Metal-Organic Frameworks for Single-Dose Engineered Enzyme-Based Prophylaxis and Treatment of Organophosphate Poisoning

Metal-Organic Frameworks for Single-Dose Engineered Enzyme-Based Prophylaxis and Treatment of Organophosphate Poisoning | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Metal-Organic Frameworks for Single-Dose Engineered Enzyme-Based Prophylaxis and Treatment of Organophosphate Poisoning Jeremiah Gassensmith, Ikeda Trashi, Orikeda Trashi, Fanrui Sha, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7799763/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Organophosphate (OP) poisoning is among the most lethal forms of chemical intoxication, commonly occurring in both warfare and agricultural settings. Although enzyme-based detoxification strategies, such as organophosphorus acid anhydrolase (OPAA), have shown promise in vitro , their rapid clearance and instability have severely limited their clinical translation. Here, we report the first in vivo study demonstrating the use of metal–organic frameworks (MOFs) as delivery depots for sustained OP detoxification. OPAA was successfully encapsulated within two MOFs—zeolitic imidazolate framework-8 (ZIF-8) and NU-1003—using biomimetic mineralization and passive diffusion strategies, respectively. These composites provided prolonged subcutaneous retention, preserved enzymatic activity, and offered complete protection in mice exposed to lethal doses of OP agents. In addition to immediate post-exposure rescue, the system also functioned as a long-acting prophylactic with minimal immunogenicity and excellent biocompatibility. This dual-use approach provides a powerful new platform for enzyme therapeutics, where both sustained activity and rapid response are required. Physical sciences/Nanoscience and technology/Nanomedicine/Drug delivery Physical sciences/Materials science/Biomaterials/Drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Organophosphates (OPs), widely used as pesticides and chemical warfare agents (e.g., sarin, VX), remain a persistent global threat to both civilian and military populations. 1 , 2 Acute exposure causes rapid systemic toxicity, as exemplified by the 1995 Tokyo subway attack, 3–6 while chronic exposure continues to affect millions of agricultural workers worldwide. 2 , 3 , 7 At the molecular level, OPs such as malathion, chlorpyrifos, and diisopropylfluorophosphate (DFP) irreversibly inhibit acetylcholinesterase, leading to cholinergic crisis, seizures, respiratory depression, and often death. 8 – 11 Standard treatments (atropine and pralidoxime, 2-PAM) require intravenous infusion, trained personnel, and often fail to prevent delayed neurological damage. 12 , 13 Their short systemic half-lives—70 min for atropine and 4 h for 2-PAM—contrast sharply with the persistence of lipophilic OPs in adipose depots, leading to long-lasting toxicity that neither therapy can realistically address during a crisis response. 14 – 18 This pharmacokinetic mismatch underscores the need for countermeasures that act both rapidly and durably, capable of functioning as a single-dose treatment or prophylactic. Catalytic bioscavengers such as organophosphate acid anhydrolase (OPAA), engineered from extremophile-derived proteins, exhibit high levels of catalytic activity against fluorine bonds, making them attractive for neutralizing fluorine-containing OPs by hydrolyzing them into benign products. 19 However, their poor pharmacokinetics (rapid clearance and low serum stability) hinder their translation into future therapies. Protein nanocarriers—including liposomes, dendrimers, and polymers—have been explored, 20–26 yet they suffer from poor shelf stability, reliance on intravenous delivery, and limited ability to match the long half-life of OPs. Metal–organic frameworks (MOFs) provide a compelling alternative. These crystalline porous materials combine high surface area, tunable pore size, and modular chemistry, making them attractive platforms for biomacromolecule stabilization and delivery. MOFs can encapsulate enzymes via biomimetic mineralization or post-synthetic infusion, protecting them from proteolysis, thermal degradation, and preserving their catalytic activity while enabling controlled release. Prior work has demonstrated MOF-mediated delivery of antibiotics, chemotherapeutics, enzymes, and vaccines, 27–30 but their potential for continuous enzyme-mediated detoxification, particularly against OPs, remains unexplored in vivo . 31 – 33 Here, we report a unique approach using a subcutaneously injectable enzyme–MOF depots that deliver sustained release of OPAA for weeks in vivo. Using two complementary strategies—biomimetic mineralization with zeolitic imidazolate framework-8 (ZIF-8) and passive loading into the large-pore NU-1003—we generated composites that remain localized at injection sites and gradually release active enzyme. These depots bridge the gap between acute rescue and long-term prophylaxis, providing rapid therapeutic response while maintaining extended protection. In lethal OP challenge models, OPAA@MOFs functioned as both prophylactic and therapeutic countermeasures, with NU-1003 in particular exhibiting minimal immunogenicity and maintaining efficacy under repeated dosing. Together, these results establish a generalizable strategy for single-dose enzyme therapeutics using a system capable of delivering bifunctional therapeutic effects. Results and Discussion Subcutaneous and intramuscular injections are long-standing routes for achieving slow release of biomolecules into circulation. Yet, building depots that can actually keep therapeutic proteins intact for weeks inside living tissue is notoriously difficult. Once deposited, proteins are immediately subjected to immune surveillance, proteolysis, and a host of clearance mechanisms that conspire to strip away activity. 34 , 35 Even the heat of the body is enough to accelerate unfolding, aggregation, and hydrolysis, gradually dismantling depot formulations that appear stable in vitro. 36 Metal–organic frameworks (MOFs) have emerged as an unusually effective way to fight back against these pressures. 37 – 40 By locking enzymes inside crystalline lattices, MOFs not only shield them from proteolysis but also dramatically extend their thermal lifetime, often to the point where refrigeration is no longer needed. Delicate assemblies that would otherwise disintegrate within hours at room temperature — such as proteoliposomes — can be stabilized for months when mineralized inside a MOF. 39 Two encapsulation strategies stand out. The first is MOF-based biomimetic mineralization, in which MOF nucleation takes place directly on the protein surface. In this setting, the macromolecule itself acts as a local template, creating a pseudo-high concentration of metal ions that catalyze crystallization. Ligand and metal ratios can be tuned to sculpt particle size. 41 Applying this approach, we purified OPAA (Figure S1 A) and encased it into a ZIF-8 shell, yielding 300–500 nm crystals that retained the faceted morphology of pristine ZIF-8 (Figs. 1 A–B ) . The second strategy is direct infiltration into pre-formed, large-pore frameworks. NU-1003, a Zr-based MOF with ~ 4.4 nm channels, is particularly suited to this purpose: water-stable, robust, and large pore apatures ideal for diffusion of large proteins. 31 – 33 , 42 – 46 Passive diffusion of OPAA into the MOF likewise did not alter the morpohology of the crystalline particles (Figs. 1 C–D). Unlike ZIF-8, NU-1003 has never been tested for enzyme delivery in vivo , making it an intriguing foil. To assess structural stability after enzyme encapsulation, powder X-ray diffraction (PXRD) was performed to evaluate the crystallinity of both MOFs. The resulting diffractograms confirmed that the MOF structures remained crystalline post-loading ( Fig. 1 E ) . To visualize cargo uptake and release from the MOFs, Cy7-labeled OPAA was synthesized and encapsulated within the MOF particles. Confocal laser scanning microscopy (CLSM) was employed to verify successful encapsulation of OPAA within the MOFs. Z-stack imaging revealed the fluorescently labeled protein localized inside the MOF structures ( Fig. 1 G; animation of z-stacks avaliable in the SI ) . To confirm that MOF encapsulation preserves OPAA’s structural integrity and enzymatic function, we first encapsulated the protein and then released it via degrading the MOF shell—a process we refer to as exfoliation—using 0.01 M phosphate-buffered saline (PBS) pH 4.5 for OPAA@ZIF-8 and 0.1 M sodium bicarbonate pH 9.5 for OPAA@NU-1003. A native gel electrophoresis (Figures S2A, F) revealed that the recovered OPAA exhibited the same electrophoretic mobility as the native enzyme, indicating no alteration in charge or size. Enzymatic assays (Figures S2B, G) further demonstrated full retention of activity after exfoliation. Long-term cargo stability was assessed by storing OPAA@MOF dry constructs at room temperature for 90 d. Subsequent SEM (Figures S2C, D) and PXRD (Figure S2E) analyses showed no significant changes in MOF morphology or crystallinity. At the same time, native gel electrophoresis and enzymatic activity assays confirmed that the encapsulated and exfoliated OPAA maintained its structure and function (Figures S2F, G). Using our fluorescently labeled OPAA, we were able to evaluate encapsulation efficiency by analyzing the supernatant from the loading or encapsulation process. The encapsulation efficiency of the protein within both MOFs was determined to be approximately 99%, as measured by fluorescence intensity (Figure S3A ). PXRD analysis confirmed that the crystallinity of the MOF remained intact following encapsulation of Cy7-labeled OPAA. UV–Vis absorbance spectra demonstrated characteristic near-infrared emission for OPAA-Cy7 that was absent in the unlabeled OPAA, confirming successful dye incorporation. Additionally, SEM imaging revealed no detectable changes in MOF morphology after encapsulation, indicating structural integrity was preserved (Figures S3B, C, D) . Prior to performing in vitro experiments with mammalian cells, we evaluated the stability of NU-1003 in commonly used laboratory buffers, cell media, and in the presence of proteins. To ensure consistent mixing, all samples were incubated at room temperature on a rotisserie shaker. ZIF-8 is known to degrade in the presence of physiological ions, particularly phosphate and carbonate. 