under the LD₅₀ dose of agents such as VX, soman, and sarin.¹² Additionally, pre-injection of 200 mg of BChE intravenously in humans can increase the LD₅₀ of nerve agents 2–5 times.¹³ Notabley, HuBChE received FDA approval in 2006 for clinical research and has since progressed to phase I clinical trials.¹⁴

CarE is found in various tissues in the human body. CarE is found in the liver and relatively stable. It can retain its activity for several months when stored at room temperature.¹⁵ In addition, carE exhibits broad specificity towards neutral OP compounds, with double molecular rate constants (K_II) greater than 106 M⁻¹ min⁻¹ for Tabun,¹⁶ Sarin, Soman,¹⁷ Diisopropyl fluorophosphate, and Paraoxon. As the OP concentration is far lower than the dissociation constant (K_d) of the complex between OP and stoichiometric scavengers or the Michaelis constant (K_m) of catalytic scavengers, the detoxification rate occurs under first-order conditions. The reaction kinetics are controlled by the product of K_II and the concentration of biological scavengers in the blood, forming the first-order rate constant. Therefore, to meet detoxification rate requirements, it is necessary to ensure a sufficiently high concentration of biological scavengers.¹⁸

Catalytic bioscavenger

Catalytic bioscavengers offer advantages over stoichiometric scavengers, as they can achieve preventive or detoxification effects at lower doses. This efficiency stems from their catalytic nature, where a single enzyme molecule can neutralize multiple OP molecules. Their catalytic rates also follow the equation,¹ where K_II = K_cat/K_m, with K_cat representing the catalytic constant and K_m representing the Michaelis constant. To achieve effective in vivo detoxification, the actual therapeutic dose of enzymes must be less than 1 mg/kg, and K_II must exceed 5 × 10⁷ M⁻¹ min⁻¹. Several naturally occurring enzymes can catalyze OP clearance, including mammalian paraoxonases, organophosphorus hydrolases from Brevundimonas, diisopropyl fluorophosphatase (DFPase) from European squid (Loligo vulgaris), and organophosphorus hydrolases from Alteromonas.¹⁰ These enzymes with catalytic hydrolysis activity for OPs are listed in Table 1. Additionally, some enzymes in the body exhibit OP hydrolysis activity, but their K_II is low and not discussed further. For example, senescence marker protein-30 (SMP30) has a K_II of 0.086 mM⁻¹ min⁻¹ for Paraoxon,¹⁹ and dipeptidyl peptidase has a K_II of only 58 mM⁻¹ min⁻¹ for Soman, which is far below the requirements for effective in vivo detoxification.²⁰

Paraoxonase (PON) has both phosphotriesterase (PTE) and arylesterase activities. Human PON-1 is the most promising catalytic scavenger candidate. It is secreted by the liver and transport to various parts of the body by low-density lipoprotein, where it activates water molecules into hydroxide ions to attack the phosphorus atom within the molecule. This process hydrolyzes OPs into diethyl phosphate and other metabolites, which are water-soluble and easily removed from the body through urine. Zhao et al.²¹ compared the preventive effects of rabbit PON1 with traditional detoxifiers, a mixture of atropine and chlorpyrifos. They found that intravenous injection of rabbit PON1 10 min before poisoning significantly reduced mortality and accelerated the recovery of cholinesterase activity in the blood. The effect was superior to that of atropine combined with chlorpyrifos, and the efficacy showed a significant positive correlation with the dose, with minimal toxic side effects. Therefore, it represents a valuable candidate for treating OP poisoning.

