Safety and biodistribution of mesenchymal stromal/stem cells and biocompatible neurotrophin-releasing polyelectrolyte nanoparticles as a preclinical study in amyotrophic lateral sclerosis (ALS) cell therapy

Safety and biodistribution of mesenchymal stromal/stem cells and biocompatible neurotrophin-releasing polyelectrolyte nanoparticles as a preclinical study in amyotrophic lateral sclerosis (ALS) cell therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Safety and biodistribution of mesenchymal stromal/stem cells and biocompatible neurotrophin-releasing polyelectrolyte nanoparticles as a preclinical study in amyotrophic lateral sclerosis (ALS) cell therapy Emilia Sinderewicz, Maria Dabkowska, Anna Sarnowska, Joanna Staszkiewicz-Chodor, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7641255/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 18 You are reading this latest preprint version Abstract Amyotrophic lateral sclerosis (ALS) is a multifactorial disease that complicates the identification of unique therapeutic targets. Stem cells and neurotrophins hold therapeutic promise due to their neuroprotective and anti-inflammatory roles. This study preclinically evaluated the safety of mesenchymal stem cells (MSCs) and neurotrophin-releasing polyelectrolyte nanoparticles (NTs) as potential adjuvant therapies in a porcine model. Four groups of castrated male pigs were used. Group I (control) received saline and pegylated NT3-BDNF nanoparticles. Group II received adipose-derived stem cells (ASCs), Group III Wharton’s jelly-derived MSCs (WJ-MSCs), each followed by NT3-BDNF nanoparticles, while Group IV underwent only spinal puncture. Treatments were administered intrathecally. Safety was assessed using MRI, hematological and biochemical parameters, and cerebrospinal fluid analysis. Cell localization was studied with iron-label staining, and tissue integrity was evaluated histologically. Biochemical tests revealed no significant blood parameter changes. C-reactive protein (CRP) levels decreased after NTs and NT–MSC combinations, indicating an anti-inflammatory effect. Biodistribution analysis showed MSC migration via cerebrospinal fluid and accumulation around the spinal cord and brain. MRI and behavioral monitoring confirmed the absence of adverse effects. These findings demonstrate that MSC therapy combined with neurotrophin-releasing nanoparticles is safe and feasible as adjunct therapy for ALS. Biological sciences/Biotechnology Health sciences/Neurology Biological sciences/Neuroscience Biological sciences/Stem cells cell therapy stem cells/mesenchymal stromal cells neurotrophin-3 brain-derived neurotrophic factor preclinical studies porcine animal model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 INTRODUCTION Amyotrophic lateral sclerosis (ALS) is driven by multiple mechanisms, including oxidative stress, mitochondrial dysfunction, RNA metabolism defects, neuroinflammation, protein aggregation, and impaired autophagy [ 1 ]. Non-neuronal cells such as astrocytes, microglia, and immune cells also contribute, either protectively or harmfully [ 2 ]. This complexity hinders the identification of unique therapeutic targets. Current drugs—riluzole and edaravone—offer only modest, short-term benefits, with no significant improvement in survival or disease course [ 3 ]. Hence, developing effective therapies remains urgent. Ongoing trials focus on genetic, immune-targeting, and stem cell approaches [ 4 ]. Mesenchymal stem/stromal cells (MSCs) show promise due to their ability to act on multiple pathways simultaneously, modulating immunity, reducing oxidative stress, and promoting regeneration. They have been explored in various neurological disorders [ 5 ]. Preclinical ALS studies have tested MSCs from sources such as bone marrow, adipose tissue, and umbilical cord, using intravenous, intrathecal, or spinal injections [ 6 ]. Rodent ALS models, especially SOD1 G93A transgenics, replicate key disease features, including motor neuron degeneration and paralysis [ 7 ]. However, interspecies differences highlight the need for more adequate large-animal models to better evaluate MSC safety, biodistribution, dosing, and efficacy. In recent years, pigs have become an important preclinical model for ALS, using both transgenic and CNS injury models [ 8 ]. Transgenic pigs with SOD1 and TDP-43 mutations show a disease course resembling human ALS [ 9 ]. SOD1G93A pigs develop motor neuron degeneration, hindlimb deficits, gliosis, and protein aggregation in an age-dependent manner [ 10 ]. Early stages feature nuclear accumulation and ubiquitinated aggregates of mutant SOD1, similar to human ALS pathology [ 11 ]. Later stages show elevated TDP-43 in blood cells and severe skeletal muscle pathology, including inflammation and necrosis [ 10 ]. Preclinical trials of ALS therapies in animal models demonstrated that stem cell transplantation is feasible and safe, regardless of delivery route, cell type, or dose. However, they showed limited or no functional improvement. Stem cell–based approaches are among the innovative strategies in ALS therapy research. Another promising direction is the use of neurotrophins, particularly brain-derived neurotrophic factor (BDNF), which supports neuronal survival, differentiation, synapse formation, and provides antioxidant protection during inflammation and hypoxia [ 12 ]. BDNF acts mainly through TrkB receptors, triggering signaling cascades crucial for cell survival. Neurotrophin-3 (NT3) further enhances BDNF activity by engaging additional Trk receptors [ 13 ]. Early ALS trials with methionyl-BDNF showed initial promise, but phase III results were disappointing due to poor protein stability and [ 14 ]. To overcome these barriers, nanotechnology-based delivery systems such as PAMAM nanoparticles, multilayer polyelectrolytes, and PEG-based carriers have been developed [ 15 – 18 ]. Given the urgent need for ALS therapies, the unique properties of MSCs, and limitations of neurotrophins alone, this study aimed to test the combined use of MSCs and neurotrophin-releasing polyelectrolyte nanoparticles in a pig model. Safety and tolerability of intrathecal administration of Wharton’s Jelly–derived MSCs (WJ-MSCs), adipose-derived stem cells (ASCs), and nanoparticles were evaluated through in vivo experiments, monitoring adverse effects, biochemical markers, cell biodistribution, and procedural outcomes in pigs—a species considered highly relevant for translation to human trials. RESULTS PHYSICOCHEMICAL CHARACTERIZATION OF PEG-ylated NT3-BDNF NANOPARTICLES Physicochemical characterization is a fundamental step in the biological evaluation of nanoparticles, as it is essential for defining or confirming their properties under specific conditions and within a given medium. These properties include object size and its distribution, aggregation and agglomeration states, surface area, surface charge, and solubility. A comprehensive assessment of these characteristics is essential for understanding the interactions of nanoparticles within biological systems and ensuring their safe and effective application. Accordingly, PEGylated NT3-BDNF nanoparticles were comprehensively analyzed using transmission electron microscopy (TEM) to determine their hydrodynamic diameter in a dry state (Figure 3B), while multiangle dynamic light scattering (MADLS) was utilized to measure their particle size in a PBS solution (Figure 3C). Regulatory agencies such as the FDA and EMA require injectable formulations to meet stringent standards for particle size and distribution. A polydispersity index (PDI) greater than 0.4 is typically unacceptable unless justified and mitigated through additional formulation strategies. The characteristic hydrodynamic diameter of the PEGylated NT3-BDNF nanoparticles obtained from MADLS studies with concentrations of each neurotrophins (ca. 13.2 mg L -1 ) was between 3.7 nm ± 1 nm, and 430 nm ± 3 nm, indicating a polydispersity index (PDI) (0.38 ± 0.02). Additionally, we determined the physicochemical parameters of the synthesized PEGylated nanoparticles and the loading of neurotrophins (NT3 and BDNF ) within 90 days of freezing using MADLS and ELISA (Figure 1C, and A). TEM and MADLS studies indicated that the diameter of the PEGylated NT3-BDNF nanoparticles was between 4 nm and 430 nm, and the polydispersity index was lower than 0.4 after 28 days of freezing. The average concentration of NT-3 and BDNF reached respectively constant values of 11 ug/ml and 13 ug/ml over 28 days of storage. Afterward, the electrophoretic mobility/zeta potential of PEGylated NT3-BDNF nanoparticles was physiochemically characterized (Figure 1D). The positively charged NT3 (5 mV ± 2.5 mV) (Dąbkowska