27 47 Interestingly, under similar conditions, we found that NU-1003 exhibits significantly greater stability, particularly in PBS buffer, after 24 h. Moreover, NU-1003 exhibited minimal morphological changes in Dulbecco’s Modified Eagle Medium (DMEM) and in complete medium DMEM + 10% FBS (fetal bovine serum), indicating greater robustness than ZIF-8. However, it was less stable than ZIF-8 in bicarbonate buffers (Figs. 2, S4) . The overall improved stability—bicarbonate notwithstanding—can be attributed to NU-1003’s strong Zr–O bonds and highly connected 8-node framework, which resists hydrolysis and structural degradation even under harsh conditions. In contrast, ZIF-8 has weaker Zn–N bonds that are more kinetically labile and degrade in comparatively milder environments. Figure 2. SEM micrographs showing the morphological stability of NU-1003 particles incubated under different conditions over time. NU-1003 was suspended in saline, 0.1 M potassium phosphate (KP) buffer (pH 7.4), 0.1 M phosphate-buffered saline (PBS, pH 7.4), complete cell culture media, and 0.1 M bicarbonate buffer (pH 9.5), imaged at 1, 4, 18, and 24 h. At each time point, samples were washed with water three times and imaged by SEM to assess particle integrity and morphological changes. Once encapsulation of OPAA within the MOFs was confirmed and their stability established, we proceeded with in vitro cytotoxicity testing in HEK293 cells, a widely used mammalian cell line. NU-1003, both enzyme-loaded and unloaded, was found to be less cytotoxic than ZIF-8 at equivalent concentrations after both 4 h and 24 h exposures. NU-1003 and OPAA@NU-1003 maintained more than 50% cell viability at both 4 h and 24 h, even at high concentrations such as 1 mg/mL, indicating that NU-1003 is effectively non-toxic at the tested concentrations. In contrast, ZIF-8 and OPAA@ZIF-8 cause a marked decline in cell viability at concentrations above 0.1 mg/mL, as previously reported. 48 Based on these findings, non-toxic concentrations were identified for both MOFs, which served as the basis for dosing in subsequent in vivo experiments ( Figs. 3 A and 3 B ) . To evaluate cellular uptake of the OPAA-loaded MOFs (OPAA@ZIF-8 and OPAA@NU-1003), we conducted uptake assays visualized using CLSM. To localize the internalized MOFs, cells were incubated with Cy7-OPAA-loaded MOFs for 4 h, then washed, fixed with 4% paraformaldehyde (PFA), blocked with 5% Bovine serum albumin (BSA), and stained with antibodies against RAB7A (late endosomes). The following day, cells were treated with appropriate secondary antibodies to visualize subcellular compartments and then counterstained with DAPI (nuclei) and Phalloidin (cytoskeletal membrane marker). CLSM imaging revealed that portions of the MOFs were internalized and localized within lysosomes, while some MOF material remained associated with the cell membrane ( Fig. 3 C ) . MOF-membrane interactions can be strong and complicate uptake studies by flow cytometry. We have previously found that rapidly washing cells in an acidic solution removes surface-bound MOFs; however, this procedure does not work on the acid-fast Zr series of MOFs. 49 We next evaluated the depot effect of both OPAA-Cy7@ZIF-8 and OPAA-Cy7@NU-1003 to assess their potential for use in treatment. OPAA is known to have a short in vivo half-life (~ 1.5 h) and is rapidly cleared from the body due to its small size. Despite exhibiting strong detoxification performance in vitro , the organophosphorus hydrolase family of proteins has shown limited efficacy in vivo against OP poisoning owing to its rapid clearance from tissues. 50 To confirm this limitation, free OPAA-Cy7 was injected subcutaneously into the flanks of mice, and fluorescence was monitored. The signal was completely absent after 2 h, confirming rapid clearance ( Fig. 4 C ) . In contrast, the MOF composites retained fluorescence in their injection sites very well. Both OPAA-Cy7@ZIF-8 and OPAA-Cy7@NU-1003 were injected subcutaneously in the flanks of mice, and fluorescence imaging was used to monitor release over time. The MOF-based formulations significantly prolonged the local retention of the enzyme. Notably, NU-1003 released OPAA-Cy7 more slowly than ZIF-8, maintaining a detectable signal for an additional 9 d compared to ZIF-8 ( Fig. 4 C ) . This prolonged release is attributed to the greater stability of NU-1003 in biological fluids as well as slower diffusion kinetics of OPAA-Cy7 from NU-1003 relative to ZIF-8. To assess the physicochemical effects of tissue residency from the MOF materials at the injection site, we examined the persistence of the materials in solid form and evaluated metal release into distal organs. Both ZIF-8 and NU-1003 initially formed stable deposits, consistent with our slow-release data. However, by day 24, ZIF-8 depots had fully degraded and were no longer detectable in the tissue, whereas NU-1003 remained present in solid form. To further investigate this, we excised MOF depots at both day 3 and day 24. On day 3, we found ZIF-8 more degraded than NU-1003, which aligns with our in vitro findings that physicochemical anions—in particular phosphate—were better able to degrade ZIF-8 than NU-1003 (Figure S5) . We then evaluated the biodistribution of released metals from NU-1003 by assessing Zr 4+ in major organs 72 h post-administration using inductively coupled plasma mass spectrometry (ICP-MS) (Figure S6) . Results indicated minimal Zr 4+ accumulation in the kidney, liver, and lungs. Notably, in contrast to ZIF-8, no detectable Zr 4+ was found in lymphatic tissue. Therapeutic Treatment Given that one of the primary causes of death from OP poisoning is the rapid onset of severe neurological symptoms—often within the first 10 min of exposure—we next evaluated whether enzyme–MOF depots could act quickly enough to function as effective post-exposure therapeutics. To mimic realistic intoxication scenarios such as accidental ingestion or occupational exposure, mice were challenged with a lethal oral dose of DFP (4 mg/kg), followed immediately by subcutaneous injection of saline, free OPAA, OPAA@ZIF-8, or OPAA@NU-1003 (Fig. 5 A). This design allowed us to directly probe both the speed and efficacy of each intervention under acute, high-stakes conditions. In addition to monitoring survival, we performed a double-blinded panel of neurobehavioral assays that quantify the hallmarks of OP toxicity: nociceptive distress, tremor, neuromuscular strength, and postural stability. These readouts are critical because survival alone does not capture the extent of long-term neuromuscular injury that often follows OP exposure. Grimace scoring ( Fig. 5 B ) showed that mice treated with saline or free OPAA exhibited signs of pain and discomfort, whereas mice treated with OPAA@ZIF-8 and especially OPAA@NU-1003 had significantly reduced grimace scores, reflecting lower distress. Similarly, tremor severity ( Fig. 5 C ) was significantly reduced in both MOF-treated groups compared to the saline and free OPAA groups. The OPAA@NU-1003 group showed the lowest tremor scores, indicating greater protection against acute neurotoxicity. Grip strength testing provided a more quantitative measure of neuromuscular protection (Fig. 5 D). In saline- and OPAA-treated cohorts, animals exhibited a significant loss of strength following DFP administration. In contrast, OPAA@ZIF-8 (p = 0.1380) and OPAA@NU-1003 (p = 0.2258) animals showed no significant decline, indicating that MOF depots preserved motor strength. To confirm this effect across groups, we compared the absolute change (Δ) in grip strength between treatments ( Figure S9 ). Both MOF depots exhibited significantly smaller declines compared to saline or OPAA controls, while the two depots did not differ from each other. Thus, the apparent “non-significance” in the within-group MOF analyses reflects the absence of neuromuscular