A Novel Method for In Vivo Gene Editing in the Brain of Guppies Using Unique Nanoparticles as Delivery Vehicles

A Novel Method for In Vivo Gene Editing in the Brain of Guppies Using Unique Nanoparticles as Delivery Vehicles | 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 Research Article A Novel Method for In Vivo Gene Editing in the Brain of Guppies Using Unique Nanoparticles as Delivery Vehicles María Camila Monsalve, Miguel A. Vergara, María Fernanda Vasquez, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6254078/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Uncovering the genomic basis of traits has advanced rapidly in evolutionary biology and neuroscience, largely through phenotypic traits with adaptive value has been advancing rapidly in evolutionary biology, neuroscience, and behavior, largely due to research using non-traditional model systems. Direct gene editing in the adult brain represents a crucial next step in linking genotype to phenotype, avoiding the confounding effects that arise from modifications during development. However, implementing these technologies beyond traditional laboratory models remains challenging due to delivery limitations. Methods We developed an intracranial microinjection protocol for adult guppies ( Poecilia reticulata ) to deliver gene-editing elements to brain cells. We designed magnetic nanoparticles functionalized with a novel translocating agent, a non-viral carrier capable of transporting linearized nucleic acids across cellular and nuclear membranes. We comprehensively assessed nanoparticle uptake, nuclear colocalization, and potential health impacts using histological analysis, liver enzyme activity assays, and behavioral assessments. Results Our functionalized nanoparticles successfully entered brain cells and colocalized with nuclei at rates exceeding 50% after two weeks, demonstrating their potential for efficient in vivo gene editing. Health assessments showed no significant brain cell death (> 80% viability), no liver toxicity (normal ALT, AST, and ALP enzyme levels), and no alterations in individual and social behaviors, confirming the nanoparticles’ biocompatibility and systemic safety. Conclusions Our results, combined with previous in vitro work demonstrating our functionalized magnetic nanoparticles are an effective delivery system for gene editing, show they can be used for safe in vivo interventions in the adult brain of P. reticulata . This protocol overcomes a major technical barrier in evolutionary biology and neuroscience, with a novel nucleic acid-carrying vehicle that can be used in vivo in the adult brain. This approach provides a versatile platform for studying the genetic mechanisms underlying behavior in small freshwater fish while helping overcome the major limitations of conducting functional studies on non-model organisms. Intracranial microinjection magnetic nanoparticles in vivo gene editing Poecilia reticulata behavioral genetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Gene editing technologies, particularly CRISPR-Cas9, have revolutionized our ability to investigate biological systems and disease mechanisms. By enabling precise genetic modifications and knockouts, these technologies have transformed our ability to study phenotypic traits, genetic diseases, and biological pathways with unprecedented ease and precision. The advent of modern sequencing technologies has strongly driven efforts to uncover the genomic basis of phenotypic traits with adaptive value ( 1 ). Moreover, the ability to use these technologies on any organism has enabled neuroscience, behavior, and physiology research to embrace emerging and diverse models. However, implementing cutting-edge gene editing techniques beyond traditional laboratory models remains technically challenging, especially for tissue-specific applications in vivo in adult organisms. Gene editing technologies have transformative potential for neuroscience and behavior research. Targeted genetic modifications can provide critical insights into the genetic underpinnings of brain function, behavioral traits, and neurological disorders. However, when studying genes that influence behavior, a significant challenge emerges: embryonic gene knockouts—the standard approach—also disrupt brain development, creating confounding effects that obscure the gene's direct role in adult neural function. To isolate a gene's role in processing behavioral stimuli and generating behaviors, tissue-specific editing in the adult brain becomes essential. Delivering gene editing elements to the adult brain presents multiple technical challenges. First, systemic administration methods typically fail to target the brain effectively due to the blood-brain barrier (BBB) ( 2 ), which regulates the transfer of exogenous substances between the bloodstream and central nervous system. Intracerebroventricular (ICV) injection offers a direct solution by delivering molecules and/or vehicles directly into brain ventricles, facilitating immediate access to neural tissue. While ICV injection techniques have been described for fish models, including zebrafish and poecilids ( 3 ), standardized protocols optimized for behavioral neuroscience ( 4 , 5 ) and gene editing applications remain underdeveloped ( 6 , 7 ). Beyond the delivery procedure, effective tissue-specific genome manipulation in adult tissues depends on appropriate carriers for the gene editing components. Tissue-specific in vivo applications of CRISPR-Cas gene editing face significant challenges in delivering editing components to target cells. While viral vectors are commonly used for in vivo gene delivery ( 8 , 9 ), they present considerable limitations, including restricted cargo capacity, random DNA integration risks, potential immunogenicity, and complex engineering requirements that make them inaccessible to many researchers ( 10 , 11 ). To overcome these obstacles, non-viral nanocarriers have emerged as promising alternatives for delivering therapeutic agents across biological barriers such as the BBB ( 12 ). For CRISPR-Cas to effectively reach brain tissue, it must be paired with a nanocarrier capable of encapsulating and protecting gene-editing components while facilitating cellular uptake, endosomal escape, and nuclear translocation—all critical for successful gene editing ( 13 ). Therefore, developing an effective non-viral delivery system represents a key milestone toward accessible in vivo gene editing in non-model organisms. Among available nanocarriers, magnetite nanoparticles (MNPs) offer distinctive advantages for CRISPR-Cas delivery, addressing key challenges of in vivo gene editing. MNPs effectively protect nucleic acids from nuclease degradation while enhancing cellular penetration ( 14 , 15 ). Their capacity for efficient genetic material loading, cellular translocation, and endosomal escape is well documented ( 16 – 18 ), as is their high hemocompatibility, low cytotoxicity, and minimal thrombogenic effects in multiple in vitro applications ( 19 , 20 ). We have developed a novel magnetite nanoparticle, specifically functionalized with nucleic acid/protein binding agents and a cell-translocating peptide (Buforin II), demonstrated to deliver large nucleic acids via a mechanism that exploits the reducing conditions of the cytoplasm to release cargo while preserving functionality. Using these nanoparticles, we have previously achieved successful in vitro CRISPR manipulations, confirming its capacity to efficiently transport editing components into target cells ( 15 , 20 ). The guppy, Poecilia reticulata , represents an ideal model to implement and test novel in vivo gene editing technologies. Studied for over a century in sexual selection and mate choice research ( 21 ), guppies have emerged as valuable models in behavioral neuroscience, genomics, and biomedical research due to their complex behaviors, experimentally tractable size, and unique adaptations ( 22 ). The application of modern technologies in this group has accelerated the contributions made by guppy research across the biological sciences. We have made massive progress in our ability to make genetic associations and to dissect genetic etiologies in behavior and neuroscience. However, our inability to perform gene editing in guppies has significantly hindered essential functional studies. Developing brain-specific gene editing capabilities in guppies and similar fish species would unlock research into the precise genetic basis of multiple behaviors, brain function, and neurological disorders, with broad implications for neuroscience, physiology, and gene therapy development. Here, we overcome these challenges by developing a custom intracranial microinjection setup and protocol optimized for guppies and other small fish and functionalized magnetic nanoparticles capable of safely and effectively delivering gene editing elements into brain cells. We aim to evaluate the efficiency of our magnetic nanoparticle delivery system by assessing cellular uptake and nuclear localization following the microinjection. We then comprehensively assess whether these nanoparticles are appropriate for in vivo use through liver enzyme analysis, cytotoxicity assays, and behavioral testing. Our protocol and nanoparticle system are designed to offer versatility across multiple CRISPR-Cas strategies in a cost-efficient manner, potentially transforming neuroscience research capabilities in non-model organisms and creating new opportunities for targeted gene editing in anatomically complex tissues like the brain. Materials and methods Nanoparticle (MNP) synthesis Magnetite nanoparticles (MNPs) were synthesized via a chemical co-precipitation method following established protocols ( 14 , 23 , 24 ). Briefly, 0.01 mol of iron (II) chloride and 0.02 mol of iron (III) chloride (2:1 Fe³⁺:Fe²⁺ molar ratio) were dissolved in 100 mL of type I water (ultrapure water with a resistivity > 18 MΩ-cm, and conductivity < 0.056 µS/cm) and cooled to 2°C. Simultaneously, 0.08 mol of NaOH were dissolved in 100 mL of type I water and cooled to the same temperature. The iron chloride solution was continuously stirred magnetically under a nitrogen atmosphere to prevent oxidation. After 10 minutes of equilibration, the NaOH solution was gradually added dropwise to the iron solution while maintaining stirring under continuous nitrogen flow, resulting in the formation of MNPs as a black precipitate. The reaction continued for an additional hour to ensure complete precipitation and crystallization. The resultant MNPs were magnetically separated using a neodymium magnet and washed three times with 0.1 M NaCl solution and twice with deionized water to remove unreacted precursors and byproducts. To prepare for further functionalization, 100 mg of synthesized MNPs was suspended in 40 mL of type I water and sonicated to ensure homogeneous dispersion. Surface silanization of nanoparticles was performed based on the protocol described in previous studies ( 20 ). First, 250 µL of tetramethylammonium hydroxide (TMAH) was added as a stabilizing agent, followed by sonication and stirring to ensure uniform distribution. The suspension was then acidified with 50 µL of glacial acetic acid, followed by another round of sonication and stirring. Finally, 1 mL of (3-aminopropyl) triethoxysilane (APTES) (20% v/v) was slowly added to the solution, and the reaction mixture was stirred at 60°C for 1 hour with continuous stirring to ensure complete surface coverage. The silanized nanoparticles (MNPs-Si) were then magnetically separated and washed thoroughly as described above to remove unreacted APTES. To obtain the pegylated magnetite nanoparticles (MNPs-PEG), 100 mg of MNPs-Si were dispersed in 40 mL of type I water and sonicated in an ultrasonic bath for 10 minutes. Polyethylene glycol (PEG) conjugation was achieved via glutaraldehyde-mediated crosslinking to the surface amines. Initially, 2 mL of 2% (v/v) glutaraldehyde solution was added to the MNPs-Si suspension and agitated for 1 hour at room temperature to activate amine groups through Schiff base formation. Then, 10 mg of NH₂-PEG₁₂-NH₂ dissolved in 5 mL of type I water were added to the activated nanoparticles. The mixture was stirred at 220 RPM for 24 hours at room temperature to ensure complete conjugation. The MNPs-PEG were magnetically collected and extensively washed with deionized water to remove unbound PEG and glutaraldehyde, as previously described. To obtain the Buforin II conjugated magnetite nanoparticles (MNPs-BUF-II), 100 mg of MNPs-PEG were dispersed in 40 mL of type I water and sonicated in an ultrasonic bath for 10 minutes. BUF-II peptide (BUF-II, TRSSRAGLQFPVGRVHRLLRK), a cell-penetrating peptide derived from histone H2A with demonstrated nuclear-localizing properties ( 23 ), was conjugated to the terminal amine of the PEG chains using carbodiimide chemistry. Specifically, 14 mg of N-[3-(dimethylamino)-propyl]-N′-ethylcarbodiimide hydrochloride (EDC) and 7 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of type I water and added to the MNPs-PEG suspension to activate carboxyl groups on the PEG chains, generating NHS-ester intermediates. After 15 minutes of activation at room temperature with stirring at 220 rpm, 1 mg of BUF-II dissolved in 1 mL of Type I water was added. The conjugation reaction proceeded for 24 hours at room temperature with continuous stirring at 220 RPM to ensure efficient peptide attachment. The final MNPs-BUF-II were magnetically collected and thoroughly washed with deionized water to remove unbound peptide and coupling reagents (Fig. 1 and Supplementary Methods - Chemicals for nanoparticles synthesis section). Animal husbandry All experimental procedures involving animals were conducted in accordance with the ethical standards and guidelines approved by the Animal Ethics Committee of Universidad de los Andes (CICUAL), under approval number C.FUA_23 − 004. Adult female guppies ( Poecilia reticulata ) were obtained from a laboratory-maintained stock and individually housed in aquaria (14.1 cm in height, 21.5 cm long at the bottom, 25.4 cm long at the top, and 10 cm in width) under controlled environmental conditions: temperature 24–26°C, conductivity 500–600 µS/cm, dissolved oxygen 5–6 ppm, and pH 6–7. Fish were fed at libitum a diet of Formula - Freshwater flakes and Artemia. Only sexually mature females were used in experiments to ensure consistency. Brain ventricle microinjection Development of Surgical Apparatus, Anesthesia and Life Support System A custom surgical bed was designed and fabricated using 3D printing to facilitate precise ICV injections (3D model provided in the supplementary files). The apparatus featured adjustable components to accommodate fish ranging from 2.5-4.0 cm in standard length while maintaining optimal positioning for dorsal cranial access. The bed was coated with biocompatible soft foam to provide a moist, non-abrasive surface, minimizing physical stress and epidermal injury during surgery. The design incorporated an open ventral section to enable optimal light transmission, enhancing visualization and ensuring stable positioning with optimal dorsal access to the cranial region under a stereomicroscope. Anesthesia was maintained using a gravity-fed perfusion system. A cannula (Jelco Seriva 24Gx19mm) was inserted into the fish’s mouth to deliver anesthetic or freshwater solutions. The system comprised two reservoirs: (I) one containing buffered tricaine methanesulfonate (MS-222, 0.2 mg/mL, buffered to pH 7.0 with Tris Buffer 1M) and (II) the other containing fresh water. Flow rates were controlled via a three-way valve system, allowing seamless transition between anesthesia and recovery phases ( 25 ) (Fig. S1 ). Needle Pulling Microinjection needles were crafted from borosilicate glass capillaries (TW 100-4, WPI) using a programmable puller (PC-100, Narishige), set with the following parameters: first pull temperature (T1): 65°C; second pull temperature (T2): 80°C; with a weight configuration of two lightweights and one heavyweight. These settings were optimized to produce needles with the required durability and thickness to reach the third ventricle while remaining thin enough to minimize damage to the surrounding brain tissue. Needle tips were manually beveled using a surgical blade under stereomicroscopic guidance to achieve consistent injection volumes. Each needle was calibrated prior to use by dispensing Evans Blue dye (0.1% w/v) droplets into mineral oil, and the resulting droplet diameter was measured microscopically (Further details in Supplementary Methods – Brain ventricle microinjection and Fig. S2 ). Microinjection Procedure Fish were fasted for 12 hours before the procedure to minimize regurgitation risk ( 26 ). Anesthesia was induced by immersion in buffered tricaine solution (MS-222, 0.3 mg/mL) until the fish exhibited loss of equilibrium, reduced opercular movement (approximately 1 opercular movement per second), and absence of response to gentile tactile stimuli (typically within 3 minutes) ( 27 ). Anesthetized fish were transferred to the custom surgical bed in ventral recumbency and immediately connected to the life support system under a stereoscope (Olympus SZX7), ensuring continuous perfusion of tricaine (MS-222, 0.25 mg/mL) at a rate of one drop every 3 seconds. The surgical procedure was limited to a maximum of 8 minutes to minimize anesthetic exposure. A craniotomy was performed to access the third ventricle for