et al. 2024) was adsorbed onto negatively charged PEG macroions (-4 mV ± 2.5 mV) (Dąbkowska et al. 2020, Dąbkowska et al. 2023) according to bulk diffusion transport. The zeta potential of the PEG-NT3 complexes was 3.6 ± 3.2 mV. As expected, the adsorption of the negatively charged PEG molecule to positively charged NT3 led to an electrokinetic charge reduction, resulting in slightly positively charged conjugates of PEG-NT3. Afterward, BDNF (-0,66 mV ± 1.91 mV) was adsorbed onto the PEG-NT3 complexes. As expected, after the adsorption of slightly negatively charged BDNF , the entire PEGylated NT3-BDNF nanoparticle surface exhibited a charge reduction reaching 1,33 mV ± 4,79 mV. Furthermore, microelectrophoretic experimental data indicated that the electrokinetic charge of the PEGylated NT3-BDNF nanoparticles became stable over 28 days. ANALYSIS OF BIOCHEMICAL PARAMETERS AND BLOOD MORPHOLOGY The analysis of selected biochemical parameters and blood counts on smears from the experimental animals was performed in an accredited veterinary laboratory on samples collected at four time points: before the first administration of cells/NaCl (1), one week after the first administration of cells/NaCl (2), one week after the second administration of cells/NaCl (3), one week after the administration of NTs (4). The results are presented in the Table 1. The analysis of the results obtained did not indicate any significant changes in the parameters analysed at the chosen time points, neither in the control group nor in the research groups. No trends were observed in the changes in the parameters related to the course of the experiment. There were few deviations from the norm, both before and during the treatments resulting from the planned experiment. As the deviations were mostly small, they do not appear to be significant from the point of view of the health of the experimental animals. In the control group higher level of leukocyte and neutrophil content were observed in two of three examined animals. However, these deviations were normalized at least after third NaCl administration. Moreover, in one animal thrombocyte level was also higher. As with the factors described above, thrombocyte levels returned to normal after the third administration of NaCl. The remaining few deviations from the norm were very close to the limit values and were therefore not considered as significant for animals health. In the WJ-MSC group a slight decrease in MCH was observed in all animals throughout the experiment, but at levels close to the lower limit of normal. In one animal, there was a slight decrease in monocytes level throughout the experiment and an increase in the level of eosinophilia were observed before the 3rd and 4th administration of the active substances. The remaining very few and small deviations from the norm did not appear to be important for animal health. In the blood morphology of the animals in the ASC group a very slight reduction in the MCH and MCV parameters was observed at all time points examined. One of the animals showed lower eosinophil levels before and after the first cell administration, but this parameter returned to normal after NTs administration. Another animal showed deviations in leukocyte, monocyte and AST levels before and after the first cell administration. These parameters were adjusted later in the further stage of experiment. In this group, one animal died before the end of the experiment, however, there was no direct connection between the death of the animal and the procedures performed. Similar to the groups described above, slight deviations from the norm in MHC and MCV levels were observed in the sham-operated group throughout the experiment. In addition, one of the animals examined had significantly increased ALT levels. However, as the measured ALT level was high both before and during the experiment, this deviation was not due to the procedures performed. C-REACTIVE PROTEIN LEVEL C-reactive protein (CRP) levels were measured in plasma (Fig.4) and cerebrospinal fluid (Fig. 5) of pigs within three examined groups: 1) control group, 2) group of pigs receiving intrathecal ASC, 3) group of pigs receiving intrathecal WJ-MSC, at four time points of the experiment: a) before the start of cells/NaCl administration, b) one week after the first administration of cells/NaCl, c) one week after the second administration of cells/NaCl, d) one week after NTs administration. CRP levels, measured before the start of the experimental procedures, did not differ significantly between the examined groups, either in plasma or CSF. One week after the first administration of cells/NaCl, an increase of CRP concentration in plasma was observed in all groups, but a statistically significant increase was measured only in the control and WJ-MSC groups (P<0.05), but not in the ASC group. After the second administration of NaCl and WJ-MSC, a decrease of CRP concentration in plasma was observed, whereas in the ASC group CRP concentration remained at the same level (P<0.05). Administration of NTs caused a significant decrease in CRP concentration in all studied groups, compared to the CRP level measured after NaCl/cells administration. In the ASC and WJ-MSC groups, the decrease was statistically significant (P<0.01). In the control group the effect was slighter (P<0.05). The observed trends of changes in CRP after NaCl/MSC/NTs administration are similar in plasma and CSF. One week after the administration of WJ-MSC, ASC or NaCl, an increased level of CRP was found in the CSF, but a statistically significant difference was found only in the control group (P<0.05). The second administration of NaCl and WJ-MSC resulted in a decrease in CSF CRP levels, whereas a further increase in CRP level was observed after the second administration of ASC. NTs administration resulted in a final decrease of CRP levels in CSF. In all groups, this effect was statistically significant (P<0.05) compared to the levels measured after the 1st and 2nd cells/ NaCl administrations. The concentration of CRP was also statistically lower after NTs administration compared to the level measured before the experiments in the CSF of animals receiving WJ-MSC. The absence of the significant differences between CRP levels measured before and after ASC and WJ-MSC administration may confirm the safety of the procedure used in the experiment. In addition, the results obtained indicate the positive effect of NTs and MSC in combination with NTs on the level of CRP in the CSF. TISSUE STAINING HE staining was used to assess the preservation of tissue and cell integrity. Analysis of the images obtained showed no deviation from the norm. Anatomical structures were preserved and the tissues contained many normal cells. Figure 6 shows examples of the structures analysed. The analysed specimens come from the brain and spinal cord of a pigs treated with WJ-MSC and neurotrophins. Staining with potassium ferrocyanide confirmed the presence of trivalent iron, which was used to label the administered cells (Fig. 7). As a result of this staining, iron ions took on a dark blue color. In order to assess the degree of MSCs deposition labeled with iron ions in the nervous system tissues during the analysis of images obtained as a result of potassium ferrocyanide staining, the signal strength was marked with numerical values (1 - 4; 1 means a weak signal in the form of few iron deposits, and 4 means a strong signal in the form of very numerous iron deposits). Analysis of histological slides from the animals' spinal cords showed the greatest presence of iron ions at the site of administration and its nearest area (end thoracic and lumbar, Fig. 7, Fig. 8B). The presence of trivalent iron was also observed in the terminal part of the spinal cord and brain, indicating the migration of labelled cells with the circulating intracellular fluid. In animals brain preparations, the highest concentrations of iron ions were found in fragments located at the periphery of the tissue on the side of the ventricles and on the side of the medulla oblongata (Fig. 7, Fig. 8A). No penetration of the administered cells through the blood-brain barrier into the tissues was observed (Fig. 7). The presence of iron ions used to label MSCs around the nervous tissue along the entire length of the CNS and the lack of penetration of the administered cells through the blood-brain barrier into the tissues. This result suggests that the presence and action of MSCs is local. Below there are sample images of the structures analysed showing staining for trivalent iron. The specimens analysed were taken from the brain and spinal cord of a pig treated with WJ-MSC and neurotrophins. Since a