decline, which becomes clear when comparing the magnitude of change across groups. To evaluate the impact of treatment on postural balance and motor coordination, an incapacitance test was performed ( Fig. 5 E ) . This test measures the difference in weight-bearing between the hind limbs. Higher ΔW values indicate imbalance and impaired motor function. Saline- and OPAA-treated mice showed significant imbalance, while OPAA@ZIF-8 and especially OPAA@NU-1003 treated mice maintained normal postural stability, indicating preserved motor control. Survival analysis ( Fig. 5 F ) showed a stark contrast between groups. All mice treated with saline or free OPAA died within 30 min after DFP exposure. In contrast, 100% of mice treated with OPAA@ZIF-8 or OPAA@NU-1003 survived the full 20-day observation period without exhibiting behavioral signs of toxicity. Histological analysis (H&E staining) was performed across all treatment groups (Figure S8) . These data collectively demonstrate that MOF-based delivery of OPAA provides complete protection against lethal challenge, resulting in 100% survival, compared to 0% survival in treated controls. Further, the surviving animals show significantly reduced neurotoxic symptoms when compared to controls—they largely maintain motor function and postural stability, even after lethal OP exposure. The NU-1003 formulation, in particular, offers enhanced protection without the confounding issue of immunogenicity, likely due to its slower and more sustained enzyme release profile as well as the absence of observable metal in the draining lymphatic tissue, as shown in Figure S6 . Another critical application of this technology is its use as a preventative countermeasure in high-risk environments, such as agriculture or military operations, where a single prophylactic dose could be administered before OP exposure. MOFs are particularly well-suited for this role, as their slow and sustained release properties enable long-term enzyme delivery, overcoming the rapid systemic clearance that limits traditional enzyme therapies and makes repeated dosing impractical. A central challenge in deploying exogenous proteins, such as OPAA, is the potential for adaptive immune responses, including the production of opsonizing IgG or T-cell activation, which can ultimately compromise therapeutic efficacy through accelerated blood clearance (ABC) or hypersensitivity reactions. Such responses are not limited to proteins; even synthetic macromolecules, including polymers like polyethylene glycol (PEG), are susceptible to immune recognition, sometimes resulting in the rapid loss of bioavailability for PEGylated drugs. While small proteins are often weakly immunogenic unless adjuvanted, the creation of long-lived depots for slow protein release raises a legitimate concern: the risk of eliciting high-titer IgG that can neutralize circulating protein and drive ABC. 51 Notably, ZIF-8 has emerged as a potent adjuvant, capable of inducing strong IgG responses against encapsulated payloads. 38 This observation is consistent with our histological data, which suggests an immune response is explicitly elicited by ZIF-8-based formulations. Given that OPAA release from MOF depots persists for over 20 d, we established a long-term prophylactic model to rigorously test both the durability of enzyme protection and the immunological consequences of repeated dosing (Fig. 6 A). Mice were given a single subcutaneous injection of saline, free OPAA, OPAA@ZIF-8, or OPAA@NU-1003 on day 0, and were left untreated for 60 d to allow clearance of any unencapsulated enzyme. On day 60, each group received a second dose of the same formulation, simulating a repeat-exposure scenario. For biologic drugs, the induction of antibodies—especially long-lived IgG—can compromise efficacy and safety by ABC or triggering hypersensitivity reactions. 52 We therefore measured anti-OPAA antibody titers before and after dosing ( Fig. 6 B ) . These data show that while OPAA@ZIF-8 strongly stimulates both an initial (IgM) and a lasting (IgG) antibody response, consistent with robust immunological memory, OPAA@NU-1003 induces only minimal IgG and little or no detectable IgM, mirroring the low immunogenicity of unencapsulated enzyme. Importantly, this lack of strong class-switched antibody response—where initial IgM is gradually replaced by longer-lived IgG—as demonstrated with NU-1003, supports its suitability for repeated or long-term prophylactic administration, minimizing the risk of depot failure owing to immune-mediated clearance. To assess the efficacy of repeated MOF-based prophylactic treatments, neurobehavioral evaluations were conducted 30 min after the DFP challenge on day 63. Tremor intensity was recorded using the Racine scale ( Fig. 6 C ) . Mice treated with saline or free OPAA at both time points exhibited high tremor scores, indicating no protection. In contrast, mice treated with OPAA@ZIF-8 and OPAA@NU-1003 exhibited significantly lower tremor scores, with the NU-1003 group showing the most pronounced suppression of tremor activity. Grip strength was measured before and after DFP exposure and normalized to body weight (Fig. 6 D). Saline- and OPAA-treated groups again showed significant post-challenge declines, whereas OPAA@ZIF-8 and OPAA@NU-1003 did not, indicating preserved motor function. To confirm this across groups, we compared the absolute change (Δ grip strength) between treatments ( Figure S9 ) and found that both MOF depots exhibited significantly smaller losses than controls, with no difference between ZIF-8 and NU-1003. Facial grimace scores were used to assess nociceptive responses ( Fig. 6 E ) . The saline and free OPAA groups exhibited high levels of facial tension. In contrast, mice treated with either MOF formulation exhibited significantly lower grimace scores, indicating a reduction in pain-related behavior. Postural balance was evaluated using an incapacitance test ( Fig. 6 F ) . Mice treated with saline or OPAA showed substantial imbalance (higher ΔW), whereas OPAA@ZIF-8 and OPAA@NU-1003 groups showed improved hind limb weight distribution. The NU-1003 group had the lowest ΔW values, indicating better motor coordination. To assess the durability of protection and the potential for ABC after repeated prophylactic dosing, we subjected mice from the cohort described in Fig. 6 to two independent OP challenges (Fig. 7 A). Each group received an initial depot injection on day 0 and a second on day 60, followed by a lethal DFP challenge (4 mg/kg) on day 63. As expected, saline- and free OPAA–treated mice did not survive beyond 30 min, confirming the acute lethality of DFP and the lack of durable protection from non-depotized enzyme. In striking contrast, all mice treated with OPAA@ZIF-8 or OPAA@NU-1003 survived the initial DFP challenge, demonstrating robust and persistent protection after repeat MOF-based prophylaxis. To thoroughly evaluate long-term depot efficacy, mice that survived were re-exposed to a second lethal dose of DFP on day 84. Animals treated with OPAA@ZIF-8 maintained 100% survival up to day 90, demonstrating the sustained release and enduring protective effect of this depot. The OPAA@NU-1003 group experienced some mortality after the second challenge; however, its performance was statistically not significant compared to OPAA@ZIF-8. Overall, MOF-based release systems showed a significant survival advantage over the controls (Fig. 7 B). Curiously, despite the strong antibody response elicited by ZIF-8, protective efficacy was preserved, indicating that circulating antibodies did not completely neutralize depot-released OPAA during the experimental period. These findings underscore the remarkable promise of MOF-based depots for sustained, repeatable prophylaxis against OP poisoning, even when immune activation occurs. They also highlight the importance of further investigating depot design and immune interplay to maximize durability and safety. To check the chronic toxicity in the organs, histology was performed, and tissues were observed for any sign of toxicity. Histological examination was conducted on major organs—including heart, lung, liver, kidney, spleen, and brain—following prophylactic treatment and subsequent DFP exposure (Figure S10) . Conclusion We developed two OPAA-loaded MOFs—OPAA@ZIF-8 and OPAA@NU-1003—for OP detoxification. NU-1003 demonstrated higher morphological stability in physiological buffers compared to ZIF-8, maintaining its structural integrity over 24 h. In vitro cytotoxicity studies showed that NU-1003 was less toxic to HEK293 cells than ZIF-8 at equivalent concentrations. In vivo fluorescence imaging confirmed that both MOFs enabled prolonged subcutaneous retention of OPAA, with NU-1003 displaying slower release kinetics. Both OPAA@ZIF-8 and OPAA@NU-1003 effectively reduced neurotoxic symptoms and significantly improved survival in mice challenged with lethal doses of DFP. Importantly, NU-1003 did not elicit any observable immune response, despite functioning as a long-term depot for the sustained release of an exogenous protein. A single prophylactic dose of MOF-formulated OPAA conferred long-term