PTE, also known as organophosphorus hydrolase (OPH), belongs to the amidohydrolase family and can be found in various bacteria such as Brevundimonas diminuta, Pseudomonas, Rhodococcus, and Pseudomonas pseudoalcaligenes. Over the past few decades, mutants of phosphotriesterase (BdPTE) from B. diminuta have been investigated as catalytic biological scavenger against OPs. The active site of this enzyme is formed by a pair of zinc ions coordinated by several imidzole group from histidine and one acetylated lysine side chain, bridged by a nucleophilic hydroxide ion. Hydrolysis of OPs occurs when the hydroxide ion nucleophilically attacks the phosphorus atom within the molecule. PTE catalyzes the hydrolysis of organophosphorus insecticides, including parathion, methyl parathion, and fenitrothion, in a relatively short time. Additionally, PTE can hydrolyze nerve agents such as Sarin, Soman, and VX, although with lower catalytic efficiency compared to less toxic insecticides. Animal studies in mice have shown that intravenous injection of 1.5 U of PTE before or immediately after exposure to parathion can protect brain AChE activity against inhibition by parathion.

OpdA is isolated from the soil bacterium Radiobacter P230. It shares 90% amino acid sequence identity with PTE at its C-terminus,²² with OpdA being 20 amino acids longer than PTE. Unlike PTE, OpdA can hydrolyze insecticides such as malathion and phosphamidon. Moreover, it exhibits a higher turnover rate for OPs with shorter side chains.

DFPase, identified in European squid, is an effective phosphotriesterase capable of hydrolyzing various toxic G-series OP nerve agents, including diisopropyl fluorophosphate (DFP), Sarin, Soman, and cyclosarin (GF).²³ Upon substrate binding, the residue D229, coordinated with a calcium at the active site, becomes activated, enabling nucleophilic attack and oxidation of the substrate into non-toxic products. However, the naturally occurring DFPase exhibits a preference for less toxic stereoisomers of its substrates, limiting its effectiveness against a broader range of OPs. To address this limitation, researchers have utilized rational design to engineer enzyme mutants that flip their enantioselectivity, enhancing their activity against more toxic OPs.²⁴

SsoPox, a member of the phosphotriesterase-like lactonase (PLL) family, has attracted attention for its ability to detoxify OPs, including pesticides and nerve agents. It achieves this by breaking down the phosphate ester bonds, converting toxic OPs into less harmful substances. For instance, in the case of the nerve agent VX or pesticides similar to diazinon, SsoPox catalyzes the hydrolysis of the P-O bond, resulting in the formation of a glycol (a molecule with two alcohol groups) and a thiol (a compound containing a sulfur-hydrogen bond), or similar less toxic products. This mechanism is crucial for mitigating the toxic effects of OPs.²⁵ While SsoPox exhibits broad substrate specificity and high thermal stability, it does have limitations. These include lower catalytic efficiency compared to other enzymes in the phosphotriesterase family and potential immunogenicity when introduced into the human body. To address these issues, researchers are employing protein engineering techniques, such as mutation and directed evolution, to enhance the catalytic efficiency of SsoPox against specific OPs and improve its substrate specificity. Additionally, novel encapsulation technologies are being explored to improve its delivery stability within the body and reduce its immunogenicity.

Pseudocatalytic bioscavengers

Cholinesterases have been utilized as long-acting therapeutic biological scavengers for unreactive organophosphorus compounds, but their success has been limited because they react with organophosphorus nerve agents in a one-to-one stoichiometric manner. Pseudo-catalytic scavenger systems employ alkyne “click” chemistry to form an intermediate substance known as “cholinesterase-polymer-oxime conjugates”. These conjugates couple nucleophilic reactivators with stoichiometric biological scavengers. Covalently linking oxime polymers to butyrylcholinesterase surfaces slows down the deactivation rate induced by the nerve agent Sarin. Furthermore, when the enzyme is inhibited by Sarin, covalently linked oximes induce intermolecular and intramolecular reactivation. Intramolecular reactivation paves the way for the development of a new generation of nerve agent scavengers, combining biological speed and selectivity with the robustness and simplicity of synthetic chemicals.²⁶

Kovarik et al.²⁷ developed a combination of AChE mutant Y337A/F338A and HI-6 for Soman detoxification. Their study revealed enhanced catalytic scavenging of Soman in mice, leading to improved treatment outcomes characterized by delayed onset of toxic symptoms. This approach also circumvented AChE aging, thereby potentially reducing the required dose of BChE for effective treatment.