qualitative analysis of the obtained images was used, the results presented at Fig. 8 are intended to indicate the CNS fragments in which iron ion deposits were identified, but not to statistically assess the signal strength between the analyzed CNS sections or between experimental groups. MRI The analysis of the MRI images after cells and NTs administration was compared with the image obtained before the start of the MSC/NTs administration procedure. In animals in the control group, no changes were observed in the MRI image after administration of the placebo compared to the image obtained before surgery. In the analysis of images gained before the start of the cell/NaCl administration procedure, no artefacts were observed in the spinal canal (Fig. 9 A). Analysis of MRI images from animals that received the MSC/NTs in the operating room and were then transported to the MRI laboratory did not reveal the presence of artefacts (Fig. 9B-E). This is probably due to the fact that the cells were suspended in a larger volume of cerebrospinal fluid and their displacement in the spinal canal under the influence of fluid circulation during transport of the animal. Analysis of MRI images obtained during cell administration using a drain under MRI guidance revealed the presence of darkening in the spinal canal, characteristic of the presence of iron (Fig.10A-D). The darkening was located approximately 10 cm above the drain insertion site, which is consistent with the recorded length of the drain in the canal.In the MRI images taken one week after the last surgical procedures, no changes were observed in the spinal canal of the pigs in any of the study groups (Fig. 9 E). During the experiment, the animal caretakers did not report any deviations from the norm that could indicate a deterioration in the health of the animals. The results obtained may indicate the safety of the method used for the intrathecal administration of the prototype medicinal product. DISCUSSION Cell therapies, with their multifactorial mechanisms, offer promising options for treating many diseases, including neurological ones. Current treatments - such as riluzole and edaravone for ALS, dopaminergic drugs for PD, and immunomodulators for MS - mainly relieve symptoms, have limited efficacy, and often cause adverse effects [ 19 ]. Despite progress in neuroprotective agents and gene therapies, their long-term effectiveness remains uncertain. These limitations, especially in rare conditions like ALS, underline the need for approaches that regenerate neural tissue and support functional recovery, with stem cell therapy presenting a potential solution. Initially, MSCs were expected to differentiate into neural cells and replace damaged motor neurons. However, later studies showed their main role is regulating the microenvironment by releasing neurotrophic factors and cytokines that modulate immune responses [ 20 ]. This supports activation of endogenous cells, stimulates neurogenesis and angiogenesis, and reduces secondary cell death through anti-inflammatory effects [ 21 , 22 ]. Administration of hUCB was shown to decrease reactive gliosis and slow disease progression, while SCF-activated bone marrow cells improved motor function and survival in ALS mice by lowering pro-inflammatory cytokines and increasing IGF-1 [ 23 ]. These findings highlight the importance of modifying the CNS microenvironment and suggest that supplementing stem cell therapy with additional trophic factors may enhance its effectiveness in ALS treatment. Preclinical animal models have shown that a number of stem cell types could be viable options for ALS therapy. These include autologous BM- and adipose-derived MSCs, Wharton's jelly-derived MSCs, granulocyte colony-stimulating factor (G-CSF)-stimulated peripheral blood stem cells (PBSCs), embryonic stem cells (ESCs), NPCs derived from fetal or adult tissues, and non-neural progenitor cells (non-NPCs) [ 24 ]. However, the optimal type for human testing remains unclear [ 25 ]. Early clinical trials have assessed the safety and potential efficacy of various stem cell sources and delivery methods, from intravenous and intra-arterial to intrathecal, intraspinal, and intracerebral administration [ 24 ]. Considering ALS heterogeneity and expert recommendations [ 26 ], we chose intrathecal administration of allogenic ASC and MSC, avoiding long autologous preparation times and the need for immunosuppression. A number of clinical trials have selected MSCs as the cell source for ALS therapy. The safety of stem cell therapy in ALS was confirmed for BM-MSC, WJ-MSC, ASC or NSC [ 27 – 31 ]. Phase I trials confirmed the safety of intraspinal NSI-566RSC NSC transplants via lumbar or cervical injections, with some improvement in ALSFRS-R scores, especially at higher doses [ 32 , 27 ]. Mazzini et al. [ 29 ] also demonstrated safe intraparenchymal administration of human NSCs, showing transient slowing of ALS progression without adverse effects. Multiple intrathecal (IT) or intravenous BM-MSC administrations were safe, reduced neuroinflammatory biomarkers, but efficacy remained uncertain due to small patient numbers [ 33 – 35 ]. Phase II trials with repeated IT BM-MSC injections showed clinical improvement related to dosing intervals [ 31 ]. Repeated IT WJ-MSCs increased median survival with no serious adverse effects [ 36 ]. Intrathecal autologous ASC injections were well tolerated, with minor CSF changes and nerve root thickening; some subjective symptom improvement was reported, but ALSFRS-R scores did not improve [ 37 ]. These studies highlight the potential of stem cells in ALS therapy but show that further optimization is needed due to lower-than-expected efficacy. Intrathecal and intramuscular autologous BM-MSCs improved disease progression by increasing neurotrophic factors and reducing inflammatory CSF biomarkers in rapidly progressing patients [ 38 ]. Despite promising results, some trials lacked statistical significance. Preclinical research explored combining MSCs with neurotrophin-releasing polyelectrolyte nanoparticles in pigs to enhance efficacy. The administration route also influences therapy outcomes. Considering effectiveness and minimal invasiveness, the intrathecal route was selected for cell and neurotrophin delivery. The first indicator of the procedure’s potential safety was the assessment of animals’ general health and well-being before, during, and after the procedures. Caretakers reported no behavioral deviations suggesting health deterioration. Safety evaluation also included potential side effects and systemic health parameters, such as blood counts. Analysis showed no significant changes in these parameters at any time point in either the control or experimental groups, and no trends affecting animal health were observed. Thus, the absence of significant differences in the measured markers before and after administration of cells/NTs/NaCl supports the safety of the method. Similar results have been documented in clinical trial of intrathecal administration of WJ-MSCs in ALS patients [ 36 ]. Among 34 patients, only one adverse event manifested as a headache without any other signs and abnormalities at neurological examination. No serious adverse effects were noted also after intrathecal injections of autologous BM-MSC [ 30 , 39 ]. Only minor adverse drug reaction was noted, manifested by pyrexia, pain and headache. Similarly, no adverse effects were observed in studies evaluating influence of MSCs administration via lumbar puncture [ 34 ]. Given the link between ALS and neuroinflammation, CRP levels in plasma and cerebrospinal fluid were measured. CSF CRP increased after the first NaCl/cell administration, likely due to procedural stress. This effect disappeared after the second administration in the control and WJ-MSC groups but increased again with ASC. Similar CRP dynamics across groups suggest stress from procedures, not cell administration, caused the changes. Notably, NTs administration reduced CSF CRP in all groups, also lowering plasma CRP compared to post-NaCl/cell levels. Our findings are in accordance with phase III of the clinical trial findings also revealed significant improvements in cerebrospinal biomarkers of neuroinflammation, neurodegeneration, and neurotrophic factor support in patients who received three intrathecal MSC-NTF treatments. The treatment was well tolerated but did not reach statistical significance [ 40 ]. Moreover, it was documented, that injections of MSCs and Lin- cells, isolated from bone marrow, induce the secretion of neurotrophic factor and diminish the inflammation in ALS patients [ 38 ]. Our results, indicating the positive effect of NTs and WJ-MSC in combination