protection, with therapeutic efficacy maintained even after a second OP challenge. Repeated MOF dosing was well tolerated, with no detectable toxicity or behavioral side effects observed over the 90 d study period. Collectively, these findings support the use of enzyme-loaded MOFs—particularly NU-1003—as a promising platform for long-acting prophylaxis and emergency treatment strategies involving engineered enzymes. Declarations All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas at Dallas under protocol 22-07 and were conducted in accordance with all relevant institutional guidelines, federal regulations, and the National Research Council’s Guide for the Care and Use of Laboratory Animals. Conflict of Interest J.J.G has no conflict of interest to declare. O.K.F. has a financial interest in NuMat Technologies, a start-up seeking to commercialize MOFs. Acknowledgments J.J.G. acknowledges support from the Welch Foundation (AT-198920190330) and the University of Texas at Dallas animal resource center (LARC) for their assistance in animal care. O.K.F. acknowledges support from Northwestern University. References Naughton, S. X. & Terry, A. V., Jr. Neurotoxicity in acute and repeated organophosphate exposure. Toxicology 408 , 101-112 (2018). https://doi.org/10.1016/j.tox.2018.08.011 Jaga, K. & Dharmani, C. Sources of exposure to and public health implications of organophosphate pesticides. Revista panamericana de salud pública 14 , 171-185 (2003). Suratman, S., Edwards, J. W. & Babina, K. Organophosphate pesticides exposure among farmworkers: pathways and risk of adverse health effects. Reviews on environmental health 30 , 65-79 (2015). Gbadamosi, M. R., Abdallah, M. 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Encapsulation of a nerve agent detoxifying enzyme by a mesoporous zirconium metal–organic framework engenders thermal and long-term stability. Journal of the American Chemical Society 138 , 8052-8055 (2016). Chen, Y., Li, P., Modica, J. A., Drout, R. J. & Farha, O. K. Acid-resistant mesoporous metal–organic framework toward oral insulin delivery: protein encapsulation, protection, and release. Journal of the American Chemical Society 140 , 5678-5681 (2018). Mian, M. R. et al. Catalytic degradation of an organophosphorus agent at Zn–OH sites in a metal–organic framework. Chemistry of Materials 32 , 6998-7004 (2020). Ma, K. et al. Fibrous Zr‐MOF nanozyme aerogels with macro‐nanoporous structure for enhanced catalytic hydrolysis of organophosphate toxins. Advanced Materials 36 , 2300951 (2024). Islamoglu, T. et al. Metal–organic frameworks against toxic chemicals. Chemical reviews 120 , 8130-8160 (2020). Luzuriaga, M. A. et al. ZIF-8 degrades in cell media, serum, and some—but not all—common laboratory buffers. Supramolecular Chemistry 31 , 485-490 (2019). https://doi.org/10.1080/10610278.2019.1616089 Kumari, S. et al. In vivo biocompatibility of ZIF-8 for slow release via intranasal administration. Chemical Science 14 , 5774-5782 (2023). Brohlin, O. R. et al. Zeolitic imidazolate framework nanoencapsulation of CpG for stabilization and enhancement of immunoadjuvancy. ACS Applied Nano Materials 5 , 13697-13704 (2022). Jackson, C. J. et al. Pharmacokinetics of OpdA, an organophosphorus hydrolase, in the African green monkey. Biochemical pharmacology 80 , 1075-1079 (2010). Lila, A. S. A., Kiwada, H. & Ishida, T. The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage. Journal of Controlled Release 172 , 38-47 (2013). Shankar, G., Pendley, C. & Stein, K. E. A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nature biotechnology 25 , 555-561 (2007). Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations Yes there is potential Competing Interest. J.J.G has no conflict of interest to declare. O.K.F. has a financial interest in NuMat Technologies, a start-up seeking to commercialize MOFs. Supplementary Files SI.docx Dataset 1 Scheme1.docx GraphicalAbstract.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7799763","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":537465335,"identity":"13de68de-38f8-48f9-ae10-6989c1135760","order_by":0,"name":"Jeremiah 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12:56:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7799763/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7799763/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96584532,"identity":"68638f1a-0758-41eb-860d-2584d1f2413a","added_by":"auto","created_at":"2025-11-24 04:01:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":715510,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of OPAA@NU-1003 and OPAA@ZIF-8. SEM micrographs showing the morphology of ZIF-8 (A), OPAA@ZIF-8 (B), NU-1003 (C), and OPAA@NU-1003 (D). (E) PXRD patterns comparing simulated and experimental diffraction profiles for ZIF-8, NU-1003, and their respective OPAA-loaded forms. (F) Confocal laser scanning microscopy (CLSM) images of OPAA-Cy7@NU-1003 (left column) and OPAA-Cy7@ZIF-8 (right column), showing brightfield (top), Cy7 fluorescence (middle), and merged channels (bottom). Scale bars: 20 µm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/a2da7e9d004b1866f39085a9.png"},{"id":96605565,"identity":"82de6ac8-01b9-4e5d-9a47-45632cb23402","added_by":"auto","created_at":"2025-11-24 09:23:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":302815,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs showing the morphological stability of NU-1003 particles incubated under different conditions over time. NU-1003 was suspended in saline, 0.1 M potassium phosphate (KP) buffer (pH 7.4), 0.1 M phosphate-buffered saline (PBS, pH 7.4), complete cell culture media, and 0.1 M bicarbonate buffer (pH 9.5), imaged at 1, 4, 18, and 24 h. At each time point, samples were washed with water three times and imaged by SEM to assess particle integrity and morphological changes.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/32fd8c2bbc9adaffbaabc6ba.png"},{"id":96584536,"identity":"f2ed1a57-10d5-436c-b353-474eef5694d4","added_by":"auto","created_at":"2025-11-24 04:01:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2999308,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity and cellular uptake analysis of OPAA-loaded MOFs. (A \u0026amp; B) Cell viability of HEK293 cells treated with increasing concentrations of OPAA@NU-1003 and OPAA@ZIF-8, as well as unloaded NU-1003 and ZIF-8, after 4 h and 24 h of exposure. (C) Confocal laser scanning microscopy (CLSM) images showing cellular uptake and colocalization of OPAA@NU-1003 with intracellular markers. Nuclei were stained with DAPI (blue), late endosomes with RAB7A (magenta), and Cy7-labeled MOFs (green). The merged image confirms the internalization and partial colocalization of MOFs with endo-lysosomal compartments—scale bars: 20 µm.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/7fd1ea6209f506627562190e.png"},{"id":96584535,"identity":"aa62dd14-8547-49e5-b533-c5344d42f530","added_by":"auto","created_at":"2025-11-24 04:01:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1206806,"visible":true,"origin":"","legend":"\u003cp\u003eNormalized Cy7 fluorescence signal from mice subcutaneously injected with OPAA-Cy7 formulations to assess depot effect over time. (A) Fluorescence retention profile of OPAA-Cy7@NU-1003 compared to free OPAA-Cy7. (B) Fluorescence retention profile of OPAA-Cy7@ZIF-8 compared to free OPAA-Cy7. Data are shown over a period of 24 days (n = 3 per group). (C) Time-resolved \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging of mice injected subcutaneously in the upper flank with free OPAA-Cy7 (top row), OPAA-Cy7@NU-1003 (middle row), or OPAA-Cy7@ZIF-8 (bottom row). Images were collected at multiple points post-injection to monitor tissue retention.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/7ebf3bebe58502149463d593.png"},{"id":96584540,"identity":"8bacf886-6de0-4c78-a09a-ec0fd5cb4f87","added_by":"auto","created_at":"2025-11-24 04:01:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":266758,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the experimental setup (A): mice were orally dosed with DFP (4 mg/kg) followed by immediate subcutaneous injection of saline, free OPAA, OPAA@ZIF-8, or OPAA@NU-1003. Grimace score (B), tremor intensity measured using the Racine scale (C), grip strength normalized to body weight (D), and incapacitance test showing hind limb weight distribution (E) were recorded 30 minutes post-treatment. Kaplan–Meier curves for each group over 20 days are shown in (F). A one-way analysis of variance (ANOVA) was used for the statistical analysis of B, C, and F. A paired t-test was applied to D, and the Mann–Whitney test was used for E.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/bd1e2697fa1607853c68689d.png"},{"id":96584541,"identity":"73fffa2a-a9bb-468d-b48e-61de8f9e5688","added_by":"auto","created_at":"2025-11-24 04:01:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1176793,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral assessments following repeated prophylactic treatment and DFP challenge. (A) Schematic presentation of the study design. (B) Serum anti-OPAA IgM and IgG titers were quantified by endpoint ELISA on days 12 and 60 to evaluate the humoral immune response elicited by each formulation.