microinjection. First, the exact injection site was identified using external neuroanatomical landmarks—specifically targeting the cranial midline at the junction between the telencephalon and optic tectum, which corresponds to the location of the third ventricle. A small incision was made in the cranial bone as previously reported ( 25 , 28 ), using a 27G x ½ needle and fine forceps (Dumont Tweezers #5). The injection needle was positioned at an approximately 30–45° angle relative to the cranial surface and inserted approximately 2 mm deep through the incision using a micromanipulator (PM 1000 Cell, MDI) to deliver the solution. Post-injection, the needle was gently withdrawn, and the three-way valve system was switched to fresh water for 1 minute to begin the recovery process. The fish was then carefully transferred to a dedicated recovery tank supplied with oxygen. Recovery was monitored continuously and assessed using standardized criteria: restoration of normal swimming axis (typically within 5–10 minutes), regular opercular movement (within 2–3 minutes), and coordinated swimming behavior (within 10–15 minutes) ( 27 ) (Fig. S3). Behavioral Tests Novel Tank Test The Novel Tank Test was performed 24 hours post-injection to assess stress responses and locomotor activity. This test exploits the natural tendency of fish to seek protection in unfamiliar environments, which typically manifests as reduced exploration and bottom-dwelling behavior. An imaginary horizontal line is used to divide the tank into upper and lower compartments (each compartment having a height of 6 cm, as the water column was maintained at 12 cm). Such division is effective because stress and anxiety-like behavior tend to increase the time spent in the bottom half of the tank ( 29 ). Tests were conducted in a trapezoidal tank (14.1 cm height x 21.5 cm bottom x 25.4 cm top x 10 cm width). Fresh water was used in each trial to avoid chemosensitive stimuli from previous subjects altering behavior. Each fish was gently transferred from its home tank using a dedicated net and placed in the center of the novel tank. After a 60-second acclimation period to minimize handling effects, behavior was recorded for 10 minutes under standardized lighting conditions. From these recordings, we extracted the following quantitative behavioral measures: time spent in the bottom compartment relative to total test time, swimming distance [cm], freezing duration relative to total test time and swimming velocity [cm/s] (Fig. S4A). Social Interaction Test The social interaction test was performed 48 hours post-injection to evaluate social motivation and recognition, functions that involve complex neural processing across multiple brain regions. Altered social behavior can indicate neurological dysfunction that might not be apparent in individual behavioral tests. The experimental setup consisted of a two-tank setup: a larger test tank (20.0 cm height x 41.0 cm length x 29.0 cm width) and a smaller stimulus tank (20.0 cm height x 5.0 cm length x 9.5 cm width) positioned adjacent to one end of the test tank. Water depth was maintained at 12 cm in both tanks. The individual being studied was placed into the larger tank, while a shoal of two female and three male guppies was placed in the smaller tank. The test tank was divided into three distinct zones: a proximity zone (8 cm width) adjacent to the stimulus tank, and two equal-sized neutral zones (16.5 cm each) extending toward the opposite end ( 30 ). Individuals were handled with a net and transferred to the tank, where they were allowed to have one minute to acclimate and then recorded for 10 minutes. Time spent in each zone [s] was measured (Fig. S4B). All data for behavioral assays was collected using a video tracking system (Noldus EthoVision XT7), with special care taken to ensure proper recording conditions for the software, as detailed in Cachat et al. ( 29 ). Validation of the ICV microinjection Before administering MNPs, we validated the ICV microinjection protocol by injecting Evans Blue dye to confirm accurate delivery into the third ventricle through visible tissue coloration upon dissection and to assess potential behavioral effects. The dye also helped verify the injection site. Additionally, we evaluated whether the ICV microinjection protocol caused any alterations in the nervous system of guppy fish through behavioral tests. To assess the effect of ICV microinjection, we injected six adult fish with Evans Blue solution and compared their behavior to non-injected control fish. We tested their behavior using standard novel tank and social interaction tests. The novel tank test took place 10 minutes after injection, and the social interaction test was conducted 48 hours later. Evaluation of nanoparticles as delivery vehicles after ICV microinjection To assess the efficiency and safety of our magnetic nanoparticles (MNPs) as delivery vehicles for gene editing applications, we administered MNPs via ICV microinjection and conducted comprehensive evaluations of their distribution, cellular uptake, and potential health effects. For all experiments in this section, we injected 600 nL of MNP solution at a concentration of 25 µg/mL directly into the third ventricle. Prior to each injection, the MNP solution was sonicated for 30 seconds and the suspended volume was immediately aspirated into the microinjection needle to ensure consistent nanoparticle delivery and prevent aggregation-related variations. Cellular Uptake The effectiveness of MNPs as gene delivery vehicles depends on their ability to reach target cells (brain cells). To evaluate the capacity of MNPs to enter brain cells and access the nucleus—a prerequisite for effective delivery of gene-editing components—we conducted detailed cellular uptake studies at two timepoints: 24 hours (short-term exposure) and 2 weeks (long-term exposure) post-injection, with four subjects per timepoint. Sample preparation : We euthanized guppies with an MS-222 overdose (0.3 mg/mL) for 10 minutes and then performed decapitation. Heads were immediately fixed in 4% paraformaldehyde (PFA) for 24 hours at 4°C to preserve ultrastructure and fluorescent labeling. Brains were carefully dissected under stereomicroscopic guidance and cryoprotected through sequential incubation in a 1:1 solution of PFA and sucrose (30% v/v), followed by a sucrose solution for approximately two hours or until they completely sank in the Eppendorf tube. The cryoprotected brains were embedded in OCT compound, flash-frozen on dry ice, and sectioned dorsally at 20 µm thickness using a cryostat (Leica CM1860). Sections were collected from three neuroanatomically distinct regions: telencephalon (TL), tissue surrounding the third ventricle (3V), and optic tectum (OT). These regions were selected based on their proximity to the injection site. For each region, 2–3 sections were collected per brain and mounted on positively charged microscope slides (Fig. S5). Prior to injection, MNPs were labeled with SYBR Green using a previously validated protocol that maintains nanoparticle functionality while providing a stable fluorescent signal. To visualize cell nuclei, tissue sections were washed three times with PBS and counterstained with 10 µM DAPI. Slides were then stored in darkness until imaging. Confocal microscopy was performed using an Olympus FV1000 system with a 60× oil immersion objective (NA 1.42). For each section, z-stacks (0.5 µm steps) were acquired across the full tissue thickness using three channels: 405 nm excitation/425–475 nm emission for DAPI (nuclei), 488 nm excitation/500–550 nm emission for SYBR Green (MNPs), and 559 nm excitation/575–625 nm emission for tissue autofluorescence (to visualize general cellular morphology). Images were analyzed using ImageJ software with custom macros to quantify MNP cellular uptake and nuclear colocalization. Background signals were removed by subtracting the average fluorescence intensity from control (non-injected) samples. Nuclear uptake efficiency was calculated as the percentage of DAPI-positive nuclei showing colocalization with SYBR-labeled MNPs, determined using the Jaccard coefficient of similarity with intensity thresholding (Further details in Supplementary Methods - Cellular uptake section and Fig. S5). Systemic Distribution We expect excess nanoparticles to exit the brain and we know these tend to accumulate in the liver where they are metabolized ( 31 ). We measured iron concentrations in brain and liver tissues 48 hours post-injection using inductively coupled plasma optical emission spectroscopy (ICP-OES). Six fish were injected with MNPs and six non-injected fish served as controls. After 48 hours, fish were euthanized as described above, and brain and liver tissues were carefully dissected. Due to the small tissue mass, organs from two fish were pooled for each analytical sample, resulting in three biological replicates per treatment group. Tissues were stored at 4°C until analysis. For sample preparation, tissues were digested in a mixture containing 0.25 mL of concentrated nitric acid (70% w/w), 0.75 mL of concentrated hydrochloric acid (37% w/w), and 0.3 mL of hydrogen peroxide (30% w/w). We then heated the mixture at 80°C for 3 hours and diluted it to a final volume of 5 mL with deionized water. Additionally, we included a group of six non-injected control samples. Iron concentration was measured using ICP-OES (Perkin Elmer Optima 8000) with the following parameters: plasma power 1300 W, nebulizer flow 0.7 L/min, auxiliary flow 0.2 L/min, and plasma flow 12 L/min. Standard curves were prepared using certified iron standard solutions (1000 mg/L, traceable to NIST) diluted to 0-500 µg/L. The detection limit for iron was 3.33 µg/L, and samples below this threshold were excluded from analysis. Final results were normalized to tissue wet weight and reported as µg Fe/mg tissue (Fig. S6). Evaluation of Nanoparticle Microinjection Effects on Fish Health To evaluate the potential effects of magnetic nanoparticles (MNPs) on animal health, we assessed hepatic function and cell viability in MNP-exposed fish. We compared control and MNP-injected fish, focusing on key hepatic enzymes and cytotoxicity indicators. Hepatic function was evaluated by measuring the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Cell viability was assessed using calcein AM and propidium iodide staining in brain tissues. These analyses provide insights into the biochemical and cellular impacts of MNP exposure on fish health. Hepatic Function Tests To assess liver damage, we measured the activity of alanine aminotransferase ALT, AST, and ALP. The study included a control group of non-injected fish and an experimental group of fish injected with MNPs seven days before sample extraction. Each group consisted of four biological replicates, with each replicate pooling three individuals. This timeframe was selected to allow nanoparticles to reach the liver and induce any detectable damage. To minimize variations in enzyme concentrations due to food intake, fish were fasted for 12 hours before sample collection. Due to the small blood volume of individual guppies, we adapted a specialized microsampling approach based on previously reported protocols for small fish ( 32 ). We euthanized guppies with an MS-222 overdose (0.3 mg/mL) for 10 minutes and then performed decapitation. The body and head were separately placed in modified chromatography columns with the cut surfaces facing downward. The columns were inserted into microcentrifuge tubes containing 1 µL of heparin (500 IU/mL) and centrifuged at 190 × g for 90 seconds followed by 1500 × g for 3 minutes. The collected blood (approximately 5–8 µL per fish) was pooled from three individuals to create a single biological replicate, yielding four replicates per treatment group. Plasma was separated by centrifugation at 2000 × g for 5 minutes and stored at -20°C until analysis. Tissue samples were collected from the same individuals, and the extracted volume from each fish was determined according to the manufacturer's protocol for each enzymatic kit. The activity of ALT, AST, and ALP was measured using commercial kits: ALT (MAK052, Sigma-Aldrich), AST (ab105135, Abcam), and ALP (ab83371, Abcam). All assays were performed according to manufacturer protocols with slight modifications to optimize for the small sample volumes available from guppies (detailed in Supplementary Methods – Hepatic health section). Each biological replicate was measured in triplicate to ensure technical reproducibility, and results were expressed as enzymatic units per liter (U/L) (Fig. S7). Cytotoxicity Assessment To directly evaluate whether MNPs cause cell death in neural tissue, we conducted in situ viability assays on brain cells two weeks post-injection. This timepoint was selected to capture both acute and subacute cytotoxic effects while allowing sufficient time for MNP distribution and cellular interactions. Three experimental groups were compared: MNP-injected fish (n = 4), saline-injected fish (n = 4, procedural control), and non-injected fish (n = 4, negative control). Following euthanasia, brains were immediately dissected in PBS and incubated in DMEM with 1:1000 calcein AM and 1:2000 propidium iodide at 37°C for 30–90 minutes. Using an Olympus FV1000 Confocal Laser Scanning Microscope (Olympus, Shinjuku, Tokyo, Japan) with a PlanApo 10× and 20×, 1.2 NA objective, we captured images. Calcein AM permeates living cells and is converted to calcein by intracellular esterases, producing bright green fluorescence in viable cells. Propidium iodide is membrane-impermeable and only enters cells with compromised membranes (dead or dying cells), where it binds to nucleic acids and emits red fluorescence. Finally, we determined the dead cell ratio by performing intensity analysis in ImageJ/Fiji v2.9.0 ( 33 ) and compared it among the experimental groups. Briefly, a custom Python script that implemented the following workflow: ( 1 ) noise reduction through morphological operations, ( 2 ) segmentation using Sobel filtering and Otsu's thresholding, ( 3 ) watershed-based cell separation, and ( 4 ) connected component labeling to count individual cells. Cell viability was calculated as the ratio of calcein-positive cells to total cells (calcein-positive plus propidium iodide-positive) expressed as a percentage. This approach provided region-specific quantification of cell viability that could be directly compared across treatment groups (Further details in Supplementary Methods - Cytotoxicity (Life and dead) section, and Fig. S8). Behavioral Effects of Nanoparticle Microinjection To assess potential neurofunctional effects of MNPs that might not be detectable through direct cytotoxicity measurements, we conducted comprehensive behavioral testing using the previously described novel tank and social interaction assays. These tests serve as sensitive functional indicators of nervous system and general health. Three experimental groups were evaluated: MNP-injected fish (n = 19), saline-injected fish (n = 4, procedural control), and non-injected fish (n = 16, negative control). The novel tank test was performed 24 hours post-injection, while the social interaction test was conducted 48 hours allowing the fish a reasonable rest period between tests. To ensure the robustness of our results, and considering the number of fish we could handle in a single injection batch, the experiment was carried out in batches, as shown in Fig. 6 . These batches were included as a random factor in all statistical models. Behavioral data for the Evans blue-injected fish were analyzed using a t-test. The other experimental groups were analyzed using linear mixed-effects models, implemented in the lme4 package (v1.1) in R. Treatment (MNP-injected, saline-injected, or non-injected) was included as a fixed effect, while batch was included as a random effect to account for potential environmental or temporal variations. When relevant Post-hoc comparisons between groups were performed with Tukey's honest significant difference test, with significance threshold set at p < 0.05. Results Validation of ICV microinjection protocol We developed a comprehensive intracerebroventricular (ICV) microinjection protocol optimized for adult Poecilia reticulata and potentially applicable to other small teleost fish. Central to this development was a custom-designed 3D-printed surgical apparatus integrated with a life support and anesthesia system (Fig. S1 and Fig. S9). This system maintains fish in optimal position for precise microinjection while providing continuous anesthesia delivery and respiratory support, critical factors for procedure success and post-operative recovery. We performed an initial validation of the ICV microinjection procedure using Evans blue dye injections, which allowed visual confirmation of injection accuracy and assessment of diffusion patterns within brain tissue (Fig. S1 0). To assess potential adverse effects of the microinjection procedure itself, we performed comprehensive behavioral testing on Evans blue-injected fish compared to non-injected controls. Here, the novel tank test, showed no significant differences between groups in anxiety-related parameters including time spent in the bottom of the tank (t = 0.891, p = 0.391) or freezing behavior (t = 0.777, p = 0.453). Similarly, locomotor function remained intact, with no significant differences in total swimming distance (t = 0.822, p = 0.428) or mean