with NTs on the level of neuroinflammation in the CSF, suggest that enhancing the neuroprotective effect by slow releasing NTs from biocompatible polyelectrolyte nanoparticles may improve the effectiveness of the adiuvant therapy of ALS. Successful stem cell therapy requires reliable outcome measures, but no in vivo assay universally confirms transplantation, incorporation, or neurotrophic factor secretion. Clinical trials track cell fate to ensure safety and prevent migration to undesirable regions [ 41 ]. To avoid interference with cell biology, we used iron as a contrast agent. Superparamagnetic iron oxide particles (SPIOs) are widely studied [ 42 ] due to fast labelling, long half-life, and high MRI resolution [ 43 ]. SPIOs do not affect stem cell survival, proliferation, or differentiation [ 44 ]. SPIO-labelled cells produce hypointense areas on T2/T2*-weighted MRI, indicating transplanted cell location. MRI during spinal cord administration showed hypointense areas above the drain site, confirming procedure effectiveness. SPIO-labelled cells, however, cannot be distinguished from hemorrhage or clots [ 45 ]. Absence of artefacts in animals transported to MRI suggests signals were not due to hemorrhage. Lack of hypointense signal in animals without a drain likely resulted from cell suspension dispersal in cerebrospinal fluid. Future strategies may use positive contrast agents, such as gadolinium (Gd-DTPA), fluorine (^19F), or manganese complexes, for clearer localization [ 46 ]. Comparing MRI before, during, and one week after procedures showed no spinal canal changes, consistent with previous studies, supporting the safety of intrathecal administration of the prototype medicinal product. Besides promising therapeutic outcomes, a key consideration in cell therapy is their fate and potential consequences. Human DNA was detected in peripheral organs but not in the brain after intravenous hUCB administration [ 47 ], prompting further studies on IV or CNS delivery. In SOD1G93A mice, few IV hUCB-HSCs reached the spinal cord, while most localized in the spleen, modulating immune response and increasing Th2 cytokines [ 22 ]. hUCB-MNCs injected into CSF remained in the subarachnoid space or lateral ventricles, likely secreting neurotrophic and anti-inflammatory factors [ 48 ]. In this study the biodistribution of mesenchymal stem/stromal cells, the preservation of tissue and cell integrity in CNS was assessed. Similarly to our previous studies, anatomical structures of the tissues and phenotype of the cells were preserved with no deviation from the norm. Moreover, the presence of the iron ions, used for MSCs labelling, were detected surrounding the nervous tissue of the spinal cord and the brain. The highest concentrations of iron ions were located at the periphery of the tissue on the side of the ventricles and on the side of the medulla oblongata. However, no penetration of the administered cells through the blood-brain barrier into the tissues was observed, which indicates that the presence and action of MSCs is local. These results seem to confirm the final location of cell migration and the safety of the method used. Furthermore, these findings substantiate the efficacy of cell intrathecal administration as a therapeutic modality for diseases of the nervous system. MATERIAL AND METHODS ETHICAL APPROVAL All methods were conducted in accordance with relevant guidelines and regulations. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by the Local Ethics Committee in Olsztyn (approval no. 36/2021) and were performed in compliance with the ARRIVE guidelines. Human mesenchymal stem/stromal cells isolated from Wharton’s Jelly of the umbilical cord (WJ-MSC) were obtained under approval of the Bioethics Committee (Resolution no. 27/2015) at the University of Warmia and Mazury in Olsztyn, Poland. Adipose tissue was collected during the liposuction procedure of Plastic Surgery Department at Orlowski’s Clinical Hospital in Warsaw. The study protocol was approved by the Institutional Review Board (IRB) at the Centre of Postgraduate Medical Education (No. 62/PB/2016) on September 14, 2016. All procedures involving human tissue were performed in accordance with relevant guidelines and regulations. Written informed consent was obtained from all donors (or their legal guardians) prior to sample collection.PROCEEDING OF THE IN VIVO EXPERIMENT In order to investigate the safety and tolerance of intrathecal administration of mesenchymal stem/stromal cells (MSC) and biocompatible nanoparticles made of polyelectrolytes releasing neurotrophins (NTs), an in vivo experiment on the animal model was conducted. During the experiment, the tested products were administered intrathecally, in the lumbar section of the spinal canal of pigs, and the number of adverse events, biochemical parameters as well as biodistribution of transplanted cells were analysed. All procedures were approved by Local Ethics Committee in Olsztyn (approval 36/2021) and were performed in accordance with the ARRIVE guidelines. The experiment was conducted on the 12 castrated male pigs with an average weight of approximately 30 kg. The animals were housed in the animal facility of the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn. The light cycle was 12 hours on and 12 hours off. The rooms were equipped with a continuous and emergency ventilation system with the capacity ensuring 15 to 20 air changes per hour. The noise level did not exceed 60 dB. The air temperature was maintained at 21 Celsius degrees, and the humidity was maintained at a level of 50-60%. The animals were provided with access to a clean place to lie down, drinking water, hay and straw. Following their transportation from the breeder to the animal facility, the boars were subjected to the required adaptation period before the start of the in vivo experiment. The animals were earmarked, divided into four research groups (n=3) and placed in the aforementioned pens (3 animals per pen). In the experiment human mesenchymal stem/stromal cells isolated from Wharton's jelly of the umbilical cord (WJ-MSC; Bioethics Committee Resolution No. 27/2015), human mesenchymal stem/stromal cells isolated from adipose tissue (ASC; Bioethical Committee at the Centre of Postgraduate Medical Education (No. 63/PB/2013) and neurotrophin-releasing nanoparticles (NTs) were used. The tested cells were administered inthratecally twice, at a dose of 5 million, with a seven-day interval between each administration. Seven days following the second administration of cells, the animals were administered 250 µl of PEGylated NT3–BDNF nanoparticles, containing 13.2 mg L −1 of each neurotrophins. The third group of animals, which served as the control group, was given 0.9% NaCl twice and NTs once, at seven-day intervals. The fourth group was the sham-operated group. This group underwent a spinal canal puncture only, four times at seven-day intervals. The experimental setup is shown below (Figure 1). Prior to each surgical procedure, the animals were premedicated and anaesthetised in the animal facility. Xylazine (Vetaxyl) was administered intramuscularly at a dosage of 3 mg/kg, ketamine intramuscularly at a dosage of 5-8 mg/kg, and atropine (Atropinum sulfuricum WZF 1 mg/mL) subcutaneously at dosage of 0.05 mg/kg. Subsequently, butorphanol (Torbugesic) was administered intravenously at a dosage of 0.3 mg/kg, propofol intravenously at a dosage of 2-4 mg/kg and the animals were intubated. After that, the animals were transported to the Emil Behring’s Experimental Medicine Centre of the Faculty of Medicine, University of Warmia and Mazury in Olsztyn, in a specially adapted car. Every time, the animals were under the supervision of a veterinarian, with access to life-sustaining/life-saving equipment. On day 0, a baseline, magnetic resonance imaging (MRI) of the spine of each pig was performed. The examinations were conducted using in T1 and T2 sequences, without contrast administration. During the study, the pigs were anaesthetised with sevoflurane 1-2.5% by inhalation. Thereafter, the pigs were then transported to the operating room. The animals were administered intrathecally in the lumbar spine with 0.5 mL of 0.9% NaCl (control group, 1), ASC and WJ-MSC (experimental groups, 2 and 3, respectively; 5 million cells suspended in 0.5 mL of sterile PBS), or only the injection was performed (sham operated group, 4). The procedure was conducted under intraoperative X-ray control. As during MRI, pigs were maintained under anaesthesia with sevoflurane 1-2.5% by inhalation. After surgery, the pigs underwent a repeat MRI imaging. After completion of the procedure, the pigs were transported to the animal facility under