(C) Tremor intensity was assessed using the Racine scale. (D) Grip strength was measured before and after DFP exposure, normalized to body weight. (E) Facial grimace scoring to evaluate nociceptive behavior. (F) Incapacitance test measuring hind limb weight distribution (ΔW) as a readout of postural stability. Statistical analysis of C and F was performed using a one-way analysis of variance (ANOVA). D was examined with a paired t-test, and E was evaluated using the Mann–Whitney test.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/de78ed9a0261f817a622d393.png"},{"id":96605596,"identity":"38320f2a-de12-4125-829e-3cec18eff857","added_by":"auto","created_at":"2025-11-24 09:23:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":478013,"visible":true,"origin":"","legend":"\u003cp\u003eLong-term survival study following repeated prophylactic treatment and dual organophosphate challenges. (A) Experimental timeline: mice received two doses of their assigned treatment (saline, OPAA, OPAA@ZIF-8, or OPAA@NU-1003) on days 0 and 60, followed by DFP (4 mg/kg) challenges on days 63 and 84. (B) Kaplan–Meier survival curves showing survival through day 90. Saline- and OPAA-treated mice did not survive the first challenge, while MOF-treated groups showed extended protection, with full survival in the OPAA@ZIF-8 group and partial survival in the OPAA@NU-1003 group after the second challenge. A one-way analysis of variance (ANOVA) was used as statistical analysis.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/e139f013a838a8d09e947e3b.png"},{"id":96708168,"identity":"2f4d5e8c-a090-40e8-8b48-cfd38c0ffef5","added_by":"auto","created_at":"2025-11-25 09:58:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9023910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/fd37ecb6-eeeb-4f2b-8d4c-a7475a4daf68.pdf"},{"id":96605888,"identity":"a9de1294-2c95-421f-b6b9-61c834bfb8d1","added_by":"auto","created_at":"2025-11-24 09:24:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2438335,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/c04073c433bd4f8d17e93ca6.docx"},{"id":96584534,"identity":"2dd776b7-c320-4ba6-a87d-0ffab4ee8190","added_by":"auto","created_at":"2025-11-24 04:01:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":516231,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/ad3c3df0f9db34b3e9ea8045.docx"},{"id":96584538,"identity":"3f38c3b3-c81e-4573-ac3a-ed66e18ee8e5","added_by":"auto","created_at":"2025-11-24 04:01:15","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":231930,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7799763/v1/84aef818c4f8d94ae3a5cbeb.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nJ.J.G has no conflict of interest to declare. O.K.F. has a financial interest in NuMat Technologies, a start-up seeking to commercialize MOFs.","formattedTitle":"Metal-Organic Frameworks for Single-Dose Engineered Enzyme-Based Prophylaxis and Treatment of Organophosphate Poisoning","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOrganophosphates (OPs), widely used as pesticides and chemical warfare agents (e.g., sarin, VX), remain a persistent global threat to both civilian and military populations.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Acute exposure causes rapid systemic toxicity, as exemplified by the 1995 Tokyo subway attack,\u003csup\u003e3\u0026ndash;6\u003c/sup\u003e while chronic exposure continues to affect millions of agricultural workers worldwide.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e At the molecular level, OPs such as malathion, chlorpyrifos, and diisopropylfluorophosphate (DFP) irreversibly inhibit acetylcholinesterase, leading to cholinergic crisis, seizures, respiratory depression, and often death.\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eStandard treatments (atropine and pralidoxime, 2-PAM) require intravenous infusion, trained personnel, and often fail to prevent delayed neurological damage.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Their short systemic half-lives\u0026mdash;70 min for atropine and 4 h for 2-PAM\u0026mdash;contrast sharply with the persistence of lipophilic OPs in adipose depots, leading to long-lasting toxicity that neither therapy can realistically address during a crisis response.\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e This pharmacokinetic mismatch underscores the need for countermeasures that act both rapidly and durably, capable of functioning as a single-dose treatment or prophylactic.\u003c/p\u003e\u003cp\u003eCatalytic bioscavengers such as organophosphate acid anhydrolase (OPAA), engineered from extremophile-derived proteins, exhibit high levels of catalytic activity against fluorine bonds, making them attractive for neutralizing fluorine-containing OPs by hydrolyzing them into benign products.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e However, their poor pharmacokinetics (rapid clearance and low serum stability) hinder their translation into future therapies. Protein nanocarriers\u0026mdash;including liposomes, dendrimers, and polymers\u0026mdash;have been explored,\u003csup\u003e20\u0026ndash;26\u003c/sup\u003e yet they suffer from poor shelf stability, reliance on intravenous delivery, and limited ability to match the long half-life of OPs.\u003c/p\u003e\u003cp\u003eMetal\u0026ndash;organic frameworks (MOFs) provide a compelling alternative. These crystalline porous materials combine high surface area, tunable pore size, and modular chemistry, making them attractive platforms for biomacromolecule stabilization and delivery. MOFs can encapsulate enzymes via biomimetic mineralization or post-synthetic infusion, protecting them from proteolysis, thermal degradation, and preserving their catalytic activity while enabling controlled release. Prior work has demonstrated MOF-mediated delivery of antibiotics, chemotherapeutics, enzymes, and vaccines,\u003csup\u003e27\u0026ndash;30\u003c/sup\u003e but their potential for continuous enzyme-mediated detoxification, particularly against OPs, remains unexplored \u003cem\u003ein vivo\u003c/em\u003e.\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eHere, we report a unique approach using a subcutaneously injectable enzyme\u0026ndash;MOF depots that deliver sustained release of OPAA for weeks in vivo. Using two complementary strategies\u0026mdash;biomimetic mineralization with zeolitic imidazolate framework-8 (ZIF-8) and passive loading into the large-pore NU-1003\u0026mdash;we generated composites that remain localized at injection sites and gradually release active enzyme. These depots bridge the gap between acute rescue and long-term prophylaxis, providing rapid therapeutic response while maintaining extended protection. In lethal OP challenge models, OPAA@MOFs functioned as both prophylactic and therapeutic countermeasures, with NU-1003 in particular exhibiting minimal immunogenicity and maintaining efficacy under repeated dosing. Together, these results establish a generalizable strategy for single-dose enzyme therapeutics using a system capable of delivering bifunctional therapeutic effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eSubcutaneous and intramuscular injections are long-standing routes for achieving slow release of biomolecules into circulation. Yet, building depots that can actually keep therapeutic proteins intact for weeks inside living tissue is notoriously difficult. Once deposited, proteins are immediately subjected to immune surveillance, proteolysis, and a host of clearance mechanisms that conspire to strip away activity.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Even the heat of the body is enough to accelerate unfolding, aggregation, and hydrolysis, gradually dismantling depot formulations that appear stable in vitro.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eMetal\u0026ndash;organic frameworks (MOFs) have emerged as an unusually effective way to fight back against these pressures.\u003csup\u003e\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e By locking enzymes inside crystalline lattices, MOFs not only shield them from proteolysis but also dramatically extend their thermal lifetime, often to the point where refrigeration is no longer needed. Delicate assemblies that would otherwise disintegrate within hours at room temperature \u0026mdash; such as proteoliposomes \u0026mdash; can be stabilized for months when mineralized inside a MOF.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eTwo encapsulation strategies stand out. The first is MOF-based biomimetic mineralization, in which MOF nucleation takes place directly on the protein surface. In this setting, the macromolecule itself acts as a local template, creating a pseudo-high concentration of metal ions that catalyze crystallization. Ligand and metal ratios can be tuned to sculpt particle size.