Subsequently, this team improved the detoxification function in Tabun or Sarin poisoning by adding bispyridinium oxime as a reactivator for BChE in pseudocatalytic scavengers.²⁸ In vitro tests indicate that approximately 50% of the activity of phosphorylated human plasma butyrylcholinesterase (BChE) can be restored within 20 min, ultimately reaching 70% of the control group’s activity. However, the pralidoxime mediated reactivation of BChE was slower, necessitating exploration of new reactivators to rapidly reactivate BChE and enhance the detoxification ability of this system while reducing the dose of BChE required.

However, there are pharmacokinetic compatibility issues between the enzyme and the reactivator in this system. Stability and permeability are two major factors affecting the absorption of biotechnology-derived drugs. The aggregation of biological macromolecules can impact both stability and permeability within the body due to increased molecular weight, thereby affecting their absorption efficiency. The increased molecular weight may also reduce the drug’s ability to cross vascular walls and cell membranes, thereby limiting its ability to reach target sites. Aggregated biological macromolecules may alter the distribution characteristics of original biological enzymes and reactivators within the human body. Their increased molecular weight can make it more difficult to penetrate biological barriers such as the BBB, potentially restricting distribution and action within specific tissues or organs The metabolic pathways of biological macromolecules obtained through polymer techniques may differ from those of the original molecules, which may affect their metabolic rate and products. Larger molecules may be more difficult for the kidneys to excrete, increasing accumulation and the risk of toxicity. While aggregation methods can improve the pharmacokinetics, pharmacodynamics, and immunological characteristics, they may also have adverse effects. Such modifications could reduce the drug’s target affinity, decrease efficacy, or increase its immunogenicity, potentially leading to adverse immune reactions.

Protein engineering of biological scavengers

Currently, biological scavengers face challenges such as high production costs, limited substrate specificity, inadequate thermal and pH stability, limited delivery methods, and short in vivo half-life. Additionally, their effectiveness observed in animal models often fails to translate directly to humans, necessitating extensive and expensive clinical trials. To address these challenges, researchers are employing various protein engineering techniques, such as rational design, random mutagenesis and directed evolution, to improve the substrate specificity and catalytic activity. Additionally, researchers are isolating variants from extreme environments or microorganisms to enhance enzyme thermal stability, pH stability, and antioxidative properties. Furthermore, nanoparticle synthesis techniques, such as cell membrane camouflage technology, and PEGylation are being utilized to overcome immunogenicity and penetrate the BBB. These advancements significantly improve the effectiveness of biological scavenger therapy and hold promise as a novel approach for treating OP poisoning in the future.

Large-scale production of biological scavengers

The production of stoichiometric scavengers, especially plasma derived BChE, is a significant limiting factor for their widespread use due to the high demand for human plasma and the high cost required for enzyme extraction and purification. New technologies have been developed that aim to lower the cost of the large production of biological scavengers. These innovations include recombinant enzyme expression, novel purification methods for serum-derived biological scavengers, gene delivery techniques for biological scavengers, and induced expression of endogenous biological scavengers. Addressing these production challenges is crucial for advancing the large-scale production of biological scavengers and making them more accessible for therapeutic applications.

Recombinant biological scavengers are primarily produced using eukaryotic systems, including yeast, insect cell, and mammalian cells. Mammalian cell expression systems, such as the AdenoVATOR system, have been explored for expressing recombinant human butyrylcholinesterase (rHuBChE) in human embryonic kidney 293T (HEK293T) cells.²⁹ This system produced rHuBChE in a tetrameric form with enzymatic and physicochemical properties similar to native HuBChE.

The U.S. Army Medical Research Institute of Chemical Defense (USAMRICD) and Pharmthene Biotechnology Company extracted large amounts of rHuBChE from transgenic goat milk, reaching up to 5 g/L, which is over a thousand times the content in human plasma.³⁰ Goats are cost-effective and have a rapid reproductive rate, making them a reliable biological source for producing rHuBChE for preventing and treating organophosphate poisoning.