general anaesthesia and in appropriate conditions, where they were awakened under the veterinary supervision and received the necessary post-operative care. The animals were kept under veterinary supervision for 24 hours after the procedure. Biochemical tests were performed on the blood samples, including morphology, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and C-reactive protein (CRP) levels. Additionally, cerebrospinal fluid was collected. The procedure was repeated after 7 days. Postoperative MRI imaging was performed. After 14 days from the beginning of the experiment, animals in the experimental and control groups received PEGylated NT3–BDNF nanoparticles, containing 13.2 mg L −1 of each neurotrophins suspended in 0.5 mL of sterile PBS, instead of the cells/0.9% NaCl. In the sham-operated group, the injection was repeated. Blood samples for biochemical tests and cerebrospinal fluid were taken each time. MRI of the spine was performed after the procedure. The animals showed no central nervous system damage, pain symptoms or other abnormalities, after any of the treatments, which may confirm the safety of the method used. After a further 7 days (21 days from the start of the experiment), MRI of the spines was repeated. Finally, the animals were euthanised with propofol and tissues were collected for assessment of cell biodistribution. Brain, spinal cord and fragments of the liver, spleen and lung located near large blood vessels were obtained. The tissues were fixed and frozen until histochemical analyses. The corpses were transferred to the corpse repository at the Department of Pathological Anatomy of the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn and then transferred to the rendering plant. CELL-BASED PRODUCT PREPARATION WJ-MSC, produced in Laboratory for Regenerative Medicine, Department of Neurosurgery, University of Warmia and Mazury in Olsztyn and ASC obtained from the Mossakowski Medical Research Institute, Polish Academy of Sciences, were thawed and seeded into flasks containing DMEM medium (Macopharma #BC0110060) supplemented with the antibiotic penicillin-streptomycin (Sigma Aldrich #P0781) 2% v/v, Human Platelet Lysate Virally inactivated MultiPL'100i (Macopharma #BC0190032) 5 % v/v and heparin (Sigma-Aldrich #H3149-250KU) 0.1% v/v. Approximately 18 hours before administration, the cells were labelled with Molday ION Rhodamine B iron preparation (BioPAL #CL-50Q02-6A-50) for magnetic resonance imaging. The preparation was added to the bottles in the amount of 10 µL/mL of medium. On the day of administration, the cells were detached using trypsin (Sigma-Aldrich #T4049), washed and prepared for administration of 5 million cells suspended in 0.5 mL of sterile PBS for each pig. NTs PREPARATION In our earlier work, we studied the PEG layers’ self-assembled arrangement (Dąbkowska et al. 2023b) in a serum-free and complex serum environment with 0.1, 1, or 5 mg L −1 concentrations of both neurotrophins. The main objective of this research was to produce and characterize PEGylated NT3–BDNF nanoparticles with 13.2 mg L −1 of each BDNF and NT3, with a particular focus on their physicochemical behavior over time in 0.15 M phosphate-buffered saline (PBS) at pH 7.4, and 37°C. To assess the stability of the obtaining nanoparticles over 28 days, we determined a range of physicochemical properties using transmission electron microscopy (TEM) and multiangle dynamic and electrophoretic light scattering (MADLS/ELS). Reagents 1. BDNF and NT3 Unfiltered stock solutions (typically 250 mg/L) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248-BDB-250/CF; R&D Systems), were prepared by dissolving lyophilized of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at -20 °C. In the text, BDNF and NT3 are collectively referred to as neurotrophins (NTs). Before each measurement, the stock solution was diluted to the desired bulk concentration, 10 mg L -1, in Ringer’s solution (pH 7.0 ± 0.6, 0.16 M) (Fresenius Kabi, Polska). The exact concentrations of these solutions were determined by the commercially available enzyme-linked immunosorbent assay (ELISA) (described in 2.3.4). The temperature of all materials remained constant at 298 ± 0.1 K. Carrier-free recombinant human BDNF (rhBDNF) and carrier-free recombinant human NT3 (rhNT3), collectively referred to as neurotrophins (NTs), were prepared as unfiltered stock solutions with a typical concentration of 250 mg/L. The lyophilized NTs of known concentrations were dissolved in phosphate-buffered saline (PBS), with a pH of 7.4 ± 0.2, 0.15 M (Biomed, Lublin, Poland) and stored at -20°C for a maximum of three months. Before each measurement, the stock solution was diluted to a bulk concentration of 13.2 mg/L in PBS solution. The exact concentrations of the solutions were determined using a commercially available enzyme-linked immunosorbent (ELISA) assay (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of the experiments was kept at a constant value equal to 298 ± 0.1 K. 2. PEG Poly(ethylene glycol) (PEG), a 4 kDa molar mass (1546569, GMP grade, Sigma Aldrich), was used without further purification to simultaneously encapsulate NT3-BDNF nanoparticles. The working solution of PEG was prepared by aseptically dissolving 2 g of PEG-4000 in 10 mL of Ringer's solution. The mixture was then gently rotated in a tube for 15 minutes at room temperature. Subsequently, the solution was filtered through a 0.22 µm filter (Millipore), creating 200 mg mL -1 PEG-4000 working stock that was further used in the preparation of PEGylated NT3-BDNF nanoparticles. Preparation of PEGylated NT3-BDNF nanoparticles PEGylated BDNF-NT3 nanoparticles were prepared at the Pomeranian Medical University in Szczecin. BDNF and NT3 were adsorbed onto the PEG surface through electrostatic interactions. Initially, the hydrodynamic diameter and electrophoretic mobility of PEG molecules in the working solution were determined. Subsequently, NT3 was adsorbed by combining 20 000 mg L -1 PEG in the working solution (as described in 2.1.2) with a 250 mg L -1 NT3 suspension (prepared as described in 2.1.1) at a ratio of 17:1. The mixture was incubated at room temperature for 900 seconds, resulting in a 13.2 mg L -1 final concentration of the NT3 protein in the PEG-NT3 suspension. The hydrodynamic diameter and electrophoretic mobility of the PEG-NT3 nanoparticles were measured, and the corresponding zeta potential was calculated. Then, 250 mg L -1 BDNF (prepared as described in Section 2.1.1) was added to the PEG-NT3 suspension at a ratio of 17:1 (PEG-NT3: BDNF). The mixture was incubated at room temperature for another 900 seconds, resulting in a final concentration of 13.2 mg L -1 BDNF protein in the PEG-NT3-BDNF mixture. After 1 500 seconds of incubation, spontaneous self-assembly of PEG-NT3-BDNF complexes occurred, referred to as “PEGylated NTs-based nanoparticles” or “PEGylated NT3-BDNF". NTs were mixed with PEG in aqueous solution without further sonication or extensive agitation. PEG chains were conjugated to nanoparticle surfaces via amide or carboxyl bonds, depending on the type of core charge of the amino acids in the protein complex NT3-BDNF and the formation of PEG amino groups and protein amino surface groups. After each component was added, samples were taken to assess the protein concentration using an ELISA (described in Section 2.4). The nanoparticles in PBS's solution were stored at -20 °C for up to two months without any detectable protein loss. The preparation of nanoparticles for in vivo experiments was carried out within a controlled laminar flow hood to ensure sterility of the entire system. Physicochemical characterization of PEGylated NTs nanoparticles 1. Multiangle dynamic light scattering (MADLS) A multifaceted approach was used to determine the concentration of PEGylated BDNF-NT3 nanoparticles. The particle size distribution was measured, the time-averaged intensity was scattered by a molecular scatterer, and the sample was subjected to multiangle dynamic light scattering using a Malvern ZetaSizer Ultra instrument (Malvern Instruments, Malvern, UK) and ZS XPLORER 3.2.0 software. MADLS and DLS (dynamic light scattering) are well-established techniques for determining the hydrodynamic size distribution of molecules or NPs dispersed in solution. The MADLS technique is amenable to studying nanomaterials' dispersion/aggregation states. The particle size distributions were obtained from the measured diffusion coefficients. The diffusion coefficient of the NPs was determined via DLS using a Zetasizer Nano ZS Malvern instrument at a final concentration of BDNF and NT3 of 13.2 mg L -1 , as described previously ( Dąbkowska et al. 2018, Dąbkowska et al. 2021). The data analysis was performed in automatic mode at 25 °C. The measured size is presented as the average value of 20 runs, with triplicate measurements within each run, described in detail elsewhere (Dąbkowska et al. 2021, Wasilewska et al. 2009). 