velocity (t = 1.196, p = 0.256) as schematically shown in Fig. S1 1A-D. The social interaction test, conducted 48 hours post-injection, revealed that the injection procedure did not affect social motivation or recognition, as evidenced by equivalent time spent in the proximity zone between Evans blue-injected fish and controls (t = -0.109, p = 0.91) (Fig. S1 1E). These comprehensive validation results demonstrate that our optimized ICV microinjection protocol provides precise targeting of the third ventricle with minimal anatomical or behavioral perturbation, establishing a reliable foundation for subsequent nanoparticle delivery experiments. Cellular Uptake The effectiveness of MNPs as gene delivery vehicles depends critically on their ability to enter target cells and access the nucleus. We assessed cellular uptake and nuclear localization of fluorescently-labeled MNPs in brain tissue at two timepoints: 24 hours (short-term) and 2 weeks (long-term) post-injection. We selected cryosections corresponding to tissues around the third ventricle for all analysis, as this is where the ICV microinjection was performed (images for each individual can be found in Fig. S12). Confocal microscopy analysis of brain sections surrounding the third ventricle revealed substantial uptake of MNPs by brain cells at both timepoints (Fig. 2 A). In the short-term exposure group, we observed a nuclear colocalization rate (defined as the percentage of nuclei showing MNP signal) of 40.9%, albeit with very large variability across individuals (SD = 26.9%). This high variability reflected considerable inter-individual differences, with nuclear uptake rates ranging from 10.9–73.6% across the subjects examined. Notably, two subjects exhibited exceptionally high nuclear uptake rates (> 50%), demonstrating that MNPs can efficiently penetrate the nuclear membrane within 24 hours of administration (Fig. 2 B). The mean nuclear colocalization rate increased to 54.6% at two weeks, with much more consistent results (SD = 7.7%) and all individual values ranging from 44–62.3%. The substantially reduced variability and higher nuclear uptake rates suggest that prolonged exposure promotes more uniform cellular and nuclear uptake across individuals. (A) Confocal microscopy images showing nuclei stained with DAPI (blue) and MNPs labeled with SYBR Green (green) in brain tissue cryoslices for injected individuals. The "Merge" panel displays the overlay of the DAPI and SYBR channels, highlighting areas where MNPs are in close proximity to the nuclei. The "Intercept" panel selectively shows regions where both signals coincide, indicating nuclear uptake of the MNPs. (B) Quantification of nuclear uptake efficiency, as a percentage of nuclei with MNPs (SYBR green) signal, in individuals injected with MNPs after 24 hours (short-term) and 2 weeks (long-term). Systemic distribution After confirming cellular uptake within the brain, we investigated whether MNPs administered via ICV injection remained confined to the central nervous system or distributed to peripheral organs. We measured iron concentrations in brain and liver tissues 48 hours post-injection using inductively coupled plasma optical emission spectroscopy (ICP-OES). As expected, MNP-injected fish exhibited higher mean iron concentrations in brain tissue compared to non-injected controls (0.091 µg/mg in the control group vs. 0.132 µg/mg in the injected group), confirming retention of the injected nanoparticles within the target organ. We also detected elevated iron levels in the liver of MNP-injected fish (0.053 µg/mg in controls vs. 0.372 µg/mg in injected fish), suggesting that a portion of the injected MNPs migrated to the liver (Fig. 3 ). The magnitude of iron concentration increase was substantially greater in the liver (+ 602%) than in the brain (+ 45%), suggesting a large fraction of nanoparticles are exiting the brain and accumulating in hepatic tissue. This liver accumulation is consistent with previous studies showing that the liver serves as the primary organ for nanoparticle clearance once they enter the bloodstream. Despite the clear trend toward higher iron concentrations in MNP-injected individuals, the differences were not statistically significant due to high variability within each treatment group (t-test: brain, t = -0.905, p = 0.41; liver, t = -1.672, p = 0.16). The observed hepatic accumulation of MNPs following ICV injection highlights the importance of evaluating potential effects this could have on the fish’ health, particularly hepatotoxicity. Three samples were lost from analysis due to the small size of livers and iron concentrations below the detection limit of the equipment. We excluded one brain and one liver sample from the MNP-injected group, and one brain sample from the control group. The exclusion of these samples did not significantly alter the overall pattern of results. Evaluation of Nanoparticle Microinjection Effects on Fish Health Given the observed distribution of MNPs to both neural tissue and liver, we conducted comprehensive health assessments focusing on potential hepatotoxicity and neurotoxicity—critical safety parameters for any nanocarrier used in in vivo applications. Hepatic function tests To evaluate potential hepatotoxic effects of MNPs, we measured the activities of three liver-specific enzymes that serve as established biomarkers of hepatocellular damage and dysfunction: alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST). Elevated activities of these enzymes in circulation typically indicate hepatocellular damage or altered liver function. Analysis of hepatic enzyme activities revealed no significant differences between MNP-injected fish and non-injected controls for any of the measured enzymes (Fig. 4 ). ALT activity showed nearly identical levels between groups (t = 0.371, p = 0.723), suggesting no hepatocellular damage following MNP exposure (Fig. 4 A). Similarly, AST activity remained unchanged (t = 0.920, p = 0.393), further indicating hepatocyte integrity was maintained despite MNP accumulation in liver tissue (Fig. 4 C). ALP activity exhibited greater variability in the MNP-injected group but did not differ significantly from controls (t = -0.684, p = 0.525) (Fig. 4 B). The observed variability may reflect individual differences in nanoparticle processing and biliary function. Importantly, none of the MNP-injected fish exhibited enzyme activities exceeding the upper reference limits established from the control group (defined as mean + 2SD), indicating absence of clinically significant hepatotoxicity. These biochemical findings demonstrate that despite the accumulation of MNPs in liver tissue observed in our systemic distribution studies, hepatic function remained uncompromised, suggesting effective biotransformation and/or elimination of the nanoparticles without associated damage. Cytotoxicity Assessment To directly evaluate potential cytotoxic effects of MNPs on neural tissue, we performed live/dead cell viability assays using calcein-AM and propidium iodide staining on brain tissues harvested two weeks post-injection. This dual-fluorescence approach allowed simultaneous visualization and quantification of viable (calcein-positive) and non-viable (propidium iodide-positive) cells across different brain regions. Confocal microscopy analysis revealed high cell viability across all experimental groups, with no significant differences in viability rates among MNP-injected, saline-injected, and non-injected treatments (ANOVA: F = 2.115, p = 0.236) (Fig. 5 ). The MNP-injected and non-injected control groups both maintained average cell viability exceeding 80% in all examined brain regions, indicating minimal cytotoxic effects from nanoparticle exposure. The saline-injected group showed slightly lower viability (> 70%), although this reduction was not statistically significant. We also assessed regional differences in cell viability across three distinct brain areas: tissue surrounding the third ventricle (3V), optic tectum (OT), and telencephalon (TL). No significant differences were detected across these regions (ANOVA: F = 1.421, p = 0.341), suggesting uniform safety profiles throughout the brain despite variable MNP distribution (Fig. 5 ). The slightly higher viability observed in MNP-injected fish compared to saline-injected controls is particularly noteworthy, suggesting that our functionalized MNPs may offer some degree of cellular protection rather than toxicity. While the mechanism for this potential protective effect requires further investigation, it may relate to the antioxidant properties of magnetite or the surface functionalization components. These cytotoxicity findings, combined with the hepatic enzyme results, provide strong evidence for the biological safety of our MNP formulation when administered via ICV injection, supporting its potential use as a delivery vehicle for in vivo gene editing applications in the brain. Behavioral assays The novel tank test, conducted 24 hours post-injection, revealed several subtle but statistically significant differences in stress-related behavior and locomotor parameters across treatment groups (Fig. 6 A-D). Analysis of bottom-dwelling behavior—a well-established anxiety indicator in fish—showed no significant difference between MNP-injected fish and non-injected controls (z-value = -0.219, p = 0.972). However, saline-injected fish exhibited significantly increased bottom-dwelling time compared to controls (z-value = 2.402, p = 0.040) (Fig. 6 A). This finding suggests that while the injection procedure itself may induce transient anxiety-like behavior, the presence of MNPs does not exacerbate this effect. Both MNP-injected and saline-injected groups displayed significantly increased freezing duration compared to non-injected controls (MNP: z-value = 2.941, p = 0.008; saline: z-value = 3.161, p = 0.004) (Fig. 6 B). This consistent effect across both injected groups suggests that increased freezing behavior is primarily attributable to the microinjection procedure rather than to the nanoparticles themselves. Total swimming distance was significantly reduced in both MNP-injected (z-value = -0.291, p = 0.010) and saline-injected (z-value = -3.186, p = 0.004) groups compared to controls, indicating decreased locomotor activity following the injection procedure (Fig. 6 C). Similarly, swimming velocity showed a non-significant decrease in the MNP-injected group (z-value = -0.614, p = 0.801) and a significant decrease in the saline-injected group (z-value = -2.506, p = 0.031) relative to controls (Fig. 6 D). Importantly, across all measured parameters, the MNP-injected group never showed more pronounced alterations than the saline-injected group, indicating that observed behavioral changes were primarily due to the microinjection procedure itself rather than specific effects of the nanoparticles. Additionally, all behavioral parameters remained within the natural variability range observed in the control population, suggesting that the detected differences, while statistically significant, do not represent severe functional impairment. The social interaction test, conducted 48 hours post-injection, revealed no significant differences in social preference across treatment groups (Fig. 6 E). Time spent in the proximity zone—a direct measure of social motivation—was statistically equivalent in MNP-injected (z-value = 0.800, p = 0.696), saline-injected (z-value = 1.036, p = 0.546), and non-injected control fish. This preservation of normal social behavior is particularly noteworthy as social cognition involves complex neural processing across multiple brain regions, including those showing high MNP accumulation in our cellular uptake studies. The absence of alterations in social behavior at 48 hours post-injection, combined with the generally mild and procedure-related changes observed in the novel tank test at 24 hours, suggests that any effects of the ICV microinjection on the nervous system are transient and resolve within 48 hours. Collectively, these behavioral findings indicate that while the microinjection procedure itself may induce mild and transient stress responses, the administration of MNPs does not cause additional behavioral alterations or damage to the nervous system. The preservation of complex social behaviors and the containment of locomotor alterations within the normal range of variability provide strong functional evidence supporting the neurocompatibility of our nanoparticle delivery system. Discussion Recent advances in genomic technologies have dramatically enhanced our understanding of the genetic architecture underlying adaptive traits in behavior, neuroscience, and neurological disorders. However, further functional tests often remain out of reach due to our inability to perform targeted genetic manipulation in adult tissues in non-traditional model organisms. Developing tools for in vivo gene editing directly in the adult brain is crucial for studying questions in behavioral neuroscience, as manipulating genes in embryos, can introduce confounding effects on brain development ( 34 , 35 ). Our study addresses this fundamental challenge by developing a complete technological platform for brain-specific gene editing in adult guppies—comprising both a standardized microinjection protocol and a biocompatible nanocarrier optimized for delivering gene editing components. A key component of this goal was to design a delivery vehicle that is both safe for in vivo use and capable of efficiently and effectively transporting gene editing elements into brain cells. With this goal in mind, we designed a magnetic nanoparticle delivery system, that we previously tested in vitro ( 15 , 20 ), and investigated the efficiency and safety of these magnetic nanoparticles as delivery vehicles in the brain, evaluating their impact on fish’s health and their nuclear uptake rates in brain cells. The first major barrier to adult brain-specific gene editing is the blood-brain barrier (BBB), which prevents systemic delivery of gene editing components to neural tissue ( 36 ). We have overcome this obstacle by developing a intracerebroventricular (ICV) microinjection protocol that provides direct access to the brain's ventricular system. Our custom-designed surgical apparatus and life support system enable precise, minimally invasive delivery. The validation data using Evans blue dye confirmed accurate targeting of the third ventricle with subsequent diffusion demonstrating the technical feasibility of our approach. Behavioral evaluation further showed that the microinjection procedure itself had minimal impact on fish health and behavior. While we observed some transient effects on locomotor parameters and stress responses at 24 hours post-injection, these alterations were mild and largely resolved by 48 hours, as evidenced by normal social behavior in the interaction test. The rapid recovery confirms that our protocol provides a viable avenue for brain-targeted interventions in small fish models. The second crucial barrier to in vivo gene editing is identifying an appropriate delivery vehicle that can efficiently transport editing components into cells and their nuclei. The choice of delivery carrier is particularly critical, requiring careful optimization to ensure both efficiency and safety. Our magnetic nanoparticles present several advantages over viral vectors, including simpler preparation, reduced immunogenicity, and greater loading capacity ( 10 , 17 , 37 ). We have previously evaluated the efficiency of our magnetic nanoparticles as delivery vehicles for CRISPR gene editing in vitro . We found them to be very efficient vehicles, binding nucleic acids efficiently and achieving transfection rates over 50% that ultimately led to a 130-fold overexpression of the target gene when combined with a CRISPRa plasmid ( 20 ). Therefore, we know our magnetic nanoparticles are capable of carrying and delivering CRISPR elements into cells, achieving successful CRISPR manipulations results. The magnetic nanoparticles we use are ideally suited for gene editing applications, as they can be easily bound with diverse CRISPR/Cas elements depending on the needs of different gene editing strategies (Fig. 1 ). They can bind thiol-modified nucleic acids, enabling conjugation to linearized CRISPR plasmids, single-guide RNA (sgRNA), and Cas9 mRNA. For ribonucleoprotein (RNP) complexes, conjugation occurs via a direct covalent bond through amine-carboxyl coupling on the Cas9 protein. This versatility ensures compatibility with multiple CRISPR delivery strategies, from plasmid-based expression systems to pre-assembled RNP complexes for immediate genome editing. The observed progression from variable uptake at 24 hours (40.9% ± 26.9%) to more consistent and higher uptake at two weeks (54.6% ± 7.7%) suggests an ongoing cellular internalization process rather than a single rapid uptake event. This temporal pattern is advantageous for gene editing applications, as it provides a sustained delivery window that may enhance editing efficiency. The reduced variability at the later timepoint further suggests that individual differences in initial uptake eventually converge to a consistent high level across subjects, an important consideration for experimental reproducibility. A promising way to address the initial variability in magnetite nanoparticle uptake and ensure sustained delivery for gene editing could involve combining targeted surface