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Applying this approach, we purified OPAA (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) and encased it into a ZIF-8 shell, yielding 300\u0026ndash;500 nm crystals that retained the faceted morphology of pristine ZIF-8 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;B\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe second strategy is direct infiltration into pre-formed, large-pore frameworks. NU-1003, a Zr-based MOF with ~\u0026thinsp;4.4 nm channels, is particularly suited to this purpose: water-stable, robust, and large pore apatures ideal for diffusion of large proteins.\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43 CR44 CR45\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Passive diffusion of OPAA into the MOF likewise did not alter the morpohology of the crystalline particles (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;D). Unlike ZIF-8, NU-1003 has never been tested for enzyme delivery \u003cem\u003ein vivo\u003c/em\u003e, making it an intriguing foil.\u003c/p\u003e\u003cp\u003eTo assess structural stability after enzyme encapsulation, powder X-ray diffraction (PXRD) was performed to evaluate the crystallinity of both MOFs. The resulting diffractograms confirmed that the MOF structures remained crystalline post-loading \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. To visualize cargo uptake and release from the MOFs, Cy7-labeled OPAA was synthesized and encapsulated within the MOF particles. Confocal laser scanning microscopy (CLSM) was employed to verify successful encapsulation of OPAA within the MOFs. Z-stack imaging revealed the fluorescently labeled protein localized inside the MOF structures \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG; animation of z-stacks avaliable in the SI\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo confirm that MOF encapsulation preserves OPAA\u0026rsquo;s structural integrity and enzymatic function, we first encapsulated the protein and then released it via degrading the MOF shell\u0026mdash;a process we refer to as exfoliation\u0026mdash;using 0.01 M phosphate-buffered saline (PBS) pH 4.5 for OPAA@ZIF-8 and 0.1 M sodium bicarbonate pH 9.5 for OPAA@NU-1003. A native gel electrophoresis \u003cb\u003e(Figures S2A, F)\u003c/b\u003e revealed that the recovered OPAA exhibited the same electrophoretic mobility as the native enzyme, indicating no alteration in charge or size. Enzymatic assays \u003cb\u003e(Figures S2B, G)\u003c/b\u003e further demonstrated full retention of activity after exfoliation.\u003c/p\u003e\u003cp\u003eLong-term cargo stability was assessed by storing OPAA@MOF dry constructs at room temperature for 90 d. Subsequent SEM \u003cb\u003e(Figures S2C, D)\u003c/b\u003e and PXRD \u003cb\u003e(Figure S2E)\u003c/b\u003e analyses showed no significant changes in MOF morphology or crystallinity. At the same time, native gel electrophoresis and enzymatic activity assays confirmed that the encapsulated and exfoliated OPAA maintained its structure and function \u003cb\u003e(Figures S2F, G).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing our fluorescently labeled OPAA, we were able to evaluate encapsulation efficiency by analyzing the supernatant from the loading or encapsulation process. The encapsulation efficiency of the protein within both MOFs was determined to be approximately 99%, as measured by fluorescence intensity \u003cb\u003e(Figure S3A\u003c/b\u003e). PXRD analysis confirmed that the crystallinity of the MOF remained intact following encapsulation of Cy7-labeled OPAA. UV\u0026ndash;Vis absorbance spectra demonstrated characteristic near-infrared emission for OPAA-Cy7 that was absent in the unlabeled OPAA, confirming successful dye incorporation. Additionally, SEM imaging revealed no detectable changes in MOF morphology after encapsulation, indicating structural integrity was preserved \u003cb\u003e(Figures S3B, C, D)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrior to performing \u003cem\u003ein vitro\u003c/em\u003e experiments with mammalian cells, we evaluated the stability of NU-1003 in commonly used laboratory buffers, cell media, and in the presence of proteins. To ensure consistent mixing, all samples were incubated at room temperature on a rotisserie shaker. ZIF-8 is known to degrade in the presence of physiological ions, particularly phosphate and carbonate.\u003csup\u003e27 47\u003c/sup\u003e Interestingly, under similar conditions, we found that NU-1003 exhibits significantly greater stability, particularly in PBS buffer, after 24 h. Moreover, NU-1003 exhibited minimal morphological changes in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) and in complete medium DMEM\u0026thinsp;+\u0026thinsp;10% FBS (fetal bovine serum), indicating greater robustness than ZIF-8. However, it was less stable than ZIF-8 in bicarbonate buffers \u003cb\u003e(Figs.\u0026nbsp;2, S4)\u003c/b\u003e. The overall improved stability\u0026mdash;bicarbonate notwithstanding\u0026mdash;can be attributed to NU-1003\u0026rsquo;s strong Zr\u0026ndash;O bonds and highly connected 8-node framework, which resists hydrolysis and structural degradation even under harsh conditions. In contrast, ZIF-8 has weaker Zn\u0026ndash;N bonds that are more kinetically labile and degrade in comparatively milder environments. \u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure 2.\u003c/b\u003e SEM micrographs showing the morphological stability of NU-1003 particles incubated under different conditions over time. NU-1003 was suspended in saline, 0.1 M potassium phosphate (KP) buffer (pH 7.4), 0.1 M phosphate-buffered saline (PBS, pH 7.4), complete cell culture media, and 0.1 M bicarbonate buffer (pH 9.5), imaged at 1, 4, 18, and 24 h. At each time point, samples were washed with water three times and imaged by SEM to assess particle integrity and morphological changes.\u003c/p\u003e\u003cp\u003eOnce encapsulation of OPAA within the MOFs was confirmed and their stability established, we proceeded with \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity testing in HEK293 cells, a widely used mammalian cell line. NU-1003, both enzyme-loaded and unloaded, was found to be less cytotoxic than ZIF-8 at equivalent concentrations after both 4 h and 24 h exposures. NU-1003 and OPAA@NU-1003 maintained more than 50% cell viability at both 4 h and 24 h, even at high concentrations such as 1 mg/mL, indicating that NU-1003 is effectively non-toxic at the tested concentrations. In contrast, ZIF-8 and OPAA@ZIF-8 cause a marked decline in cell viability at concentrations above 0.1 mg/mL, as previously reported.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e Based on these findings, non-toxic concentrations were identified for both MOFs, which served as the basis for dosing in subsequent \u003cem\u003ein vivo\u003c/em\u003e experiments \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo evaluate cellular uptake of the OPAA-loaded MOFs (OPAA@ZIF-8 and OPAA@NU-1003), we conducted uptake assays visualized using CLSM. To localize the internalized MOFs, cells were incubated with Cy7-OPAA-loaded MOFs for 4 h, then washed, fixed with 4% paraformaldehyde (PFA), blocked with 5% Bovine serum albumin (BSA), and stained with antibodies against RAB7A (late endosomes). The following day, cells were treated with appropriate secondary antibodies to visualize subcellular compartments and then counterstained with DAPI (nuclei) and Phalloidin (cytoskeletal membrane marker). CLSM imaging revealed that portions of the MOFs were internalized and localized within lysosomes, while some MOF material remained associated with the cell membrane \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. MOF-membrane interactions can be strong and complicate uptake studies by flow cytometry. We have previously found that rapidly washing cells in an acidic solution removes surface-bound MOFs; however, this procedure does not work on the acid-fast Zr series of MOFs.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next evaluated the depot effect of both OPAA-Cy7@ZIF-8 and OPAA-Cy7@NU-1003 to assess their potential for use in treatment. OPAA is known to have a short \u003cem\u003ein vivo\u003c/em\u003e half-life (~\u0026thinsp;1.5 h) and is rapidly cleared from the body due to its small size. Despite exhibiting strong detoxification performance \u003cem\u003ein vitro\u003c/em\u003e, the organophosphorus hydrolase family of proteins has shown limited efficacy \u003cem\u003ein vivo\u003c/em\u003e against OP poisoning owing to its rapid clearance from tissues.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e To confirm this limitation, free OPAA-Cy7 was injected subcutaneously into the flanks of mice, and fluorescence was monitored. The signal was completely absent after 2 h, confirming rapid clearance \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eIn contrast, the MOF composites retained fluorescence in their injection sites very well. Both OPAA-Cy7@ZIF-8 and OPAA-Cy7@NU-1003 were injected subcutaneously in the flanks of mice, and fluorescence imaging was used to monitor release over time. The MOF-based formulations significantly prolonged the local retention of the enzyme. Notably, NU-1003 released OPAA-Cy7 more slowly than ZIF-8, maintaining a detectable signal for an additional 9 d compared to ZIF-8 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e)\u003c/span\u003e. This prolonged release is attributed to the greater stability of NU-1003 in biological fluids as well as slower diffusion kinetics of OPAA-Cy7 from NU-1003 relative to ZIF-8.