2. Electrophoretic light scattering (ELS) The zeta potential and polydispersity index of the PEGylated NT3-BDNF nanoparticles were determined by laser Doppler velocimetry (LDV) at 25 °C with a Malvern ZetaSizer Ultra Particle Analyzer through diffusion coefficient (D) and electrophoretic mobility (μe) measurements. The LDV method introduced by Adamczyk et al. is based on measuring ζ-potential/microphoretic mobility changes during the adsorption of tested proteins/particles on a model colloid particle (Adamczyk et al. 2011). The electrophoretic mobility was recalculated to the ζ-potential using the Henry equation, which is valid for higher ionic strengths in which the polarization of the electric double layer is relevant (the double-layer thickness decreases than the protein dimension). 3. Transmission electron microscopy (TEM) A JEOL JSM-7500F electron microscope working in transmission mode (TEM) was used to evaluate the morphology and size distribution of the PEGylated NT3-BDNF nanoparticle suspension (in 0.15 M PBS solution). Stock suspensions of nanoparticles (NPs) containing 13.2 mg/L BDNF and NT3 proteins were dispersed on a copper grid covered with carbon film to prepare the samples for microscopic imaging. After the PBS solution had evaporated, dark and bright field images of the NPs were taken. The micrographs were analyzed using MultiScan 6.08 software (a computer scanning system). All the images represent direct detection from the sample surfaces, with no coating or contrast applied. The size of the PEGylated NT3-BDNF nanoparticles was determined using ImageJ software by gathering the number and coordinates of a minimum of 300 nanoparticles. The counting of PEGylated NT3-BDNF nanoparticle dimensions involved a manual process that involved comparing the initial image and a modified version obtained through digital image filters. Specifically, the alteration of the picture background was instrumental in this method. By applying these filters, we assessed the accuracy of the particle analysis using the software mentioned above. 4. Enzyme-linked immunosorbent assay (ELISA) The concentration of the neurotrophins was measured with immunoenzymatic test ELISA (cat no. DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY248, DY267, R&D Systems, Minneapolis, MN, USA). The test involves the use of immobilized biotinylated antibodies specific to fragments of the investigated protein. After the studied material was applied to the antibody-coated surface, streptavidin-conjugated horseradish peroxidase was added, and the reaction substrate was incubated with the samples at λ = 540 nm and λ = 450 nm. The absorbance was read with a Varioskan LUX Plate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of each protein was determined in relation to an appropriate, freshly prepared standard curve (part of the kit). ANALYSIS OF BLOOD COUNTS AND BIOCHEMICAL PARAMETERS The analysis of selected biochemical parameters and blood counts on smears from the experimental animals was performed in the accredited veterinary laboratory on samples taken at four time points: before the first administration of cells/NaCl (1), one week after the first administration of cells/NaCl (2), one week after the second administration of cells/NaCl (3), one week after the administration of NTs/NaCl (4). Levels of liver enzymes (AST, ALT) and blood morphological parameters (MCV, MCH, MCHC, hematocrit, hemoglobin content, number per volume unit and the percentage of erythrocytes, platelets, leukocytes, monocytes, neutrophils, basophils, eosinophils) were analysed. ANALYSIS OF CRP LEVEL IN PLASMA AND CELEBROSPINAL FLUID C-reactive protein (CRP) levels were measured in the plasma and cerebrospinal fluid (CSF) of pigs in three research groups: 1) after intrathecal administration of 0.9% NaCl (control group), 2) after intrathecal administration of 5 million ASC, 3) after intrathecal administration of 5 million WJ-MSC. The CRP levels were measured: (1) before the start of the procedure, (2) one week after the first administration of cells/NaCl, (3) one week after the second administration of cells/NaCl, (4) one week after the administration of NTs. A Pig CRP ELISA kit was used (Abcam, #ab205089) was used to determine CRP levels. ANALYSIS OF MSC BIODISTRIBUTION IN THE NERVOUS TISSUE To assess the biodistribution of mesenchymal stem/stromal cells in the tissues of the experimental animals, the collected tissues were subjected to histochemical analyses. The tissues, fixed in 4% buffered paraformaldehyde and permeated with 30% sucrose, were frozen and cut into 10 μm sections using a cryostat. The brain was divided into 4 sections, according to the diagram below (Fig. 2): In anticipation of cell migration from the CSF, the preparations were sectioned to obtain samples from the surfaces closest to the areas where CSF circulates. Section I was cut from the side of the cerebral ventricle, section II from the lateral side, section III from the side of the medulla oblongata and section IV from the top. The spinal cord from the lower thoracic sections (at T11) to the cauda equina was dissected together with the nerve roots and divided into sections according to the course of the spinal nerves. The sections were embedded in freezing medium and sectioned in the transverse plane in a caudal direction. The sections obtained were subjected to topographic staining with haematoxylin and eosin (HE) and trivalent iron staining with 2% potassium ferrocyanide. The preparations were analysed using an Olympus BX51 microscope. MRI All animals participating in the study underwent magnetic resonance imaging. Imaging was performed before the start of the surgical procedures, each time after the administration of the cell-based product, NTs or placebo, and one week after the last procedure (before the animals were sacrificed). MRI imaging was performed using Siemens Magnetom Prisma Fit 3T. The scan protocol for spine analysis included the following sequences: T2-TSE-COR, T2-TSE-SAG, T2-FL2D-SAG-HEMO, T2-TSE-TRA, T2-SPC-SAG-ISO-1.0MM. CONCLUSION This study evaluated the safety of MSCs combined with neurotrophin-releasing polyelectrolyte nanoparticles as a preclinical ALS therapy. The procedure caused no side effects, and neurotrophins reduced inflammatory markers in cerebrospinal fluid and plasma, suggesting improved efficacy by modulating the CNS microenvironment. Intrathecal administration was safe, and biodistribution analysis showed cell movement within the CNS, around the spinal cord and brain, without crossing the blood-brain barrier. These findings indicate that cell therapy with neurotrophins is a promising alternative to traditional or genetically modified approaches, using readily available autologous or allogeneic cells. Further clinical trials are needed to assess efficacy in ALS patients. Declarations Acknowledgements Not applicable. Author contributions KJW, AS, and BM conceived the study concept and design; MD, JSCh, DM, ID, MCh, and MS conducted the experiments; PH and ID managed the pig herd, performed animal procedures, and assessed disease symptoms; KJW, MM, AS, and EP analyzed the data; and ES and KJW wrote the manuscript. All authors read, edited, and approved the final version of the manuscript. Funding This research was funded by the Medical Research Agency, Poland (No: 2020/ABM/01/00014-00). The clinical procedures and histopathological analyses were conducted at the Regenerative Medicine Laboratory of the Faculty of Medicine, Medical College of the University of Warmia and Mazury in Olsztyn. Data availability Data is provided within the manuscript. Conflicts of Interest The authors declare no competing interests. Consent for publication Not applicable. References Sabatelli, M., Conte, A. & Zollino, M. Clinical and genetic heterogeneity of amyotrophic lateral sclerosis. Clin. Genet. 83 , 408–416 (2013). Deda, H. et al. Treatment of amyotrophic lateral sclerosis patients by autologous bone marrow-derived hematopoietic stem cell transplantation: a 1-year follow-up. Cytotherapy 11 , 18–25 (2009). Witzel, S. et al. German Motor Neuron Disease Network (MND-NET). 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1","display":"","copyAsset":false,"role":"figure","size":312893,"visible":true,"origin":"","legend":"\u003cp\u003eThe scheme of in vivo experiment. Group I: 0.9% NaCl administered \u0026nbsp;inthratecally (i.t.) on day 0 and 7, NTs administered i.t. on day 14, n=3; Group II: ASC administrated i.t. on day 0 and 7, NTs administrated i.t. on day 14, n=3; Group III: WJ-MSC administered i.t. on day 0 and 7, NTs administered i.t.on day 14, n=3; Group IV: sham operated; n=3. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/1c1e93474d7076246127e730.png"},{"id":93338273,"identity":"ca08bc73-8b9f-447e-bb68-20fe0265f696","added_by":"auto","created_at":"2025-10-12 14:20:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14530,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of brain division for histochemical analysis. The icons have been sourced from the Microsoft Office stock images, which have been used under licence, and have been modified by the author.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/3b466b2feadb6fc0f41c096a.png"},{"id":93339917,"identity":"3e02c032-7c64-47da-827c-d27b59e4fac8","added_by":"auto","created_at":"2025-10-12 14:28:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":295614,"visible":true,"origin":"","legend":"\u003cp\u003ePhysicochemical characterization of PEGylated NT3-BDNF nanoparticles over 28 days (40 320 min) after formulation. The particle size distribution was measured by intensity (C), the zeta potential was measured against PBS buffer (D), and the loading of BDNF and NT3 was measured via ELISA (A). All syntheses were performed six times, and the error bars represent the mean ± standard deviation (SD). The individual points indicate the distribution of NP diameters from 8 samples. TEM images of nanoparticles deposited from 0.15 M at pH 7.4: 24 h after formulation (B) corresponding to a 50,000x magnification. The scale bar is 100 nm. Note that the dispersed type of PEGylated NT3-BDNF nanoparticles was maintained for 28 days.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/2a5eb686ada8e89fc84fbaca.png"},{"id":93338270,"identity":"3051d3a5-ba79-4ac9-a60c-ac292fb48d67","added_by":"auto","created_at":"2025-10-12 14:20:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76479,"visible":true,"origin":"","legend":"\u003cp\u003eCRP concentration in plasma of the pigs receiving NaCl (control group, black bars), pigs receiving ASC (white bars), pigs receiving WJ-MSC (grey bars) in a four timepoints of the experiment: a) before starting the cell/NaCl administration (0), b) one week after the first administration of cells/NaCl (I), c) one week after the second administration of cells/NaCl (II), d) one week after NTs administration (III). The statistical analysis was determined by a two-way ANOVA followed by Tukey’s multiple comparison test (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e). Asterisks indicate differences in the CRP concentration between examining timepoints (*p\u003cem\u003e \u0026lt; \u003c/em\u003e0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/8c1c55ac322418fbc30b01a0.png"},{"id":93338279,"identity":"7c84d7d4-6cc0-43cd-89de-2dfa56b210c3","added_by":"auto","created_at":"2025-10-12 14:20:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":72189,"visible":true,"origin":"","legend":"\u003cp\u003eCRP concentration in CSF of the pigs receiving NaCl (control group, black bars), pigs receiving ASC (white bars), pigs receiving WJ-MSC (grey bars) in a four timepoints of the experiment: a) before starting the cells/NaCl administration (0), b) one week after the first administration of cells/NaCl (I), c) one week after the second administration of cells/NaCl (II), d) one week after NTs administration (III). The statistical analysis was determined by a two-way ANOVA followed by Tukey’s multiple comparison test (p\u0026lt;0.05). Asterisks indicate differences in the CRP concentration between examining timepoints (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/8b2fe8fc908b005a9f896a05.png"},{"id":93338274,"identity":"fe949685-58c0-4775-9c8f-1894296cb788","added_by":"auto","created_at":"2025-10-12 14:20:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":781834,"visible":true,"origin":"","legend":"\u003cp\u003eThe topographic staining with haematoxylin and eosin of brain and spinal cord of a pigs treated with WJ-MSC and neurotrophins. B I – B IV denote the following brain fragments: section I - cut from the side of the cerebral ventricle, section II from the lateral side, section III from the side of the medulla oblongata and section IV from the top. The exaplems of spinal cord (SC) tissue originate from the thoracic section (T11). 4x, 10x, 20x, 40x - symbols indicating the magnification factor.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/fedb203cc715873d595fde55.png"},{"id":93339919,"identity":"475d6b17-2437-4a66-b3cf-734e499802cf","added_by":"auto","created_at":"2025-10-12 14:28:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":607807,"visible":true,"origin":"","legend":"\u003cp\u003eThe staining with potassium ferrocyanide of brain and spinal cord of a pigs treated with WJ-MSC and neurotrophins. B I – B IV denote the following brain fragments: section I - cut from the side of the cerebral ventricle, section II from the lateral side, section III from the side of the medulla oblongata and section IV from the top. The examples of spinal cord (SC) tissue originate from the thoracic section (T11). 4x, 10x, 20x, 40x - symbols indicating the magnification factor. Iron deposition is indicated by blue arrows.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/c3136aef143832108adaeb38.png"},{"id":93338293,"identity":"b62630fd-0e0a-4d14-b246-729a225fee21","added_by":"auto","created_at":"2025-10-12 14:20:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":45428,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 8A. Degree of MSCs deposition labeled with iron ions in the brain of pigs receiving NaCl (black bars), ASC (white bars) or WJ-MSC (grey bars). B I – B IV denote the following brain fragments: I – sections cut from the side of the cerebral ventricle, II – sections from the lateral side, III – sections from the side of the medulla oblongata and IV – sections cut from the top.\u003c/p\u003e\n\u003cp\u003eFig. 8B. Degree of MSCs deposition labeled with iron ions in the brain of pigs receiving NaCl (black bars), ASC (white bars) or WJ-MSC (grey bars). T11-14 denote the fragments of the spinal cord located at the level of the subsequent thoracic vertebrae; L1-L5 fragments of the spinal cord located at the height of subsequent lumbar vertebrae; S1-S4 fragments of the spinal cord located at the height of subsequent sacral vertebrae.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/238206eb37b93b3b15318fd6.png"},{"id":93338285,"identity":"3316fee0-7a55-4fb6-ab39-2607e4118133","added_by":"auto","created_at":"2025-10-12 14:20:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":386929,"visible":true,"origin":"","legend":"\u003cp\u003eMRI imaging of cells into the cerebrospinal fluid before (A), and after first (B), second (C) MCS and NTs (D) administration, and one week after the last surgical procedure (E), taken in the saggital plane.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/560665eae0948c47418e3cd8.png"},{"id":93342683,"identity":"e37c8407-f998-4210-a2c7-cc81c83bb630","added_by":"auto","created_at":"2025-10-12 14:44:24","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":618543,"visible":true,"origin":"","legend":"\u003cp\u003eMRI imaging after intrathecal infusion of WJ-MSC cells into the cerebrospinal fluid: images show artifacts in the cerebrospinal fluid (marked with a red frame and/or arrow); The analysed images were presented in the T2 image in three planes: A – sagittal plane; B – coronal plane; C – sagittal plane with \u003cdel\u003e_\u003c/del\u003ehemo expansion, allowing for the visualization of iron; D – transverse plane.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/61f0da74995c2bfa1977429c.png"},{"id":103765549,"identity":"3c023d63-849b-4486-a24c-8e79ca455839","added_by":"auto","created_at":"2026-03-02 16:04:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4118920,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/923a03a7-20fb-43eb-980b-e7826b717d71.pdf"},{"id":93338268,"identity":"f5a86313-9839-4d42-a81c-23cb3ce9c0aa","added_by":"auto","created_at":"2025-10-12 14:20:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":58409,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7641255/v1/d310a54f05bd348c44351418.