modifications, external stimulation, and time-dependent release strategies. Functionalizing nanoparticles with cell-penetrating peptides or other ligands can enhance early and robust internalization, while applying external magnetic fields or focused ultrasound helps localize and concentrate them in desired tissues, thus mitigating individual differences in uptake ( 17 , 38 ). Taken together, these measures could not only extend the editing window but also improve reproducibility by converging on a similarly high-level internalization profile across subjects. Nanoparticle dynamics tend to change in a physiological fluid and in a live system, becoming impossible to predict from in vitro data ( 39 , 40 ). Finding comparable nuclear uptake rates in vivo and in vitro speaks of the robust properties of the nanocarrier. For any in vivo gene editing approach, safety is paramount. The magnetic nanoparticles we tested do not induce significant cytotoxicity, as indicated by the high cell viability observed in the brain tissue of MNP-injected fish (> 80% at 25 µg/mL). The maintenance of cell viability despite substantial nuclear uptake confirms that our nanoparticles can access the nucleus without compromising cellular integrity—a delicate balance essential for functional gene editing. Our in vivo results show slightly lower viability compared to previous in vitro studies on nervous system cells ( 41 ). This slight difference may be attributed to cell death occurring during tissue processing, including dissection and staining, rather than the injection of nanoparticles themselves. In general, there is abundant evidence showing that appropriately sized and coated magnetite nanoparticles exhibit low cytotoxicity ( 42 , 43 ). Moreover, our magnetic nanoparticles are uniquely functionalized with PEG and Buforin II, which may have contributed to their low toxicity profile. Furthermore, we find fish injected with magnetic nanoparticles do not show altered indicators of hepatic health compared to controls, suggesting these nanocarriers do not impact the fish health. We found that despite the migration of excess nanoparticles to the liver, the hepatic enzyme examination showed no elevation in ALT, AST, or ALP activities in MNP-injected fish, indicating absence of liver damage despite nanoparticle accumulation. We chose to examine the liver as it has been previously shown to have an important role in nanoparticle metabolism and clearance, and MNPs are known to predominantly accumulate in the liver when they circulate systemically ( 31 , 44 , 45 ). The activity of enzymes ALP, AST and ALT is known to increase during liver stress conditions and serve as standardized biomarkers to analyze liver condition in vertebrates ( 46 , 47 ) and has been previously used to estimate the impact of metal nanoparticles in fish (48). We studied injected fish’ behavior as an indicator of their general health, and in particular as a proxy of their nervous system health. We did not see any changes in the social behavior of injected fish suggesting these fish are healthy and suffered no neurological damage from the procedure or the injection of nanoparticles. We did observe slight differences in some measures of the locomotor activity of injected fish when we performed a novel tank test. However, these differences remained within the range of variation of the control group, suggesting these are not extreme changes in behavior. Also, for those measures for which we observed significant differences, the differences were observed for both the saline-injected and MNP-injected fish, suggesting that the ICV injection procedure itself may contribute to slight stress or anxiety-like responses that are most likely transient. While our study demonstrates the safety and cellular uptake efficiency of our nanoparticle delivery system, several limitations should be addressed in future work. First, even if we have demonstrated successful gene editing in vitro using this delivery system, we have not yet done so in vivo ( 15 , 20 ). Future studies should include the evaluation of gene knockout efficiency, gene editing strategy comparison and evaluation of off-target modifications. Second, the observed variability in cellular uptake at early timepoints suggests there is still potential for optimization to achieve a more consistent initial delivery. Factors such as nanoparticle concentration, injection volume, and surface functionalization could be further refined to enhance reproducibility. Additionally, the mechanisms underlying the progressive increase in nuclear localization over time merit further investigation, as understanding these dynamics could inform optimal timing for experimental interventions. Conclusion We have developed a novel magnetic nanoparticle ideally suited for the delivery of gene editing elements across diverse applications. In this study, we adapted intracranial microinjection protocols, designed a custom 3D microinjection setup, and demonstrated that our innovative magnetic nanocarrier is safe for in vivo applications. It does not affect the health or behavior of the injected fish and reaches the brain cell nuclei as effectively as in previous in vitro tests, showing similar performance. Combined with our previous work, our findings indicate that the magnetic nanoparticles we have designed possess all the properties required for in vivo tissue-specific gene editing and can be used with our ICV protocol to perform gene editing in the guppy brain. To date, delivery methods have been the primary obstacle to implementing gene editing strategies beyond traditional laboratory model species. Developing an efficient, safe, and versatile vehicle for brain-specific in vivo gene editing in non-model organisms could revolutionize neuroscience and behavioral genetics research, as well as enhance our understanding of neurological disorders. The magnetic nanoparticles presented here can be synthesized in standard molecular biology laboratories and conjugated to different guides or plasmids with relative ease, enabling targeted gene editing by researchers in many different areas and working with diverse systems. Abbreviations BBB: Blood-brain barrier ICV: Intracerebroventricular MNPs: Magnetite nanoparticles TMAH: Tetramethylammonium hydroxide MNPs-Si: Silanized nanoparticles MNPs-PEG: Pegylated magnetite nanoparticles PEG: Polyethylene glycol MNPs-BUF-II: Buforin II conjugated magnetite nanoparticles CICUAL: Animal Ethics Committee of Universidad de los Andes PFA: Paraformaldehyde TL: Telencephalon 3V: Third ventricle OT: Optic tectum ICP-OES: Inductively coupled plasma optical emission spectroscopy ALT: Alanine aminotransferase AST: Aspartate aminotransferase ALP: Alkaline phosphatase CRISPRko: CRISPR-based gene knockout CRISPRa: CRISPR-based gene activation CRISPRi: CRISPR-based gene interference RNP: ribonucleoprotein sgRNA: single guide RNA Declarations Ethics approval and consent to participate Our work followed the international guidelines for the use of animals in research and was approved by the University of Los Andes Institutional Animal Care and Laboratory Use Committee-CICUAL (Ethical approval C.FUA_23-004). Consent for publication Not applicable Availability of data and materials All supplementary figures, tables, and methods are provided with the manuscript. The 3D printing design for the custom microinjection setup is available in the supplementary files. Competing interests The authors declare that they have no competing interests Funding This work was supported by the Vice-presidency for Research and Creation (grant no. FAPA P19.246922.005) and the Department of Biomedical Engineering from Los Andes University, Colombia. Authors' contributions M.C.M. and M.A.V. Performed most of the experiments, designed the methodology, analyzed the data, wrote and edited the manuscript. M.F.V. Performed the cellular uptake assay and contributed with data analysis. L.A. Contributed to design the ICV injection methodology and standardized all parameters. A.M. Performed hepatic health assays. N.I.B. Conceived the project and acquired funds. J.C and N.I.B. 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Regulatory Toxicology and Pharmacology. 2018 Dec;100:127–33. De Jong WH, Van Der Ven LTM, Sleijffers A, Park MVDZ, Jansen EHJM, Van Loveren H, et al. Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats. Biomaterials. 2013 Nov;34(33):8333–43. Additional Declarations No competing interests reported. Supplementary Files MicroinjectionSetup.stl SupplementaryMaterials.docx Cite Share Download PDF Status: Posted 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. 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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-6254078","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431554141,"identity":"be287e78-64b7-4a3e-8e89-afad77ea078b","order_by":0,"name":"María Camila Monsalve","email":"","orcid":"","institution":"Universidad de Los Andes","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Camila","lastName":"Monsalve","suffix":""},{"id":431554142,"identity":"019c9154-4600-4dfc-82bd-87356301a4c2","order_by":1,"name":"Miguel A. Vergara","email":"","orcid":"","institution":"Universidad de Los Andes","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"A.","lastName":"Vergara","suffix":""},{"id":431554143,"identity":"3952460f-737e-43fe-81ed-967ea038b01e","order_by":2,"name":"María Fernanda Vasquez","email":"","orcid":"","institution":"Universidad de Los Andes","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Fernanda","lastName":"Vasquez","suffix":""},{"id":431554144,"identity":"8ab3e526-d83b-4d22-842f-03430cae0d07","order_by":3,"name":"Laura Ávila","email":"","orcid":"","institution":"Universidad de Los Andes","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Ávila","suffix":""},{"id":431554145,"identity":"b1fb68a2-e566-4a58-a0be-55e70cc981e9","order_by":4,"name":"Alejandro Martínez","email":"","orcid":"","institution":"Universidad de Los Andes","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Martínez","suffix":""},{"id":431554146,"identity":"c85f2386-aa51-4fe7-af79-350768cd3a1c","order_by":5,"name":"Juan C. Cruz","email":"","orcid":"","institution":"Universidad de Los Andes","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"C.","lastName":"Cruz","suffix":""},{"id":431554147,"identity":"e517b126-95d4-49e3-b744-e3bd2d4dc1c1","order_by":6,"name":"Natasha I. Bloch","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYFACHsYDDAwHGPgZQCQDM1hMgoAWBrAWyQaStRgcAPOI0GLOwHvgwIeaO9HGN3IPHvi5w1pOvoH54G0eBjs5XFosG/gSDs449ix32428hIO9Z9KNDQ6wJVvzMCQb49JicIDH4DBvw2GglhyDA7xthxM3MPCYSQNdm9hASMvmGTkGB/+2Ha6f38D/jTgtGyRygIy2wwkMB3jY8Gs5DPbL4dwZZ94YHJZtSzfccJjN2HKOAR6/HO89+OBDzeHc/vYc449v26zl5dubH954U4E7xKARgSFigFPDKBgFo2AUjAIiAAC4CF2uIAvYtgAAAABJRU5ErkJggg==","orcid":"","institution":"Universidad de Los Andes","correspondingAuthor":true,"prefix":"","firstName":"Natasha","middleName":"I.","lastName":"Bloch","suffix":""}],"badges":[],"createdAt":"2025-03-18 14:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6254078/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6254078/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79652811,"identity":"c06ccd77-c025-4866-a817-df59dac1047d","added_by":"auto","created_at":"2025-04-01 08:13:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":172030,"visible":true,"origin":"","legend":"\u003cp\u003eMagnetite nanoparticles developed. These nanoparticles offer the versatility to bids diverse gene editing elements for different CRISPR-Cas strategies. Linearized plasmid (A) and mRNA (B) can be used in CRISPR-based gene knockout (CRISPRko), activation (CRISPRa), or interference (CRISPRi). These magnetic nanoparticles can also be bound to the Cas9 ribonucleoprotein (RNP) and sgRNA (C), enabling immediate activity upon delivery.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/dcd46c2529f6f19ae014b5ef.png"},{"id":79652813,"identity":"b0104484-386e-4ba8-a53c-a4d9222f8ef4","added_by":"auto","created_at":"2025-04-01 08:13:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":202443,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of nuclei with colocalized nanoparticles in short-exposure and long-exposure groups\u003cbr\u003e\n (A) Confocal microscopy images showing nuclei stained with DAPI (blue) and MNPs labeled with SYBR Green (green) in brain tissue cryoslices for injected individuals. The \"Merge\" panel displays the overlay of the DAPI and SYBR channels, highlighting areas where MNPs are in close proximity to the nuclei. The \"Intercept\" panel selectively shows regions where both signals coincide, indicating nuclear uptake of the MNPs. (B) Quantification of nuclear uptake efficiency, as a percentage of nuclei with MNPs (SYBR green) signal, in individuals injected with MNPs after 24 hours (short-term) and 2 weeks (long-term).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/b8985dae37f406815914dd76.png"},{"id":79652812,"identity":"67e3af14-af67-4424-b934-f117d394d46a","added_by":"auto","created_at":"2025-04-01 08:13:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31408,"visible":true,"origin":"","legend":"\u003cp\u003eIron levels in the brain and liver of control (non-injected) and MNP-injected groups. We measured iron concentrations using inductively coupled plasma optical emission spectroscopy (ICP-OES) in brains and livers collected 48 hours post-injection.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/df3eb1b29286d29ecef41227.png"},{"id":79651771,"identity":"07e5ba4e-9498-45e4-b392-7ee0e290b26d","added_by":"auto","created_at":"2025-04-01 08:05:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":216816,"visible":true,"origin":"","legend":"\u003cp\u003eActivities for liver enzymes measured as indicators of hepatic health in control (non-injected) and MNP-injected fish (A) Alanine Aminotransferase (ALT) activity [U/L]. (B) Alkaline Phosphatase (ALP) activity [U/L]. (C) Aspartate Aminotransferase (AST) activity [U/L].\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/f0ca09d578a45817cda4c158.png"},{"id":79651775,"identity":"82d059f9-f22b-40f7-a5e1-60f5039500c6","added_by":"auto","created_at":"2025-04-01 08:05:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14756,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability percentages, calculated as \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;, in the tissue around the third ventricle (3V), optic tectum (OT), and telencephalon (TL) assessed using calcein-AM and propidium iodide, “Life-dead” staining two weeks post- ICV injection.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/21024be362149d8e3020f1f1.png"},{"id":79651777,"identity":"9d76079f-43c8-436b-94f1-afecf533e975","added_by":"auto","created_at":"2025-04-01 08:05:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":189990,"visible":true,"origin":"","legend":"\u003cp\u003eNovel tank test (measured 24h after injection in tank I): (A) Time spent in the bottom compartment relative to total test time, (B) Freezing duration relative to total test time, (C) Swimming distance [cm], and(D) Swimming velocity [cm/s]. Social interaction test (measured 48h after injection in tank II): (E) Time spent in the proximity zone relative to total test. For each experimental group we show the results of each experimental batch separately.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/95bf32e5822c3918ca03ded8.png"},{"id":84884195,"identity":"fd1157c4-ac6a-4294-a3dd-fb3beb7ce08c","added_by":"auto","created_at":"2025-06-18 11:23:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1661921,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/56e94fd2-279b-4dc7-be91-e8d78bad1909.pdf"},{"id":79651773,"identity":"b371fd44-09cb-43d0-9ce7-111cd25506c0","added_by":"auto","created_at":"2025-04-01 08:05:43","extension":"stl","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":658484,"visible":true,"origin":"","legend":"","description":"","filename":"MicroinjectionSetup.stl","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/dde06b54d83afa24af661ffc.stl"},{"id":79651786,"identity":"931ad0fd-c06a-42a8-bdbf-d572e1d7bda8","added_by":"auto","created_at":"2025-04-01 08:05:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3830316,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6254078/v1/b5cb58749c72896fed0a3c90.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel Method for In Vivo Gene Editing in the Brain of Guppies Using Unique Nanoparticles as Delivery Vehicles","fulltext":[{"header":"Background","content":"\u003cp\u003eGene editing technologies, particularly CRISPR-Cas9, have revolutionized our ability to investigate biological systems and disease mechanisms. By enabling precise genetic modifications and knockouts, these technologies have transformed our ability to study phenotypic traits, genetic diseases, and biological pathways with unprecedented ease and precision. The advent of modern sequencing technologies has strongly driven efforts to uncover the genomic basis of phenotypic traits with adaptive value (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Moreover, the ability to use these technologies on any organism has enabled neuroscience, behavior, and physiology research to embrace emerging and diverse models. However, implementing cutting-edge gene editing techniques beyond traditional laboratory models remains technically challenging, especially for tissue-specific applications \u003cem\u003ein vivo\u003c/em\u003e in adult organisms.