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the physicochemical effects of tissue residency from the MOF materials at the injection site, we examined the persistence of the materials in solid form and evaluated metal release into distal organs. Both ZIF-8 and NU-1003 initially formed stable deposits, consistent with our slow-release data. However, by day 24, ZIF-8 depots had fully degraded and were no longer detectable in the tissue, whereas NU-1003 remained present in solid form. To further investigate this, we excised MOF depots at both day 3 and day 24. On day 3, we found ZIF-8 more degraded than NU-1003, which aligns with our \u003cem\u003ein vitro\u003c/em\u003e findings that physicochemical anions\u0026mdash;in particular phosphate\u0026mdash;were better able to degrade ZIF-8 than NU-1003 \u003cb\u003e(Figure S5)\u003c/b\u003e. We then evaluated the biodistribution of released metals from NU-1003 by assessing Zr\u003csup\u003e4+\u003c/sup\u003e in major organs 72 h post-administration using inductively coupled plasma mass spectrometry (ICP-MS) \u003cb\u003e(Figure S6)\u003c/b\u003e. Results indicated minimal Zr\u003csup\u003e4+\u003c/sup\u003e accumulation in the kidney, liver, and lungs. Notably, in contrast to ZIF-8, no detectable Zr\u003csup\u003e4+\u003c/sup\u003e was found in lymphatic tissue.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTherapeutic Treatment\u003c/h2\u003e\u003cp\u003eGiven that one of the primary causes of death from OP poisoning is the rapid onset of severe neurological symptoms\u0026mdash;often within the first 10 min of exposure\u0026mdash;we next evaluated whether enzyme\u0026ndash;MOF depots could act quickly enough to function as effective post-exposure therapeutics. To mimic realistic intoxication scenarios such as accidental ingestion or occupational exposure, mice were challenged with a lethal oral dose of DFP (4 mg/kg), followed immediately by subcutaneous injection of saline, free OPAA, OPAA@ZIF-8, or OPAA@NU-1003 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThis design allowed us to directly probe both the speed and efficacy of each intervention under acute, high-stakes conditions. In addition to monitoring survival, we performed a double-blinded panel of neurobehavioral assays that quantify the hallmarks of OP toxicity: nociceptive distress, tremor, neuromuscular strength, and postural stability. These readouts are critical because survival alone does not capture the extent of long-term neuromuscular injury that often follows OP exposure.\u003c/p\u003e\u003cp\u003eGrimace scoring \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e showed that mice treated with saline or free OPAA exhibited signs of pain and discomfort, whereas mice treated with OPAA@ZIF-8 and especially OPAA@NU-1003 had significantly reduced grimace scores, reflecting lower distress. Similarly, tremor severity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e was significantly reduced in both MOF-treated groups compared to the saline and free OPAA groups. The OPAA@NU-1003 group showed the lowest tremor scores, indicating greater protection against acute neurotoxicity.\u003c/p\u003e\u003cp\u003eGrip strength testing provided a more quantitative measure of neuromuscular protection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In saline- and OPAA-treated cohorts, animals exhibited a significant loss of strength following DFP administration. In contrast, OPAA@ZIF-8 (p\u0026thinsp;=\u0026thinsp;0.1380) and OPAA@NU-1003 (p\u0026thinsp;=\u0026thinsp;0.2258) animals showed no significant decline, indicating that MOF depots preserved motor strength. To confirm this effect across groups, we compared the absolute change (Δ) in grip strength between treatments (\u003cb\u003eFigure S9\u003c/b\u003e). Both MOF depots exhibited significantly smaller declines compared to saline or OPAA controls, while the two depots did not differ from each other. Thus, the apparent \u0026ldquo;non-significance\u0026rdquo; in the within-group MOF analyses reflects the absence of neuromuscular decline, which becomes clear when comparing the magnitude of change across groups.\u003c/p\u003e\u003cp\u003eTo evaluate the impact of treatment on postural balance and motor coordination, an incapacitance test was performed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. This test measures the difference in weight-bearing between the hind limbs. Higher ΔW values indicate imbalance and impaired motor function. Saline- and OPAA-treated mice showed significant imbalance, while OPAA@ZIF-8 and especially OPAA@NU-1003 treated mice maintained normal postural stability, indicating preserved motor control.\u003c/p\u003e\u003cp\u003eSurvival analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e showed a stark contrast between groups. All mice treated with saline or free OPAA died within 30 min after DFP exposure. In contrast, 100% of mice treated with OPAA@ZIF-8 or OPAA@NU-1003 survived the full 20-day observation period without exhibiting behavioral signs of toxicity. Histological analysis (H\u0026amp;E staining) was performed across all treatment groups \u003cb\u003e(Figure S8)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThese data collectively demonstrate that MOF-based delivery of OPAA provides complete protection against lethal challenge, resulting in 100% survival, compared to 0% survival in treated controls. Further, the surviving animals show significantly reduced neurotoxic symptoms when compared to controls\u0026mdash;they largely maintain motor function and postural stability, even after lethal OP exposure. The NU-1003 formulation, in particular, offers enhanced protection without the confounding issue of immunogenicity, likely due to its slower and more sustained enzyme release profile as well as the absence of observable metal in the draining lymphatic tissue, as shown in \u003cb\u003eFigure S6\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnother critical application of this technology is its use as a preventative countermeasure in high-risk environments, such as agriculture or military operations, where a single prophylactic dose could be administered before OP exposure. MOFs are particularly well-suited for this role, as their slow and sustained release properties enable long-term enzyme delivery, overcoming the rapid systemic clearance that limits traditional enzyme therapies and makes repeated dosing impractical.\u003c/p\u003e\u003cp\u003eA central challenge in deploying exogenous proteins, such as OPAA, is the potential for adaptive immune responses, including the production of opsonizing IgG or T-cell activation, which can ultimately compromise therapeutic efficacy through accelerated blood clearance (ABC) or hypersensitivity reactions. Such responses are not limited to proteins; even synthetic macromolecules, including polymers like polyethylene glycol (PEG), are susceptible to immune recognition, sometimes resulting in the rapid loss of bioavailability for PEGylated drugs. While small proteins are often weakly immunogenic unless adjuvanted, the creation of long-lived depots for slow protein release raises a legitimate concern: the risk of eliciting high-titer IgG that can neutralize circulating protein and drive ABC.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e Notably, ZIF-8 has emerged as a potent adjuvant, capable of inducing strong IgG responses against encapsulated payloads.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e This observation is consistent with our histological data, which suggests an immune response is explicitly elicited by ZIF-8-based formulations.\u003c/p\u003e\u003cp\u003eGiven that OPAA release from MOF depots persists for over 20 d, we established a long-term prophylactic model to rigorously test both the durability of enzyme protection and the immunological consequences of repeated dosing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Mice were given a single subcutaneous injection of saline, free OPAA, OPAA@ZIF-8, or OPAA@NU-1003 on day 0, and were left untreated for 60 d to allow clearance of any unencapsulated enzyme. On day 60, each group received a second dose of the same formulation, simulating a repeat-exposure scenario.\u003c/p\u003e\u003cp\u003eFor biologic drugs, the induction of antibodies\u0026mdash;especially long-lived IgG\u0026mdash;can compromise efficacy and safety by ABC or triggering hypersensitivity reactions.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e We therefore measured anti-OPAA antibody titers before and after dosing \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. These data show that while OPAA@ZIF-8 strongly stimulates both an initial (IgM) and a lasting (IgG) antibody response, consistent with robust immunological memory, OPAA@NU-1003 induces only minimal IgG and little or no detectable IgM, mirroring the low immunogenicity of unencapsulated enzyme. Importantly, this lack of strong class-switched antibody response\u0026mdash;where initial IgM is gradually replaced by longer-lived IgG\u0026mdash;as demonstrated with NU-1003, supports its suitability for repeated or long-term prophylactic administration, minimizing the risk of depot failure owing to immune-mediated clearance.