Challenges in plasma purification arise from the turbidity and high-fat content in samples from voluntary blood donors, interfering with enzyme activity tests. Techniques such as plasma defatting have been developed to solve the issue of interference caused by lipids in enzymatic activity tests and simultaneously reduce the cost of purifying plasma BChE.³¹

Gene delivery methods can replace high-cost enzyme purification schemes. Adenovirus is used to deliver genes into hepatocytes, turning them into protein production factories that continuously produce therapeutic proteins to prevent organophosphate poisoning. Studies have demonstrated the potential of adenovirus mediated HuBChE gene therapy in resisting the toxicity of chemical warfare nerve agents.^32,33

Inducing the upregulation of endogenous cholinesterases, such as AChE, by using chemical compounds offers an alternative approach to purified enzyme therapy. For example, Forskolin has been shown to significantly enhance AChE expression in mouse neuroblastoma cells and human lung cells, providing protection against OP toxicity.³⁴

Improvement of substrate Spectrum and activity of biological scavengers

Currently, the substrate spectrum of most catalytic bioscavengers is relatively narrow, with a preference for reacting with the less toxic enantiomers. Moreover, the activity of most wild type (WT) catalytic bioscavengers do not meet the requirements for OP poisoning treatment. Rational design and directed evolution have been employed to modify the active sites, enhancing catalytic efficiency, broadening substrate specificity, improving enzyme reactivation (anti-aging), and reversing the stereo selectivity of WT enzymes.³⁵ Harvey et al.³⁶ developed a PTE mutant, H254G/H259W/L303T, which reverses the natural stereo specificity of PTE and preferentially catalyzes the hydrolysis of the highly toxic isomer of cyclosarin, GF. Job et al.³⁷ replaced two unpaired cysteine residues in BdPTE with multiple inert amino acids, resulting in improved catalytic activity against VX and other substrates. Bigley et al.³⁸ screened a library of 28,800 PTE variants and identified variants with K_II values for VX increased by over 1,500 times, with one variant exhibiting a 9,200-fold increase in activity relative to WT PTE. Jackson et al.³⁹ redesigned the active site of PTE based on a binding model with VX, creating favorable electrostatic interactions for hydrolysis. By constructing a saturation mutagenesis library, they identified three PTE mutants effective against V-type toxins, with K_II values as high as 5 × 10⁶ M⁻¹ min⁻¹. Most studies are primarily focused on PTE and PON, with high catalytic activity mutants showing potential for treating acute OP poisoning, as shown in Table 2. Goldsmith et al.⁴⁰ found that single mutants of BChE, such as G117D, G117E, and L286H, as well as several double and triple mutants based on G117H, could hydrolyze diethoxyphosphoryl sulfur choline, possibly due to His117 inducing water molecules to attack the phosphoserine bond, leading to enzyme reactivation and enhanced activity against multiple OPs. Sue et al.⁴¹ identified an OPAA mutant, Y212F/V342L, exhibiting stereo specificity for Sarin, and a phosphotriesterase mutant, GWT, with opposite stereo specificity, demonstrating that amino acid residue mutations can alter enzyme stereo selectivity and enhance the detoxification capacity of biological scavenger against highly toxic enantiomers.

Stability enhancement of bioscavenger

Enhancing the thermal stability, antioxidant properties, and in vivo circulation stability of bioscavengers is pivotal for their detoxification effectiveness. Thermal stability improvement can be achieved through two main approaches. The first one is isolating enzymes with superior thermal stability from thermophilic archaea, and the other one is to screen for heat-resistant mutants via directed evolution.

The enzyme SsoPox, derived from hyperthermophilic archaeon Sulfolobus solfataricus, exhibits good thermal stability. Through a combination of protein engineering, its activity and specificity have been fine-tuned to resemble that of PTE while exhibiting enhanced stability. Goldenzweig et al.⁴² engineered an AChE variant with 51 amino acid mutations, resulting in enhanced core packing, surface polarity, and backbone rigidity. Compared to WT hAChE, its expression level in Escherichia coli was much higher and exhibited a 20 °C higher thermal stability, without significant changes in the enzyme’s active site as determined by crystallography.