\u003c/p\u003e \u003cp\u003eGene editing technologies have transformative potential for neuroscience and behavior research. Targeted genetic modifications can provide critical insights into the genetic underpinnings of brain function, behavioral traits, and neurological disorders. However, when studying genes that influence behavior, a significant challenge emerges: embryonic gene knockouts\u0026mdash;the standard approach\u0026mdash;also disrupt brain development, creating confounding effects that obscure the gene's direct role in adult neural function. To isolate a gene's role in processing behavioral stimuli and generating behaviors, tissue-specific editing in the adult brain becomes essential.\u003c/p\u003e \u003cp\u003eDelivering gene editing elements to the adult brain presents multiple technical challenges. First, systemic administration methods typically fail to target the brain effectively due to the blood-brain barrier (BBB) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), which regulates the transfer of exogenous substances between the bloodstream and central nervous system. Intracerebroventricular (ICV) injection offers a direct solution by delivering molecules and/or vehicles directly into brain ventricles, facilitating immediate access to neural tissue. While ICV injection techniques have been described for fish models, including zebrafish and poecilids (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), standardized protocols optimized for behavioral neuroscience (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) and gene editing applications remain underdeveloped (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Beyond the delivery procedure, effective tissue-specific genome manipulation in adult tissues depends on appropriate carriers for the gene editing components.\u003c/p\u003e \u003cp\u003eTissue-specific \u003cem\u003ein vivo\u003c/em\u003e applications of CRISPR-Cas gene editing face significant challenges in delivering editing components to target cells. While viral vectors are commonly used for \u003cem\u003ein vivo\u003c/em\u003e gene delivery (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), they present considerable limitations, including restricted cargo capacity, random DNA integration risks, potential immunogenicity, and complex engineering requirements that make them inaccessible to many researchers (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). To overcome these obstacles, non-viral nanocarriers have emerged as promising alternatives for delivering therapeutic agents across biological barriers such as the BBB (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). For CRISPR-Cas to effectively reach brain tissue, it must be paired with a nanocarrier capable of encapsulating and protecting gene-editing components while facilitating cellular uptake, endosomal escape, and nuclear translocation\u0026mdash;all critical for successful gene editing (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Therefore, developing an effective non-viral delivery system represents a key milestone toward accessible \u003cem\u003ein vivo\u003c/em\u003e gene editing in non-model organisms.\u003c/p\u003e \u003cp\u003eAmong available nanocarriers, magnetite nanoparticles (MNPs) offer distinctive advantages for CRISPR-Cas delivery, addressing key challenges of \u003cem\u003ein vivo\u003c/em\u003e gene editing. MNPs effectively protect nucleic acids from nuclease degradation while enhancing cellular penetration (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Their capacity for efficient genetic material loading, cellular translocation, and endosomal escape is well documented (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), as is their high hemocompatibility, low cytotoxicity, and minimal thrombogenic effects in multiple \u003cem\u003ein vitro\u003c/em\u003e applications (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). We have developed a novel magnetite nanoparticle, specifically functionalized with nucleic acid/protein binding agents and a cell-translocating peptide (Buforin II), demonstrated to deliver large nucleic acids via a mechanism that exploits the reducing conditions of the cytoplasm to release cargo while preserving functionality. Using these nanoparticles, we have previously achieved successful \u003cem\u003ein vitro\u003c/em\u003e CRISPR manipulations, confirming its capacity to efficiently transport editing components into target cells (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe guppy, \u003cem\u003ePoecilia reticulata\u003c/em\u003e, represents an ideal model to implement and test novel \u003cem\u003ein vivo\u003c/em\u003e gene editing technologies. Studied for over a century in sexual selection and mate choice research (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), guppies have emerged as valuable models in behavioral neuroscience, genomics, and biomedical research due to their complex behaviors, experimentally tractable size, and unique adaptations (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The application of modern technologies in this group has accelerated the contributions made by guppy research across the biological sciences. We have made massive progress in our ability to make genetic associations and to dissect genetic etiologies in behavior and neuroscience. However, our inability to perform gene editing in guppies has significantly hindered essential functional studies. Developing brain-specific gene editing capabilities in guppies and similar fish species would unlock research into the precise genetic basis of multiple behaviors, brain function, and neurological disorders, with broad implications for neuroscience, physiology, and gene therapy development.\u003c/p\u003e \u003cp\u003eHere, we overcome these challenges by developing a custom intracranial microinjection setup and protocol optimized for guppies and other small fish and functionalized magnetic nanoparticles capable of safely and effectively delivering gene editing elements into brain cells. We aim to evaluate the efficiency of our magnetic nanoparticle delivery system by assessing cellular uptake and nuclear localization following the microinjection. We then comprehensively assess whether these nanoparticles are appropriate for \u003cem\u003ein vivo\u003c/em\u003e use through liver enzyme analysis, cytotoxicity assays, and behavioral testing. Our protocol and nanoparticle system are designed to offer versatility across multiple CRISPR-Cas strategies in a cost-efficient manner, potentially transforming neuroscience research capabilities in non-model organisms and creating new opportunities for targeted gene editing in anatomically complex tissues like the brain.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle (MNP) synthesis\u003c/h2\u003e \u003cp\u003eMagnetite nanoparticles (MNPs) were synthesized via a chemical co-precipitation method following established protocols (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Briefly, 0.01 mol of iron (II) chloride and 0.02 mol of iron (III) chloride (2:1 Fe\u0026sup3;⁺:Fe\u0026sup2;⁺ molar ratio) were dissolved in 100 mL of type I water (ultrapure water with a resistivity\u0026thinsp;\u0026gt;\u0026thinsp;18 MΩ-cm, and conductivity\u0026thinsp;\u0026lt;\u0026thinsp;0.056 \u0026micro;S/cm) and cooled to 2\u0026deg;C. Simultaneously, 0.08 mol of NaOH were dissolved in 100 mL of type I water and cooled to the same temperature. The iron chloride solution was continuously stirred magnetically under a nitrogen atmosphere to prevent oxidation. After 10 minutes of equilibration, the NaOH solution was gradually added dropwise to the iron solution while maintaining stirring under continuous nitrogen flow, resulting in the formation of MNPs as a black precipitate. The reaction continued for an additional hour to ensure complete precipitation and crystallization. The resultant MNPs were magnetically separated using a neodymium magnet and washed three times with 0.1 M NaCl solution and twice with deionized water to remove unreacted precursors and byproducts.\u003c/p\u003e \u003cp\u003eTo prepare for further functionalization, 100 mg of synthesized MNPs was suspended in 40 mL of type I water and sonicated to ensure homogeneous dispersion. Surface silanization of nanoparticles was performed based on the protocol described in previous studies (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). First, 250 \u0026micro;L of tetramethylammonium hydroxide (TMAH) was added as a stabilizing agent, followed by sonication and stirring to ensure uniform distribution. The suspension was then acidified with 50 \u0026micro;L of glacial acetic acid, followed by another round of sonication and stirring. Finally, 1 mL of (3-aminopropyl) triethoxysilane (APTES) (20% v/v) was slowly added to the solution, and the reaction mixture was stirred at 60\u0026deg;C for 1 hour with continuous stirring to ensure complete surface coverage. The silanized nanoparticles (MNPs-Si) were then magnetically separated and washed thoroughly as described above to remove unreacted APTES.\u003c/p\u003e \u003cp\u003eTo obtain the pegylated magnetite nanoparticles (MNPs-PEG), 100 mg of MNPs-Si were dispersed in 40 mL of type I water and sonicated in an ultrasonic bath for 10 minutes. Polyethylene glycol (PEG) conjugation was achieved via glutaraldehyde-mediated crosslinking to the surface amines. Initially, 2 mL of 2% (v/v) glutaraldehyde solution was added to the MNPs-Si suspension and agitated for 1 hour at room temperature to activate amine groups through Schiff base formation. Then, 10 mg of NH₂-PEG₁₂-NH₂ dissolved in 5 mL of type I water were added to the activated nanoparticles. The mixture was stirred at 220 RPM for 24 hours at room temperature to ensure complete conjugation. The MNPs-PEG were magnetically collected and extensively washed with deionized water to remove unbound PEG and glutaraldehyde, as previously described.\u003c/p\u003e \u003cp\u003eTo obtain the Buforin II conjugated magnetite nanoparticles (MNPs-BUF-II), 100 mg of MNPs-PEG were dispersed in 40 mL of type I water and sonicated in an ultrasonic bath for 10 minutes. BUF-II peptide (BUF-II, TRSSRAGLQFPVGRVHRLLRK), a cell-penetrating peptide derived from histone H2A with demonstrated nuclear-localizing properties (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), was conjugated to the terminal amine of the PEG chains using carbodiimide chemistry. Specifically, 14 mg of N-[3-(dimethylamino)-propyl]-N\u0026prime;-ethylcarbodiimide hydrochloride (EDC) and 7 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of type I water and added to the MNPs-PEG suspension to activate carboxyl groups on the PEG chains, generating NHS-ester intermediates. After 15 minutes of activation at room temperature with stirring at 220 rpm, 1 mg of BUF-II dissolved in 1 mL of Type I water was added. The conjugation reaction proceeded for 24 hours at room temperature with continuous stirring at 220 RPM to ensure efficient peptide attachment. The final MNPs-BUF-II were magnetically collected and thoroughly washed with deionized water to remove unbound peptide and coupling reagents (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Methods - Chemicals for nanoparticles synthesis section).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal husbandry\u003c/h3\u003e\n\u003cp\u003e All experimental procedures involving animals were conducted in accordance with the ethical standards and guidelines approved by the Animal Ethics Committee of Universidad de los Andes (CICUAL), under approval number C.FUA_23\u0026thinsp;\u0026minus;\u0026thinsp;004. Adult female guppies (\u003cem\u003ePoecilia reticulata\u003c/em\u003e) were obtained from a laboratory-maintained stock and individually housed in aquaria (14.1 cm in height, 21.5 cm long at the bottom, 25.4 cm long at the top, and 10 cm in width) under controlled environmental conditions: temperature 24\u0026ndash;26\u0026deg;C, conductivity 500\u0026ndash;600 \u0026micro;S/cm, dissolved oxygen 5\u0026ndash;6 ppm, and pH 6\u0026ndash;7. Fish were fed \u003cem\u003eat libitum\u003c/em\u003e a diet of Formula - Freshwater flakes and Artemia. Only sexually mature females were used in experiments to ensure consistency.\u003c/p\u003e\n\u003ch3\u003eBrain ventricle microinjection\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of Surgical Apparatus, Anesthesia and Life Support System\u003c/h2\u003e \u003cp\u003eA custom surgical bed was designed and fabricated using 3D printing to facilitate precise ICV injections (3D model provided in the supplementary files). The apparatus featured adjustable components to accommodate fish ranging from 2.5-4.0 cm in standard length while maintaining optimal positioning for dorsal cranial access. The bed was coated with biocompatible soft foam to provide a moist, non-abrasive surface, minimizing physical stress and epidermal injury during surgery. The design incorporated an open ventral section to enable optimal light transmission, enhancing visualization and ensuring stable positioning with optimal dorsal access to the cranial region under a stereomicroscope.\u003c/p\u003e \u003cp\u003eAnesthesia was maintained using a gravity-fed perfusion system. A cannula (Jelco Seriva 24Gx19mm) was inserted into the fish\u0026rsquo;s mouth to deliver anesthetic or freshwater solutions. The system comprised two reservoirs: (I) one containing buffered tricaine methanesulfonate (MS-222, 0.2 mg/mL, buffered to pH 7.0 with Tris Buffer 1M) and (II) the other containing fresh water. Flow rates were controlled via a three-way valve system, allowing seamless transition between anesthesia and recovery phases (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNeedle Pulling\u003c/h3\u003e\n\u003cp\u003eMicroinjection needles were crafted from borosilicate glass capillaries (TW 100-4, WPI) using a programmable puller (PC-100, Narishige), set with the following parameters: first pull temperature (T1): 65\u0026deg;C; second pull temperature (T2): 80\u0026deg;C; with a weight configuration of two lightweights and one heavyweight. These settings were optimized to produce needles with the required durability and thickness to reach the third ventricle while remaining thin enough to minimize damage to the surrounding brain tissue. Needle tips were manually beveled using a surgical blade under stereomicroscopic guidance to achieve consistent injection volumes. Each needle was calibrated prior to use by dispensing Evans Blue dye (0.1% w/v) droplets into mineral oil, and the resulting droplet diameter was measured microscopically (Further details in Supplementary Methods \u0026ndash; Brain ventricle microinjection and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicroinjection Procedure\u003c/h2\u003e \u003cp\u003eFish were fasted for 12 hours before the procedure to minimize regurgitation risk (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Anesthesia was induced by immersion in buffered tricaine solution (MS-222, 0.3 mg/mL) until the fish exhibited loss of equilibrium, reduced opercular movement (approximately 1 opercular movement per second), and absence of response to gentile tactile stimuli (typically within 3 minutes) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Anesthetized fish were transferred to the custom surgical bed in ventral recumbency and immediately connected to the life support system under a stereoscope (Olympus SZX7), ensuring continuous perfusion of tricaine (MS-222, 0.25 mg/mL) at a rate of one drop every 3 seconds. The surgical procedure was limited to a maximum of 8 minutes to minimize anesthetic exposure. A craniotomy was performed to access the third ventricle for microinjection. First, the exact injection site was identified using external neuroanatomical landmarks\u0026mdash;specifically targeting the cranial midline at the junction between the telencephalon and optic tectum, which corresponds to the location of the third ventricle. A small incision was made in the cranial bone as previously reported (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e), using a 27G x \u0026frac12; needle and fine forceps (Dumont Tweezers #5). The injection needle was positioned at an approximately 30\u0026ndash;45\u0026deg; angle relative to the cranial surface and inserted approximately 2 mm deep through the incision using a micromanipulator (PM 1000 Cell, MDI) to deliver the solution. Post-injection, the needle was gently withdrawn, and the three-way valve system was switched to fresh water for 1 minute to begin the recovery process. The fish was then carefully transferred to a dedicated recovery tank supplied with oxygen. Recovery was monitored continuously and assessed using standardized criteria: restoration of normal swimming axis (typically within 5\u0026ndash;10 minutes), regular opercular movement (within 2\u0026ndash;3 minutes), and coordinated swimming behavior (within 10\u0026ndash;15 minutes) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e) (Fig. S3).