\u003c/p\u003e\u003cp\u003eTo assess the efficacy of repeated MOF-based prophylactic treatments, neurobehavioral evaluations were conducted 30 min after the DFP challenge on day 63. Tremor intensity was recorded using the Racine scale \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Mice treated with saline or free OPAA at both time points exhibited high tremor scores, indicating no protection. In contrast, mice treated with OPAA@ZIF-8 and OPAA@NU-1003 exhibited significantly lower tremor scores, with the NU-1003 group showing the most pronounced suppression of tremor activity.\u003c/p\u003e\u003cp\u003eGrip strength was measured before and after DFP exposure and normalized to body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Saline- and OPAA-treated groups again showed significant post-challenge declines, whereas OPAA@ZIF-8 and OPAA@NU-1003 did not, indicating preserved motor function. To confirm this across groups, we compared the absolute change (Δ grip strength) between treatments (\u003cb\u003eFigure S9\u003c/b\u003e) and found that both MOF depots exhibited significantly smaller losses than controls, with no difference between ZIF-8 and NU-1003.\u003c/p\u003e\u003cp\u003eFacial grimace scores were used to assess nociceptive responses \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. The saline and free OPAA groups exhibited high levels of facial tension. In contrast, mice treated with either MOF formulation exhibited significantly lower grimace scores, indicating a reduction in pain-related behavior. Postural balance was evaluated using an incapacitance test \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Mice treated with saline or OPAA showed substantial imbalance (higher ΔW), whereas OPAA@ZIF-8 and OPAA@NU-1003 groups showed improved hind limb weight distribution. The NU-1003 group had the lowest ΔW values, indicating better motor coordination.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the durability of protection and the potential for ABC after repeated prophylactic dosing, we subjected mice from the cohort described in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e to two independent OP challenges (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Each group received an initial depot injection on day 0 and a second on day 60, followed by a lethal DFP challenge (4 mg/kg) on day 63. As expected, saline- and free OPAA\u0026ndash;treated mice did not survive beyond 30 min, confirming the acute lethality of DFP and the lack of durable protection from non-depotized enzyme. In striking contrast, all mice treated with OPAA@ZIF-8 or OPAA@NU-1003 survived the initial DFP challenge, demonstrating robust and persistent protection after repeat MOF-based prophylaxis.\u003c/p\u003e\u003cp\u003eTo thoroughly evaluate long-term depot efficacy, mice that survived were re-exposed to a second lethal dose of DFP on day 84. Animals treated with OPAA@ZIF-8 maintained 100% survival up to day 90, demonstrating the sustained release and enduring protective effect of this depot. The OPAA@NU-1003 group experienced some mortality after the second challenge; however, its performance was statistically not significant compared to OPAA@ZIF-8. Overall, MOF-based release systems showed a significant survival advantage over the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eCuriously, despite the strong antibody response elicited by ZIF-8, protective efficacy was preserved, indicating that circulating antibodies did not completely neutralize depot-released OPAA during the experimental period. These findings underscore the remarkable promise of MOF-based depots for sustained, repeatable prophylaxis against OP poisoning, even when immune activation occurs. They also highlight the importance of further investigating depot design and immune interplay to maximize durability and safety.\u003c/p\u003e\u003cp\u003eTo check the chronic toxicity in the organs, histology was performed, and tissues were observed for any sign of toxicity. Histological examination was conducted on major organs\u0026mdash;including heart, lung, liver, kidney, spleen, and brain\u0026mdash;following prophylactic treatment and subsequent DFP exposure \u003cb\u003e(Figure S10)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe developed two OPAA-loaded MOFs\u0026mdash;OPAA@ZIF-8 and OPAA@NU-1003\u0026mdash;for OP detoxification. NU-1003 demonstrated higher morphological stability in physiological buffers compared to ZIF-8, maintaining its structural integrity over 24 h. \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity studies showed that NU-1003 was less toxic to HEK293 cells than ZIF-8 at equivalent concentrations. \u003cem\u003eIn vivo\u003c/em\u003e fluorescence imaging confirmed that both MOFs enabled prolonged subcutaneous retention of OPAA, with NU-1003 displaying slower release kinetics. Both OPAA@ZIF-8 and OPAA@NU-1003 effectively reduced neurotoxic symptoms and significantly improved survival in mice challenged with lethal doses of DFP. Importantly, NU-1003 did not elicit any observable immune response, despite functioning as a long-term depot for the sustained release of an exogenous protein. A single prophylactic dose of MOF-formulated OPAA conferred long-term protection, with therapeutic efficacy maintained even after a second OP challenge. Repeated MOF dosing was well tolerated, with no detectable toxicity or behavioral side effects observed over the 90 d study period. Collectively, these findings support the use of enzyme-loaded MOFs\u0026mdash;particularly NU-1003\u0026mdash;as a promising platform for long-acting prophylaxis and emergency treatment strategies involving engineered enzymes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAll animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas at Dallas under protocol 22-07 and were conducted in accordance with all relevant institutional guidelines, federal regulations, and the National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eJ.J.G has no conflict of interest to declare. O.K.F. has a financial interest in NuMat Technologies, a start-up seeking to commercialize MOFs.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eJ.J.G. acknowledges support from the Welch Foundation (AT-198920190330) and the University of Texas at Dallas animal resource center (LARC) for their assistance in animal care. O.K.F. acknowledges support from Northwestern University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNaughton, S. X. \u0026amp; Terry, A. V., Jr. Neurotoxicity in acute and repeated organophosphate exposure. \u003cem\u003eToxicology\u003c/em\u003e \u003cstrong\u003e408\u003c/strong\u003e, 101-112 (2018). https://doi.org/10.1016/j.tox.2018.08.011\u003c/li\u003e\n\u003cli\u003eJaga, K. \u0026amp; Dharmani, C. 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Although enzyme-based detoxification strategies, such as organophosphorus acid anhydrolase (OPAA), have shown promise \u003cem\u003ein vitro\u003c/em\u003e, their rapid clearance and instability have severely limited their clinical translation. Here, we report the first \u003cem\u003ein vivo\u003c/em\u003e study demonstrating the use of metal–organic frameworks (MOFs) as delivery depots for sustained OP detoxification. OPAA was successfully encapsulated within two MOFs—zeolitic imidazolate framework-8 (ZIF-8) and NU-1003—using biomimetic mineralization and passive diffusion strategies, respectively. These composites provided prolonged subcutaneous retention, preserved enzymatic activity, and offered complete protection in mice exposed to lethal doses of OP agents. In addition to immediate post-exposure rescue, the system also functioned as a long-acting prophylactic with minimal immunogenicity and excellent biocompatibility. This dual-use approach provides a powerful new platform for enzyme therapeutics, where both sustained activity and rapid response are required.\u003c/p\u003e","manuscriptTitle":"Metal-Organic Frameworks for Single-Dose Engineered Enzyme-Based Prophylaxis and Treatment of Organophosphate Poisoning","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 04:01:10","doi":"10.21203/rs.3.rs-7799763/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e6f9427d-2120-47fb-9242-2e5caaf9bd09","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57158738,"name":"Physical sciences/Nanoscience and technology/Nanomedicine/Drug delivery"},{"id":57158739,"name":"Physical sciences/Materials science/Biomaterials/Drug delivery"}],"tags":[],"updatedAt":"2025-11-24T04:01:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 04:01:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7799763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7799763","identity":"rs-7799763","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}