Substituting residues susceptible to oxidation, such as Cys and Met, can improve the enzyme’s antioxidant properties. Job et al.³⁷ engineered a variant of BdPTE by replacing two free Cys residues of the enzyme with Ala, resulting in enhanced antioxidant properties, as well as a 2 °C increase in thermal stability.

Injection of protein solutions of bioscavenger via intravenous route can lead to partial enzyme inactivation. Additionally, due to immunogenicity, foreign protein has a short half-life, resulting in a reduced detoxification effect of bioscavengers. To address these limitations, researchers have explored several strategies. One promising approach involves polymer modification, such as PEGylation. This technique increases the molecule’s size, making it more difficult for immune factors to recognize and clear it from the body, extending the bioscavenger’s half-life and allowing for sustained detoxification activity. Another innovative approach utilizes RBC membrane engineering. RBCs are modified to encapsulate the bioscavenger proteins within their membranes. This strategy leverages the natural circulation and extended lifespan of RBCs, providing a safe and biocompatible carrier system that protects the bioscavenger from premature degradation and prolongs its presence in the bloodstream, enhancing its overall detoxification potential.

Polyethylene glycol (PEG) modification of enzymes can increase the hydrodynamic volume of the enzyme, allowing it to maintain activity in the plasma for an extended period. Chilukuri et al. evaluated the pharmacokinetics of rHuBChE, natural serum derived HuBChE, and PEGylated rHuBChE in mice.²⁹ They found that the half-life time of HuBChE was 18.3 h, while PEGylated rHuBChE was 36.2 h, demonstrating that PEG modification can significantly prolong the half-life of rHuBChE. Sun et al.⁴³ through successive injections of HuBChE and HuBChE modified with PEG-5K or PEG-20K in mice, found that injection of PEGylated HuBChE had higher stability and weaker immune response compared to unmodified BChE. Cohen et al.⁴⁴ found that pre-administration of PEGylated recombinant enzyme via intravenous or intramuscular injection 20 h prior could effectively protect mice against VX, as demonstrated by a 1.3–1.5-fold increase of LD₅₀. Due to the existence of a BBB, delivery of BChE via peripheral routes can not reach the central nervous system. Gaydess et al.⁴⁵ utilized a drug delivery system based on cross-linked polyethylene glycol and polyethyleneimine nanoscale network to successfully deliver BChE to the central nervous system. This delivery technology greatly improves the tissue penetration and utilization of bioscavengers.

The RBC membrane engineering delivery system combines enzymes with biocompatible RBC membranes to improve the drug’s half-life in circulation. Smith et al.⁴⁶ developed an RBC-PEG-BChE delivery system, as shown in Fig. 2A, where each RBC can deliver over 2 million BChE molecules. They conducted in vitro activity evaluation using patient whole blood and found that compared to natural BChE, RBC-PEG-BChE had lower hemolysis rates and better cell tolerance. Pang et al.¹¹ also developed biomimetic nanoparticles composed of a polymer core surrounded by RBC membranes. Through in vitro studies, they demonstrated that biomimetic nanoparticles retained the membrane-bound AChE enzyme activity and could bind to OPs, counteracting their inhibitory effects on cholinesterases in vivo. The mechanism underlying RBC delivery system is illustrated in Fig. 2B.

PASylation technology involves the fusion or chemical conjugation of proteins, peptides, and small-molecule with naturally disordered biopolymer made of small L-amino acids, proline (Pro), alanine (Ala), and/or serine (Ser). This proline/alanine-rich sequence (PAS) is highly soluble in physiological solutions and stably adopts a random coil conformation,⁴⁷ thereby expanding the hydrodynamic volume, as shown in Fig. 2C. PAS is closest to PEG in terms of its uncharged and random-coil structure. Compared to PEG, the features of chemical conjugation and gene fusion allowed by PASylation make the manufacture of conjugates easy and inexpensive, especially for protein and peptide conjugates with pharmacological activity. Köhler et al.⁴⁸ synthesized a delayed plasma clearance complex (BdPTE-4) and a dual-specificity complex (BdPTE-7) widening the substrate spectrum using PAS protein modification technology. They evaluated the complexes’ hydrolytic ability towards VX and their detoxification ability in plasma and found that both exhibited better hydrolytic activity. Notably, BdPTE-7 showed good hydrolytic activity against both VX enantiomers, completely hydrolyzing 40 ng/mL of P(−)VX enantiomer within 45 min.