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBehavioral Tests\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eNovel Tank Test\u003c/h2\u003e \u003cp\u003eThe Novel Tank Test was performed 24 hours post-injection to assess stress responses and locomotor activity. This test exploits the natural tendency of fish to seek protection in unfamiliar environments, which typically manifests as reduced exploration and bottom-dwelling behavior. An imaginary horizontal line is used to divide the tank into upper and lower compartments (each compartment having a height of 6 cm, as the water column was maintained at 12 cm). Such division is effective because stress and anxiety-like behavior tend to increase the time spent in the bottom half of the tank (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Tests were conducted in a trapezoidal tank (14.1 cm height x 21.5 cm bottom x 25.4 cm top x 10 cm width). Fresh water was used in each trial to avoid chemosensitive stimuli from previous subjects altering behavior. Each fish was gently transferred from its home tank using a dedicated net and placed in the center of the novel tank. After a 60-second acclimation period to minimize handling effects, behavior was recorded for 10 minutes under standardized lighting conditions. From these recordings, we extracted the following quantitative behavioral measures: time spent in the bottom compartment relative to total test time, swimming distance [cm], freezing duration relative to total test time and swimming velocity [cm/s] (Fig. S4A).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSocial Interaction Test\u003c/h2\u003e \u003cp\u003eThe social interaction test was performed 48 hours post-injection to evaluate social motivation and recognition, functions that involve complex neural processing across multiple brain regions. Altered social behavior can indicate neurological dysfunction that might not be apparent in individual behavioral tests. The experimental setup consisted of a two-tank setup: a larger test tank (20.0 cm height x 41.0 cm length x 29.0 cm width) and a smaller stimulus tank (20.0 cm height x 5.0 cm length x 9.5 cm width) positioned adjacent to one end of the test tank. Water depth was maintained at 12 cm in both tanks. The individual being studied was placed into the larger tank, while a shoal of two female and three male guppies was placed in the smaller tank. The test tank was divided into three distinct zones: a proximity zone (8 cm width) adjacent to the stimulus tank, and two equal-sized neutral zones (16.5 cm each) extending toward the opposite end (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Individuals were handled with a net and transferred to the tank, where they were allowed to have one minute to acclimate and then recorded for 10 minutes. Time spent in each zone [s] was measured (Fig. S4B).\u003c/p\u003e \u003cp\u003eAll data for behavioral assays was collected using a video tracking system (Noldus EthoVision XT7), with special care taken to ensure proper recording conditions for the software, as detailed in Cachat et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eValidation of the ICV microinjection\u003c/h2\u003e \u003cp\u003eBefore administering MNPs, we validated the ICV microinjection protocol by injecting Evans Blue dye to confirm accurate delivery into the third ventricle through visible tissue coloration upon dissection and to assess potential behavioral effects. The dye also helped verify the injection site. Additionally, we evaluated whether the ICV microinjection protocol caused any alterations in the nervous system of guppy fish through behavioral tests. To assess the effect of ICV microinjection, we injected six adult fish with Evans Blue solution and compared their behavior to non-injected control fish. We tested their behavior using standard novel tank and social interaction tests. The novel tank test took place 10 minutes after injection, and the social interaction test was conducted 48 hours later.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of nanoparticles as delivery vehicles after ICV microinjection\u003c/h2\u003e \u003cp\u003eTo assess the efficiency and safety of our magnetic nanoparticles (MNPs) as delivery vehicles for gene editing applications, we administered MNPs via ICV microinjection and conducted comprehensive evaluations of their distribution, cellular uptake, and potential health effects. For all experiments in this section, we injected 600 nL of MNP solution at a concentration of 25 \u0026micro;g/mL directly into the third ventricle. Prior to each injection, the MNP solution was sonicated for 30 seconds and the suspended volume was immediately aspirated into the microinjection needle to ensure consistent nanoparticle delivery and prevent aggregation-related variations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCellular Uptake\u003c/h2\u003e \u003cp\u003eThe effectiveness of MNPs as gene delivery vehicles depends on their ability to reach target cells (brain cells). To evaluate the capacity of MNPs to enter brain cells and access the nucleus\u0026mdash;a prerequisite for effective delivery of gene-editing components\u0026mdash;we conducted detailed cellular uptake studies at two timepoints: 24 hours (short-term exposure) and 2 weeks (long-term exposure) post-injection, with four subjects per timepoint.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSample preparation\u003c/em\u003e: We euthanized guppies with an MS-222 overdose (0.3 mg/mL) for 10 minutes and then performed decapitation. Heads were immediately fixed in 4% paraformaldehyde (PFA) for 24 hours at 4\u0026deg;C to preserve ultrastructure and fluorescent labeling. Brains were carefully dissected under stereomicroscopic guidance and cryoprotected through sequential incubation in a 1:1 solution of PFA and sucrose (30% v/v), followed by a sucrose solution for approximately two hours or until they completely sank in the Eppendorf tube. The cryoprotected brains were embedded in OCT compound, flash-frozen on dry ice, and sectioned dorsally at 20 \u0026micro;m thickness using a cryostat (Leica CM1860). Sections were collected from three neuroanatomically distinct regions: telencephalon (TL), tissue surrounding the third ventricle (3V), and optic tectum (OT). These regions were selected based on their proximity to the injection site. For each region, 2\u0026ndash;3 sections were collected per brain and mounted on positively charged microscope slides (Fig. S5).\u003c/p\u003e \u003cp\u003ePrior to injection, MNPs were labeled with SYBR Green using a previously validated protocol that maintains nanoparticle functionality while providing a stable fluorescent signal. To visualize cell nuclei, tissue sections were washed three times with PBS and counterstained with 10 \u0026micro;M DAPI. Slides were then stored in darkness until imaging. Confocal microscopy was performed using an Olympus FV1000 system with a 60\u0026times; oil immersion objective (NA 1.42). For each section, z-stacks (0.5 \u0026micro;m steps) were acquired across the full tissue thickness using three channels: 405 nm excitation/425\u0026ndash;475 nm emission for DAPI (nuclei), 488 nm excitation/500\u0026ndash;550 nm emission for SYBR Green (MNPs), and 559 nm excitation/575\u0026ndash;625 nm emission for tissue autofluorescence (to visualize general cellular morphology). Images were analyzed using ImageJ software with custom macros to quantify MNP cellular uptake and nuclear colocalization. Background signals were removed by subtracting the average fluorescence intensity from control (non-injected) samples. Nuclear uptake efficiency was calculated as the percentage of DAPI-positive nuclei showing colocalization with SYBR-labeled MNPs, determined using the Jaccard coefficient of similarity with intensity thresholding (Further details in Supplementary Methods - Cellular uptake section and Fig. S5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSystemic Distribution\u003c/h2\u003e \u003cp\u003eWe expect excess nanoparticles to exit the brain and we know these tend to accumulate in the liver where they are metabolized (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e). We measured iron concentrations in brain and liver tissues 48 hours post-injection using inductively coupled plasma optical emission spectroscopy (ICP-OES). Six fish were injected with MNPs and six non-injected fish served as controls. After 48 hours, fish were euthanized as described above, and brain and liver tissues were carefully dissected. Due to the small tissue mass, organs from two fish were pooled for each analytical sample, resulting in three biological replicates per treatment group. Tissues were stored at 4\u0026deg;C until analysis. For sample preparation, tissues were digested in a mixture containing 0.25 mL of concentrated nitric acid (70% w/w), 0.75 mL of concentrated hydrochloric acid (37% w/w), and 0.3 mL of hydrogen peroxide (30% w/w). We then heated the mixture at 80\u0026deg;C for 3 hours and diluted it to a final volume of 5 mL with deionized water. Additionally, we included a group of six non-injected control samples. Iron concentration was measured using ICP-OES (Perkin Elmer Optima 8000) with the following parameters: plasma power 1300 W, nebulizer flow 0.7 L/min, auxiliary flow 0.2 L/min, and plasma flow 12 L/min. Standard curves were prepared using certified iron standard solutions (1000 mg/L, traceable to NIST) diluted to 0-500 \u0026micro;g/L. The detection limit for iron was 3.33 \u0026micro;g/L, and samples below this threshold were excluded from analysis. Final results were normalized to tissue wet weight and reported as \u0026micro;g Fe/mg tissue (Fig. S6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Nanoparticle Microinjection Effects on Fish Health\u003c/h2\u003e \u003cp\u003eTo evaluate the potential effects of magnetic nanoparticles (MNPs) on animal health, we assessed hepatic function and cell viability in MNP-exposed fish. We compared control and MNP-injected fish, focusing on key hepatic enzymes and cytotoxicity indicators. Hepatic function was evaluated by measuring the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Cell viability was assessed using calcein AM and propidium iodide staining in brain tissues. These analyses provide insights into the biochemical and cellular impacts of MNP exposure on fish health.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHepatic Function Tests\u003c/h2\u003e \u003cp\u003eTo assess liver damage, we measured the activity of alanine aminotransferase ALT, AST, and ALP. The study included a control group of non-injected fish and an experimental group of fish injected with MNPs seven days before sample extraction. Each group consisted of four biological replicates, with each replicate pooling three individuals. This timeframe was selected to allow nanoparticles to reach the liver and induce any detectable damage. To minimize variations in enzyme concentrations due to food intake, fish were fasted for 12 hours before sample collection.\u003c/p\u003e \u003cp\u003eDue to the small blood volume of individual guppies, we adapted a specialized microsampling approach based on previously reported protocols for small fish (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e). We euthanized guppies with an MS-222 overdose (0.3 mg/mL) for 10 minutes and then performed decapitation. The body and head were separately placed in modified chromatography columns with the cut surfaces facing downward. The columns were inserted into microcentrifuge tubes containing 1 \u0026micro;L of heparin (500 IU/mL) and centrifuged at 190 \u0026times; g for 90 seconds followed by 1500 \u0026times; g for 3 minutes. The collected blood (approximately 5\u0026ndash;8 \u0026micro;L per fish) was pooled from three individuals to create a single biological replicate, yielding four replicates per treatment group. Plasma was separated by centrifugation at 2000 \u0026times; g for 5 minutes and stored at -20\u0026deg;C until analysis. Tissue samples were collected from the same individuals, and the extracted volume from each fish was determined according to the manufacturer's protocol for each enzymatic kit. The activity of ALT, AST, and ALP was measured using commercial kits: ALT (MAK052, Sigma-Aldrich), AST (ab105135, Abcam), and ALP (ab83371, Abcam). All assays were performed according to manufacturer protocols with slight modifications to optimize for the small sample volumes available from guppies (detailed in Supplementary Methods \u0026ndash; Hepatic health section). Each biological replicate was measured in triplicate to ensure technical reproducibility, and results were expressed as enzymatic units per liter (U/L) (Fig. S7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity Assessment\u003c/h2\u003e \u003cp\u003eTo directly evaluate whether MNPs cause cell death in neural tissue, we conducted in situ viability assays on brain cells two weeks post-injection. This timepoint was selected to capture both acute and subacute cytotoxic effects while allowing sufficient time for MNP distribution and cellular interactions. Three experimental groups were compared: MNP-injected fish (n\u0026thinsp;=\u0026thinsp;4), saline-injected fish (n\u0026thinsp;=\u0026thinsp;4, procedural control), and non-injected fish (n\u0026thinsp;=\u0026thinsp;4, negative control). Following euthanasia, brains were immediately dissected in PBS and incubated in DMEM with 1:1000 calcein AM and 1:2000 propidium iodide at 37\u0026deg;C for 30\u0026ndash;90 minutes. Using an Olympus FV1000 Confocal Laser Scanning Microscope (Olympus, Shinjuku, Tokyo, Japan) with a PlanApo 10\u0026times; and 20\u0026times;, 1.2 NA objective, we captured images. Calcein AM permeates living cells and is converted to calcein by intracellular esterases, producing bright green fluorescence in viable cells. Propidium iodide is membrane-impermeable and only enters cells with compromised membranes (dead or dying cells), where it binds to nucleic acids and emits red fluorescence. Finally, we determined the dead cell ratio by performing intensity analysis in ImageJ/Fiji v2.9.0 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and compared it among the experimental groups. Briefly, a custom Python script that implemented the following workflow: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) noise reduction through morphological operations, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) segmentation using Sobel filtering and Otsu's thresholding, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) watershed-based cell separation, and (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) connected component labeling to count individual cells. Cell viability was calculated as the ratio of calcein-positive cells to total cells (calcein-positive plus propidium iodide-positive) expressed as a percentage. This approach provided region-specific quantification of cell viability that could be directly compared across treatment groups (Further details in Supplementary Methods - Cytotoxicity (Life and dead) section, and Fig. S8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral Effects of Nanoparticle Microinjection\u003c/h2\u003e \u003cp\u003eTo assess potential neurofunctional effects of MNPs that might not be detectable through direct cytotoxicity measurements, we conducted comprehensive behavioral testing using the previously described novel tank and social interaction assays. These tests serve as sensitive functional indicators of nervous system and general health. Three experimental groups were evaluated: MNP-injected fish (n\u0026thinsp;=\u0026thinsp;19), saline-injected fish (n\u0026thinsp;=\u0026thinsp;4, procedural control), and non-injected fish (n\u0026thinsp;=\u0026thinsp;16, negative control). The novel tank test was performed 24 hours post-injection, while the social interaction test was conducted 48 hours allowing the fish a reasonable rest period between tests. To ensure the robustness of our results, and considering the number of fish we could handle in a single injection batch, the experiment was carried out in batches, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. These batches were included as a random factor in all statistical models.\u003c/p\u003e \u003cp\u003eBehavioral data for the Evans blue-injected fish were analyzed using a t-test. The other experimental groups were analyzed using linear mixed-effects models, implemented in the lme4 package (v1.1) in R. Treatment (MNP-injected, saline-injected, or non-injected) was included as a fixed effect, while batch was included as a random effect to account for potential environmental or temporal variations. When relevant Post-hoc comparisons between groups were performed with Tukey's honest significant difference test, with significance threshold set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eValidation of ICV microinjection protocol\u003c/h2\u003e \u003cp\u003eWe developed a comprehensive intracerebroventricular (ICV) microinjection protocol optimized for adult \u003cem\u003ePoecilia reticulata\u003c/em\u003e and potentially applicable to other small teleost fish. Central to this development was a custom-designed 3D-printed surgical apparatus integrated with a life support and anesthesia system (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig. S9). This system maintains fish in optimal position for precise microinjection while providing continuous anesthesia delivery and respiratory support, critical factors for procedure success and post-operative recovery. We performed an initial validation of the ICV microinjection procedure using Evans blue dye injections, which allowed visual confirmation of injection accuracy and assessment of diffusion patterns within brain tissue (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e0). To assess potential adverse effects of the microinjection procedure itself, we performed comprehensive behavioral testing on Evans blue-injected fish compared to non-injected controls. Here, the novel tank test, showed no significant differences between groups in anxiety-related parameters including time spent in the bottom of the tank (t\u0026thinsp;=\u0026thinsp;0.891, p\u0026thinsp;=\u0026thinsp;0.391) or freezing behavior (t\u0026thinsp;=\u0026thinsp;0.777, p\u0026thinsp;=\u0026thinsp;0.453). Similarly, locomotor function remained intact, with no significant differences in total swimming distance (t\u0026thinsp;=\u0026thinsp;0.822, p\u0026thinsp;=\u0026thinsp;0.428) or mean velocity (t\u0026thinsp;=\u0026thinsp;1.196, p\u0026thinsp;=\u0026thinsp;0.256) as schematically shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e1A-D. The social interaction test, conducted 48 hours post-injection, revealed that the injection procedure did not affect social motivation or recognition, as evidenced by equivalent time spent in the proximity zone between Evans blue-injected fish and controls (t = -0.109, p\u0026thinsp;=\u0026thinsp;0.91) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e1E).\u003c/p\u003e \u003cp\u003eThese comprehensive validation results demonstrate that our optimized ICV microinjection protocol provides precise targeting of the third ventricle with minimal anatomical or behavioral perturbation, establishing a reliable foundation for subsequent nanoparticle delivery experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCellular Uptake\u003c/h2\u003e \u003cp\u003eThe effectiveness of MNPs as gene delivery vehicles depends critically on their ability to enter target cells and access the nucleus. We assessed cellular uptake and nuclear localization of fluorescently-labeled MNPs in brain tissue at two timepoints: 24 hours (short-term) and 2 weeks (long-term) post-injection. We selected cryosections corresponding to tissues around the third ventricle for all analysis, as this is where the ICV microinjection was performed (images for each individual can be found in Fig. S12).\u003c/p\u003e \u003cp\u003eConfocal microscopy analysis of brain sections surrounding the third ventricle revealed substantial uptake of MNPs by brain cells at both timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the short-term exposure group, we observed a nuclear colocalization rate (defined as the percentage of nuclei showing MNP signal) of 40.9%, albeit with very large variability across individuals (SD\u0026thinsp;=\u0026thinsp;26.9%). This high variability reflected considerable inter-individual differences, with nuclear uptake rates ranging from 10.9\u0026ndash;73.6% across the subjects examined. Notably, two subjects exhibited exceptionally high nuclear uptake rates (\u0026gt;\u0026thinsp;50%), demonstrating that MNPs can efficiently penetrate the nuclear membrane within 24 hours of administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The mean nuclear colocalization rate increased to 54.6% at two weeks, with much more consistent results (SD\u0026thinsp;=\u0026thinsp;7.7%) and all individual values ranging from 44\u0026ndash;62.3%. The substantially reduced variability and higher nuclear uptake rates suggest that prolonged exposure promotes more uniform cellular and nuclear uptake across individuals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Confocal microscopy images showing nuclei stained with DAPI (blue) and MNPs labeled with SYBR Green (green) in brain tissue cryoslices for injected individuals. The \"Merge\" panel displays the overlay of the DAPI and SYBR channels, highlighting areas where MNPs are in close proximity to the nuclei. The \"Intercept\" panel selectively shows regions where both signals coincide, indicating nuclear uptake of the MNPs. (B) Quantification of nuclear uptake efficiency, as a percentage of nuclei with MNPs (SYBR green) signal, in individuals injected with MNPs after 24 hours (short-term) and 2 weeks (long-term).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSystemic distribution\u003c/h2\u003e \u003cp\u003eAfter confirming cellular uptake within the brain, we investigated whether MNPs administered via ICV injection remained confined to the central nervous system or distributed to peripheral organs. We measured iron concentrations in brain and liver tissues 48 hours post-injection using inductively coupled plasma optical emission spectroscopy (ICP-OES). As expected, MNP-injected fish exhibited higher mean iron concentrations in brain tissue compared to non-injected controls (0.091 \u0026micro;g/mg in the control group vs. 0.132 \u0026micro;g/mg in the injected group), confirming retention of the injected nanoparticles within the target organ. We also detected elevated iron levels in the liver of MNP-injected fish (0.053 \u0026micro;g/mg in controls vs. 0.372 \u0026micro;g/mg in injected fish), suggesting that a portion of the injected MNPs migrated to the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The magnitude of iron concentration increase was substantially greater in the liver (+\u0026thinsp;602%) than in the brain (+\u0026thinsp;45%), suggesting a large fraction of nanoparticles are exiting the brain and accumulating in hepatic tissue. This liver accumulation is consistent with previous studies showing that the liver serves as the primary organ for nanoparticle clearance once they enter the bloodstream. Despite the clear trend toward higher iron concentrations in MNP-injected individuals, the differences were not statistically significant due to high variability within each treatment group (t-test: brain, t = -0.905, p\u0026thinsp;=\u0026thinsp;0.41; liver, t = -1.672, p\u0026thinsp;=\u0026thinsp;0.16).\u003c/p\u003e \u003cp\u003eThe observed hepatic accumulation of MNPs following ICV injection highlights the importance of evaluating potential effects this could have on the fish\u0026rsquo; health, particularly hepatotoxicity. Three samples were lost from analysis due to the small size of livers and iron concentrations below the detection limit of the equipment. We excluded one brain and one liver sample from the MNP-injected group, and one brain sample from the control group. The exclusion of these samples did not significantly alter the overall pattern of results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Nanoparticle Microinjection Effects on Fish Health\u003c/h2\u003e \u003cp\u003eGiven the observed distribution of MNPs to both neural tissue and liver, we conducted comprehensive health assessments focusing on potential hepatotoxicity and neurotoxicity\u0026mdash;critical safety parameters for any nanocarrier used in \u003cem\u003ein vivo\u003c/em\u003e applications.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eHepatic function tests\u003c/h2\u003e \u003cp\u003eTo evaluate potential hepatotoxic effects of MNPs, we measured the activities of three liver-specific enzymes that serve as established biomarkers of hepatocellular damage and dysfunction: alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST). Elevated activities of these enzymes in circulation typically indicate hepatocellular damage or altered liver function.\u003c/p\u003e \u003cp\u003eAnalysis of hepatic enzyme activities revealed no significant differences between MNP-injected fish and non-injected controls for any of the measured enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). ALT activity showed nearly identical levels between groups (t\u0026thinsp;=\u0026thinsp;0.371, p\u0026thinsp;=\u0026thinsp;0.723), suggesting no hepatocellular damage following MNP exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, AST activity remained unchanged (t\u0026thinsp;=\u0026thinsp;0.920, p\u0026thinsp;=\u0026thinsp;0.393), further indicating hepatocyte integrity was maintained despite MNP accumulation in liver tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eALP activity exhibited greater variability in the MNP-injected group but did not differ significantly from controls (t = -0.684, p\u0026thinsp;=\u0026thinsp;0.525) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The observed variability may reflect individual differences in nanoparticle processing and biliary function. Importantly, none of the MNP-injected fish exhibited enzyme activities exceeding the upper reference limits established from the control group (defined as mean\u0026thinsp;+\u0026thinsp;2SD), indicating absence of clinically significant hepatotoxicity.\u003c/p\u003e \u003cp\u003eThese biochemical findings demonstrate that despite the accumulation of MNPs in liver tissue observed in our systemic distribution studies, hepatic function remained uncompromised, suggesting effective biotransformation and/or elimination of the nanoparticles without associated damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity Assessment\u003c/h2\u003e \u003cp\u003eTo directly evaluate potential cytotoxic effects of MNPs on neural tissue, we performed live/dead cell viability assays using calcein-AM and propidium iodide staining on brain tissues harvested two weeks post-injection. This dual-fluorescence approach allowed simultaneous visualization and quantification of viable (calcein-positive) and non-viable (propidium iodide-positive) cells across different brain regions.\u003c/p\u003e \u003cp\u003eConfocal microscopy analysis revealed high cell viability across all experimental groups, with no significant differences in viability rates among MNP-injected, saline-injected, and non-injected treatments (ANOVA: F\u0026thinsp;=\u0026thinsp;2.115, p\u0026thinsp;=\u0026thinsp;0.236) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The MNP-injected and non-injected control groups both maintained average cell viability exceeding 80% in all examined brain regions, indicating minimal cytotoxic effects from nanoparticle exposure. The saline-injected group showed slightly lower viability (\u0026gt;\u0026thinsp;70%), although this reduction was not statistically significant.\u003c/p\u003e \u003cp\u003eWe also assessed regional differences in cell viability across three distinct brain areas: tissue surrounding the third ventricle (3V), optic tectum (OT), and telencephalon (TL). No significant differences were detected across these regions (ANOVA: F\u0026thinsp;=\u0026thinsp;1.421, p\u0026thinsp;=\u0026thinsp;0.341), suggesting uniform safety profiles throughout the brain despite variable MNP distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe slightly higher viability observed in MNP-injected fish compared to saline-injected controls is particularly noteworthy, suggesting that our functionalized MNPs may offer some degree of cellular protection rather than toxicity. While the mechanism for this potential protective effect requires further investigation, it may relate to the antioxidant properties of magnetite or the surface functionalization components.\u003c/p\u003e \u003cp\u003eThese cytotoxicity findings, combined with the hepatic enzyme results, provide strong evidence for the biological safety of our MNP formulation when administered via ICV injection, supporting its potential use as a delivery vehicle for in vivo gene editing applications in the brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eBehavioral assays\u003c/h2\u003e \u003cp\u003eThe novel tank test, conducted 24 hours post-injection, revealed several subtle but statistically significant differences in stress-related behavior and locomotor parameters across treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003eAnalysis of bottom-dwelling behavior\u0026mdash;a well-established anxiety indicator in fish\u0026mdash;showed no significant difference between MNP-injected fish and non-injected controls (z-value = -0.219, p\u0026thinsp;=\u0026thinsp;0.972). However, saline-injected fish exhibited significantly increased bottom-dwelling time compared to controls (z-value\u0026thinsp;=\u0026thinsp;2.402, p\u0026thinsp;=\u0026thinsp;0.040) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This finding suggests that while the injection procedure itself may induce transient anxiety-like behavior, the presence of MNPs does not exacerbate this effect.\u003c/p\u003e \u003cp\u003eBoth MNP-injected and saline-injected groups displayed significantly increased freezing duration compared to non-injected controls (MNP: z-value\u0026thinsp;=\u0026thinsp;2.941, p\u0026thinsp;=\u0026thinsp;0.008; saline: z-value\u0026thinsp;=\u0026thinsp;3.161, p\u0026thinsp;=\u0026thinsp;0.004) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This consistent effect across both injected groups suggests that increased freezing behavior is primarily attributable to the microinjection procedure rather than to the nanoparticles themselves.\u003c/p\u003e \u003cp\u003eTotal swimming distance was significantly reduced in both MNP-injected (z-value = -0.291, p\u0026thinsp;=\u0026thinsp;0.010) and saline-injected (z-value = -3.186, p\u0026thinsp;=\u0026thinsp;0.004) groups compared to controls, indicating decreased locomotor activity following the injection procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Similarly, swimming velocity showed a non-significant decrease in the MNP-injected group (z-value = -0.614, p\u0026thinsp;=\u0026thinsp;0.801) and a significant decrease in the saline-injected group (z-value = -2.506, p\u0026thinsp;=\u0026thinsp;0.031) relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eImportantly, across all measured parameters, the MNP-injected group never showed more pronounced alterations than the saline-injected group, indicating that observed behavioral changes were primarily due to the microinjection procedure itself rather than specific effects of the nanoparticles. Additionally, all behavioral parameters remained within the natural variability range observed in the control population, suggesting that the detected differences, while statistically significant, do not represent severe functional impairment.\u003c/p\u003e \u003cp\u003eThe social interaction test, conducted 48 hours post-injection, revealed no significant differences in social preference across treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Time spent in the proximity zone\u0026mdash;a direct measure of social motivation\u0026mdash;was statistically equivalent in MNP-injected (z-value\u0026thinsp;=\u0026thinsp;0.800, p\u0026thinsp;=\u0026thinsp;0.696), saline-injected (z-value\u0026thinsp;=\u0026thinsp;1.036, p\u0026thinsp;=\u0026thinsp;0.546), and non-injected control fish. This preservation of normal social behavior is particularly noteworthy as social cognition involves complex neural processing across multiple brain regions, including those showing high MNP accumulation in our cellular uptake studies.\u003c/p\u003e \u003cp\u003eThe absence of alterations in social behavior at 48 hours post-injection, combined with the generally mild and procedure-related changes observed in the novel tank test at 24 hours, suggests that any effects of the ICV microinjection on the nervous system are transient and resolve within 48 hours.\u003c/p\u003e \u003cp\u003eCollectively, these behavioral findings indicate that while the microinjection procedure itself may induce mild and transient stress responses, the administration of MNPs does not cause additional behavioral alterations or damage to the nervous system. The preservation of complex social behaviors and the containment of locomotor alterations within the normal range of variability provide strong functional evidence supporting the neurocompatibility of our nanoparticle delivery system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecent advances in genomic technologies have dramatically enhanced our understanding of the genetic architecture underlying adaptive traits in behavior, neuroscience, and neurological disorders. However, further functional tests often remain out of reach due to our inability to perform targeted genetic manipulation in adult tissues in non-traditional model organisms. Developing tools for \u003cem\u003ein vivo\u003c/em\u003e gene editing directly in the adult brain is crucial for studying questions in behavioral neuroscience, as manipulating genes in embryos, can introduce confounding effects on brain development (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Our study addresses this fundamental challenge by developing a complete technological platform for brain-specific gene editing in adult guppies\u0026mdash;comprising both a standardized microinjection protocol and a biocompatible nanocarrier optimized for delivering gene editing components. A key component of this goal was to design a delivery vehicle that is both safe for \u003cem\u003ein vivo\u003c/em\u003e use and capable of efficiently and effectively transporting gene editing elements into brain cells. With this goal in mind, we designed a magnetic nanoparticle delivery system, that we previously tested \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and investigated the efficiency and safety of these magnetic nanoparticles as delivery vehicles in the brain, evaluating their impact on fish\u0026rsquo;s health and their nuclear uptake rates in brain cells.\u003c/p\u003e \u003cp\u003eThe first major barrier to adult brain-specific gene editing is the blood-brain barrier (BBB), which prevents systemic delivery of gene editing components to neural tissue (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e). We have overcome this obstacle by developing a intracerebroventricular (ICV) microinjection protocol that provides direct access to the brain's ventricular system. Our custom-designed surgical apparatus and life support system enable precise, minimally invasive delivery. The validation data using Evans blue dye confirmed accurate targeting of the third ventricle with subsequent diffusion demonstrating the technical feasibility of our approach.