Improvement of delivery methods for bioscavengers

In addition to ingestion via the digestive tract, OPs can enter the human body through inhalation and skin absorption, as shown in Fig. 3A. Targeted local delivery method can effectively prevent poisoning. Researchers have improved the delivery methods of bioscavengers to achieve detoxification at the site of OP exposure, such as inhalation delivery, to neutralize OPs promptly and enhance preventive effects. Rosenberg et al.⁴⁹ reported a novel pulmonary delivery strategy using a vibrating mesh nebulizer (VMN) to deliver aerosolized recombinant human aer-rHuBChE into the lungs at a high rate, depositing in the lungs. By neutralizing OPs in situ, it prevents them from entering the systemic circulation, thus avoiding immune reactions in the body. This method can achieve a deposition rate of 50%–70% of BChE in the lungs of adults, forming an effective pulmonary biological barrier. In 2019, the team improved this aerosol delivery method by customizing a mesh spray system for macaques (Fig. 3B), extending the duration of lung deposition protection to 5 days.⁵⁰ Gordon et al.⁵¹ formed in situ water-soluble polyurethane and cholinesterase (ChEs) polymers in a porous polyurethane foam (Fig. 3C). The polymer can rapidly undergo in situ copolymerization at room temperature, and the ChEs within it exhibit high activity and stability. This decontaminating polyurethane foam can be used for skin decontamination of individules exposed to pesticides or Sarin nerve gas and other nerve agents.

Conclusion

Existing preventive and therapeutic measures for OPs provide short-term relief for patients but have lower cure rates, can cause side effects, and cannot completely eliminate residual OPs. Bioscavengers have shown significant efficacy in the prevention and treatment of OP poisoning and are one of the directions for the development of rescue solutions for OP poisoning.

Despite technological advancements, practical applications of bioscavengers for organophosphorus compound (OP) detoxification face challenges. Most catalytic bioscavengers have a relatively narrow substrate spectrum for organophosphorus compounds and exhibit insufficient activity against highly toxic isomers. Natural bioscavengers have inadequate stability within the boy, resulting in low utilization rates and requiring large doses. The lack of clinical trial data hampers the accurate extrapolation of detoxification effects in humans from animal studies. Nanotechnology and protein engineering are being continuously applied to this field, demonstrating the potential for developing highly efficient, stable, easily produced, and readily storable bioscavengers. Furthermore, research should explore new delivery methods to enhance the stability of bioscavengers within the body and reduce their immunogenicity. Finally, conducting more preclinical and clinical trials will be crucial for validating the safety and efficacy of these novel bioscavengers and advancing this field.

Author contributions

Hexi Li and Cong Lu are responsible for developing overarching research goals and aims, generating other research outputs, conducting comprehensive analysis of data, and writing of original draft. Zhenmin Liu and Pan Li are responsible for conducting the research and investigation process. Fengshun Xiang was responsible for constructing and revising the structure of the manuscript and writing the description of some concepts, while Bo Liu, Hongjuan Wang and Jie Chang were responsible for the visualization and data presentation of the article. Youwei Chen is responsible for reviewing and editing the writing. Jingfei Chen is responsible for ensuring the accuracy of descriptions and providing final approval of the version to be published. Youwei Chen and Jingfei Chen are both corresponding authors, and their contributions are equally significant.

Funding

This study was supported by the funding from The Youth Independent Scientific Innovation Fund Program (YK-202322).

Conflict of interest statement. The authors declare no competing interests.

Data availability

Data sharing is not applicable to this article as no new data were created or analyzed.

References

Author notes