\u003c/p\u003e \u003cp\u003eBehavioral evaluation further showed that the microinjection procedure itself had minimal impact on fish health and behavior. While we observed some transient effects on locomotor parameters and stress responses at 24 hours post-injection, these alterations were mild and largely resolved by 48 hours, as evidenced by normal social behavior in the interaction test. The rapid recovery confirms that our protocol provides a viable avenue for brain-targeted interventions in small fish models.\u003c/p\u003e \u003cp\u003eThe second crucial barrier to \u003cem\u003ein vivo\u003c/em\u003e gene editing is identifying an appropriate delivery vehicle that can efficiently transport editing components into cells and their nuclei. The choice of delivery carrier is particularly critical, requiring careful optimization to ensure both efficiency and safety. Our magnetic nanoparticles present several advantages over viral vectors, including simpler preparation, reduced immunogenicity, and greater loading capacity (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe have previously evaluated the efficiency of our magnetic nanoparticles as delivery vehicles for CRISPR gene editing \u003cem\u003ein vitro\u003c/em\u003e. We found them to be very efficient vehicles, binding nucleic acids efficiently and achieving transfection rates over 50% that ultimately led to a 130-fold overexpression of the target gene when combined with a CRISPRa plasmid (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Therefore, we know our magnetic nanoparticles are capable of carrying and delivering CRISPR elements into cells, achieving successful CRISPR manipulations results. The magnetic nanoparticles we use are ideally suited for gene editing applications, as they can be easily bound with diverse CRISPR/Cas elements depending on the needs of different gene editing strategies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). They can bind thiol-modified nucleic acids, enabling conjugation to linearized CRISPR plasmids, single-guide RNA (sgRNA), and Cas9 mRNA. For ribonucleoprotein (RNP) complexes, conjugation occurs via a direct covalent bond through amine-carboxyl coupling on the Cas9 protein. This versatility ensures compatibility with multiple CRISPR delivery strategies, from plasmid-based expression systems to pre-assembled RNP complexes for immediate genome editing.\u003c/p\u003e \u003cp\u003eThe observed progression from variable uptake at 24 hours (40.9% \u0026plusmn; 26.9%) to more consistent and higher uptake at two weeks (54.6% \u0026plusmn; 7.7%) suggests an ongoing cellular internalization process rather than a single rapid uptake event. This temporal pattern is advantageous for gene editing applications, as it provides a sustained delivery window that may enhance editing efficiency. The reduced variability at the later timepoint further suggests that individual differences in initial uptake eventually converge to a consistent high level across subjects, an important consideration for experimental reproducibility. A promising way to address the initial variability in magnetite nanoparticle uptake and ensure sustained delivery for gene editing could involve combining targeted surface modifications, external stimulation, and time-dependent release strategies. Functionalizing nanoparticles with cell-penetrating peptides or other ligands can enhance early and robust internalization, while applying external magnetic fields or focused ultrasound helps localize and concentrate them in desired tissues, thus mitigating individual differences in uptake (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Taken together, these measures could not only extend the editing window but also improve reproducibility by converging on a similarly high-level internalization profile across subjects. Nanoparticle dynamics tend to change in a physiological fluid and in a live system, becoming impossible to predict from \u003cem\u003ein vitro\u003c/em\u003e data (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Finding comparable nuclear uptake rates \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e speaks of the robust properties of the nanocarrier.\u003c/p\u003e \u003cp\u003eFor any \u003cem\u003ein vivo\u003c/em\u003e gene editing approach, safety is paramount. The magnetic nanoparticles we tested do not induce significant cytotoxicity, as indicated by the high cell viability observed in the brain tissue of MNP-injected fish (\u0026gt;\u0026thinsp;80% at 25 \u0026micro;g/mL). The maintenance of cell viability despite substantial nuclear uptake confirms that our nanoparticles can access the nucleus without compromising cellular integrity\u0026mdash;a delicate balance essential for functional gene editing. Our \u003cem\u003ein vivo\u003c/em\u003e results show slightly lower viability compared to previous \u003cem\u003ein vitro\u003c/em\u003e studies on nervous system cells (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e). This slight difference may be attributed to cell death occurring during tissue processing, including dissection and staining, rather than the injection of nanoparticles themselves. In general, there is abundant evidence showing that appropriately sized and coated magnetite nanoparticles exhibit low cytotoxicity (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Moreover, our magnetic nanoparticles are uniquely functionalized with PEG and Buforin II, which may have contributed to their low toxicity profile.\u003c/p\u003e \u003cp\u003eFurthermore, we find fish injected with magnetic nanoparticles do not show altered indicators of hepatic health compared to controls, suggesting these nanocarriers do not impact the fish health. We found that despite the migration of excess nanoparticles to the liver, the hepatic enzyme examination showed no elevation in ALT, AST, or ALP activities in MNP-injected fish, indicating absence of liver damage despite nanoparticle accumulation. We chose to examine the liver as it has been previously shown to have an important role in nanoparticle metabolism and clearance, and MNPs are known to predominantly accumulate in the liver when they circulate systemically (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The activity of enzymes ALP, AST and ALT is known to increase during liver stress conditions and serve as standardized biomarkers to analyze liver condition in vertebrates (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e) and has been previously used to estimate the impact of metal nanoparticles in fish (48).\u003c/p\u003e \u003cp\u003eWe studied injected fish\u0026rsquo; behavior as an indicator of their general health, and in particular as a proxy of their nervous system health. We did not see any changes in the social behavior of injected fish suggesting these fish are healthy and suffered no neurological damage from the procedure or the injection of nanoparticles. We did observe slight differences in some measures of the locomotor activity of injected fish when we performed a novel tank test. However, these differences remained within the range of variation of the control group, suggesting these are not extreme changes in behavior. Also, for those measures for which we observed significant differences, the differences were observed for both the saline-injected and MNP-injected fish, suggesting that the ICV injection procedure itself may contribute to slight stress or anxiety-like responses that are most likely transient.\u003c/p\u003e \u003cp\u003eWhile our study demonstrates the safety and cellular uptake efficiency of our nanoparticle delivery system, several limitations should be addressed in future work. First, even if we have demonstrated successful gene editing \u003cem\u003ein vitro\u003c/em\u003e using this delivery system, we have not yet done so \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Future studies should include the evaluation of gene knockout efficiency, gene editing strategy comparison and evaluation of off-target modifications. Second, the observed variability in cellular uptake at early timepoints suggests there is still potential for optimization to achieve a more consistent initial delivery. Factors such as nanoparticle concentration, injection volume, and surface functionalization could be further refined to enhance reproducibility. Additionally, the mechanisms underlying the progressive increase in nuclear localization over time merit further investigation, as understanding these dynamics could inform optimal timing for experimental interventions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have developed a novel magnetic nanoparticle ideally suited for the delivery of gene editing elements across diverse applications. In this study, we adapted intracranial microinjection protocols, designed a custom 3D microinjection setup, and demonstrated that our innovative magnetic nanocarrier is safe for in vivo applications. It does not affect the health or behavior of the injected fish and reaches the brain cell nuclei as effectively as in previous in vitro tests, showing similar performance. Combined with our previous work, our findings indicate that the magnetic nanoparticles we have designed possess all the properties required for in vivo tissue-specific gene editing and can be used with our ICV protocol to perform gene editing in the guppy brain.\u003c/p\u003e \u003cp\u003eTo date, delivery methods have been the primary obstacle to implementing gene editing strategies beyond traditional laboratory model species. Developing an efficient, safe, and versatile vehicle for brain-specific in vivo gene editing in non-model organisms could revolutionize neuroscience and behavioral genetics research, as well as enhance our understanding of neurological disorders. The magnetic nanoparticles presented here can be synthesized in standard molecular biology laboratories and conjugated to different guides or plasmids with relative ease, enabling targeted gene editing by researchers in many different areas and working with diverse systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBBB: Blood-brain barrier\u003c/p\u003e\n\u003cp\u003eICV: Intracerebroventricular\u003c/p\u003e\n\u003cp\u003eMNPs: Magnetite nanoparticles\u003c/p\u003e\n\u003cp\u003eTMAH: Tetramethylammonium hydroxide\u003c/p\u003e\n\u003cp\u003eMNPs-Si: Silanized nanoparticles \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMNPs-PEG: Pegylated magnetite nanoparticles\u003c/p\u003e\n\u003cp\u003ePEG: Polyethylene glycol\u003c/p\u003e\n\u003cp\u003eMNPs-BUF-II: Buforin II conjugated magnetite nanoparticles\u003c/p\u003e\n\u003cp\u003eCICUAL: Animal Ethics Committee of Universidad de los Andes\u003c/p\u003e\n\u003cp\u003ePFA: Paraformaldehyde\u003c/p\u003e\n\u003cp\u003eTL: Telencephalon\u003c/p\u003e\n\u003cp\u003e3V: Third ventricle\u003c/p\u003e\n\u003cp\u003eOT: Optic tectum\u003c/p\u003e\n\u003cp\u003eICP-OES: Inductively coupled plasma optical emission spectroscopy\u003c/p\u003e\n\u003cp\u003eALT: Alanine aminotransferase\u003c/p\u003e\n\u003cp\u003eAST: Aspartate aminotransferase\u003c/p\u003e\n\u003cp\u003eALP: Alkaline phosphatase\u003c/p\u003e\n\u003cp\u003eCRISPRko: CRISPR-based gene knockout\u003c/p\u003e\n\u003cp\u003eCRISPRa: CRISPR-based gene activation\u003c/p\u003e\n\u003cp\u003eCRISPRi: CRISPR-based gene interference\u003c/p\u003e\n\u003cp\u003eRNP: ribonucleoprotein\u003c/p\u003e\n\u003cp\u003esgRNA: single guide RNA\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOur work followed the international guidelines for the use of animals in research and was approved by the University of Los Andes Institutional Animal Care and Laboratory Use Committee-CICUAL (Ethical approval C.FUA_23-004).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll supplementary figures, tables, and methods are provided with the manuscript. The 3D printing design for the custom microinjection setup is available in the supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Vice-presidency for Research and Creation (grant no. FAPA P19.246922.005) and the Department of Biomedical Engineering from Los Andes University, Colombia.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors' contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eM.C.M. and M.A.V. Performed most of the experiments, designed the methodology, analyzed the data, wrote and edited the manuscript. M.F.V. Performed the cellular uptake assay and contributed with data analysis. L.A. Contributed to design the ICV injection methodology and standardized all parameters. A.M. Performed hepatic health assays. N.I.B. Conceived the project and acquired funds. J.C and N.I.B. Developed the conceptual framework, supervised and validated the project, wrote, reviewed and edited the manuscript. All authors have read the manuscript and agreed to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Valentina Quezada for her mentorship and guidance on nanoparticle-related aspects of this study and Paula Guzmán for her help with experiments. We are grateful to Verónica Akle and Yeferzon Ardila for their insights and support, as well as to Simón Reader for his valuable advice during the initial stages of our injection design. Additionally, we thank Liz Riveros for her invaluable help with fish husbandry and assistance throughout the project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSella G, Barton NH. Thinking About the Evolution of Complex Traits in the Era of Genome-Wide Association Studies. 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Biomaterials. 2013 Nov;34(33):8333\u0026ndash;43. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intracranial microinjection, magnetic nanoparticles, in vivo gene editing, Poecilia reticulata, behavioral genetics","lastPublishedDoi":"10.21203/rs.3.rs-6254078/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6254078/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUncovering the genomic basis of traits has advanced rapidly in evolutionary biology and neuroscience, largely through phenotypic traits with adaptive value has been advancing rapidly in evolutionary biology, neuroscience, and behavior, largely due to research using non-traditional model systems. Direct gene editing in the adult brain represents a crucial next step in linking genotype to phenotype, avoiding the confounding effects that arise from modifications during development. However, implementing these technologies beyond traditional laboratory models remains challenging due to delivery limitations.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe developed an intracranial microinjection protocol for adult guppies (\u003cem\u003ePoecilia reticulata\u003c/em\u003e) to deliver gene-editing elements to brain cells. We designed magnetic nanoparticles functionalized with a novel translocating agent, a non-viral carrier capable of transporting linearized nucleic acids across cellular and nuclear membranes. We comprehensively assessed nanoparticle uptake, nuclear colocalization, and potential health impacts using histological analysis, liver enzyme activity assays, and behavioral assessments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur functionalized nanoparticles successfully entered brain cells and colocalized with nuclei at rates exceeding 50% after two weeks, demonstrating their potential for efficient \u003cem\u003ein vivo\u003c/em\u003e gene editing. Health assessments showed no significant brain cell death (\u0026gt;\u0026thinsp;80% viability), no liver toxicity (normal ALT, AST, and ALP enzyme levels), and no alterations in individual and social behaviors, confirming the nanoparticles\u0026rsquo; biocompatibility and systemic safety.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results, combined with previous \u003cem\u003ein vitro\u003c/em\u003e work demonstrating our functionalized magnetic nanoparticles are an effective delivery system for gene editing, show they can be used for safe \u003cem\u003ein vivo\u003c/em\u003e interventions in the adult brain of \u003cem\u003eP. reticulata\u003c/em\u003e. This protocol overcomes a major technical barrier in evolutionary biology and neuroscience, with a novel nucleic acid-carrying vehicle that can be used \u003cem\u003ein vivo\u003c/em\u003e in the adult brain. This approach provides a versatile platform for studying the genetic mechanisms underlying behavior in small freshwater fish while helping overcome the major limitations of conducting functional studies on non-model organisms.\u003c/p\u003e","manuscriptTitle":"A Novel Method for In Vivo Gene Editing in the Brain of Guppies Using Unique Nanoparticles as Delivery Vehicles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 08:05:38","doi":"10.21203/rs.3.rs-6254078/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff830845-04d8-4fd4-abf0-8ffe29ff7b66","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-18T11:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-01 08:05:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6254078","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6254078","identity":"rs-6254078","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}