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Safety and biodistribution of mesenchymal stromal/stem cells and biocompatible neurotrophin-releasing polyelectrolyte nanoparticles as a preclinical study in amyotrophic lateral sclerosis (ALS) cell therapy","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is driven by multiple mechanisms, including oxidative stress, mitochondrial dysfunction, RNA metabolism defects, neuroinflammation, protein aggregation, and impaired autophagy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Non-neuronal cells such as astrocytes, microglia, and immune cells also contribute, either protectively or harmfully [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This complexity hinders the identification of unique therapeutic targets. Current drugs\u0026mdash;riluzole and edaravone\u0026mdash;offer only modest, short-term benefits, with no significant improvement in survival or disease course [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hence, developing effective therapies remains urgent. Ongoing trials focus on genetic, immune-targeting, and stem cell approaches [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMesenchymal stem/stromal cells (MSCs) show promise due to their ability to act on multiple pathways simultaneously, modulating immunity, reducing oxidative stress, and promoting regeneration. They have been explored in various neurological disorders [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Preclinical ALS studies have tested MSCs from sources such as bone marrow, adipose tissue, and umbilical cord, using intravenous, intrathecal, or spinal injections [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRodent ALS models, especially SOD1 G93A transgenics, replicate key disease features, including motor neuron degeneration and paralysis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, interspecies differences highlight the need for more adequate large-animal models to better evaluate MSC safety, biodistribution, dosing, and efficacy.\u003c/p\u003e\u003cp\u003eIn recent years, pigs have become an important preclinical model for ALS, using both transgenic and CNS injury models [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Transgenic pigs with SOD1 and TDP-43 mutations show a disease course resembling human ALS [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. SOD1G93A pigs develop motor neuron degeneration, hindlimb deficits, gliosis, and protein aggregation in an age-dependent manner [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Early stages feature nuclear accumulation and ubiquitinated aggregates of mutant SOD1, similar to human ALS pathology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Later stages show elevated TDP-43 in blood cells and severe skeletal muscle pathology, including inflammation and necrosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePreclinical trials of ALS therapies in animal models demonstrated that stem cell transplantation is feasible and safe, regardless of delivery route, cell type, or dose. However, they showed limited or no functional improvement.\u003c/p\u003e\u003cp\u003eStem cell\u0026ndash;based approaches are among the innovative strategies in ALS therapy research. Another promising direction is the use of neurotrophins, particularly brain-derived neurotrophic factor (BDNF), which supports neuronal survival, differentiation, synapse formation, and provides antioxidant protection during inflammation and hypoxia [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. BDNF acts mainly through TrkB receptors, triggering signaling cascades crucial for cell survival. Neurotrophin-3 (NT3) further enhances BDNF activity by engaging additional Trk receptors [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Early ALS trials with methionyl-BDNF showed initial promise, but phase III results were disappointing due to poor protein stability and [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To overcome these barriers, nanotechnology-based delivery systems such as PAMAM nanoparticles, multilayer polyelectrolytes, and PEG-based carriers have been developed [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the urgent need for ALS therapies, the unique properties of MSCs, and limitations of neurotrophins alone, this study aimed to test the combined use of MSCs and neurotrophin-releasing polyelectrolyte nanoparticles in a pig model. Safety and tolerability of intrathecal administration of Wharton\u0026rsquo;s Jelly\u0026ndash;derived MSCs (WJ-MSCs), adipose-derived stem cells (ASCs), and nanoparticles were evaluated through in vivo experiments, monitoring adverse effects, biochemical markers, cell biodistribution, and procedural outcomes in pigs\u0026mdash;a species considered highly relevant for translation to human trials.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003ePHYSICOCHEMICAL CHARACTERIZATION OF PEG-ylated NT3-BDNF NANOPARTICLES\u003c/p\u003e\n\u003cp\u003ePhysicochemical characterization is a fundamental step in the biological evaluation of nanoparticles, as it is essential for defining or confirming their properties under specific conditions and within a given medium. \u0026nbsp;These properties include object size and its distribution, aggregation and agglomeration states, surface area, surface charge, and solubility. A comprehensive assessment of these characteristics is essential for understanding the interactions of nanoparticles within biological systems and ensuring their safe and effective application. \u0026nbsp;Accordingly, PEGylated NT3-BDNF nanoparticles were comprehensively analyzed using transmission electron microscopy (TEM) to determine their hydrodynamic diameter in a dry state (Figure 3B), while multiangle dynamic light scattering (MADLS) was utilized to measure their particle size in a PBS solution (Figure 3C).\u003c/p\u003e\n\u003cp\u003eRegulatory agencies such as the FDA and EMA require injectable formulations to meet stringent standards for particle size and distribution. A polydispersity index (PDI) greater than 0.4 is typically unacceptable unless justified and mitigated through additional formulation strategies. The characteristic hydrodynamic diameter of the PEGylated NT3-BDNF nanoparticles obtained from MADLS studies with concentrations of each \u0026nbsp; neurotrophins (ca. 13.2 mg L\u003csup\u003e-1\u003c/sup\u003e) was between 3.7 nm \u0026plusmn; 1 nm, and 430 nm \u0026plusmn; 3 nm, indicating a polydispersity index (PDI) (0.38 \u0026plusmn; 0.02).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, we determined the physicochemical parameters of the synthesized PEGylated nanoparticles and the loading of \u0026nbsp;neurotrophins (NT3 and BDNF ) within 90 days of freezing using MADLS and ELISA (Figure 1C, and A). TEM and MADLS studies indicated that the diameter of the PEGylated NT3-BDNF nanoparticles was between 4 nm and 430 nm, and the polydispersity index was lower than 0.4 after 28 days of freezing. The average concentration of NT-3 and BDNF reached respectively constant values of 11 ug/ml and 13 ug/ml over 28 days of storage. Afterward, the electrophoretic mobility/zeta potential of PEGylated NT3-BDNF nanoparticles was physiochemically characterized (Figure 1D). The positively charged NT3 (5 mV \u0026plusmn; 2.5 mV) (Dąbkowska et al. 2024) was adsorbed onto negatively charged PEG macroions (-4 mV \u0026plusmn; 2.5 mV) (Dąbkowska et al. 2020, Dąbkowska et al. 2023) according to bulk diffusion transport. The zeta potential of the PEG-NT3 complexes was 3.6 \u0026plusmn; 3.2 mV. As expected, the adsorption of the negatively charged PEG molecule to positively charged NT3 led to an electrokinetic charge reduction, resulting in slightly positively charged conjugates of PEG-NT3. Afterward, BDNF (-0,66 mV \u0026plusmn; 1.91 mV) was adsorbed onto the PEG-NT3 complexes. As expected, after the adsorption of slightly negatively charged BDNF , the entire PEGylated NT3-BDNF nanoparticle \u0026nbsp;surface exhibited a charge reduction reaching 1,33 mV \u0026plusmn; 4,79 mV. Furthermore, microelectrophoretic experimental data indicated that the electrokinetic charge of the PEGylated NT3-BDNF nanoparticles became stable over 28 days.\u003c/p\u003e\n\u003cp\u003eANALYSIS OF BIOCHEMICAL PARAMETERS AND BLOOD MORPHOLOGY\u003c/p\u003e\n\u003cp\u003eThe analysis of selected biochemical parameters and blood counts on smears from the experimental animals was performed in an accredited veterinary laboratory on samples collected at four time points: before the first administration of cells/NaCl (1), one week after the first administration of cells/NaCl (2), one week after the second administration of cells/NaCl (3), one week after the administration of NTs (4). The results are presented in the Table 1.\u003c/p\u003e\n\u003cp\u003eThe analysis of the results obtained did not indicate any significant changes in the parameters analysed at the chosen time points, neither in the control group nor in the research groups. No trends were observed in the changes in the parameters related to the course of the experiment. There were few deviations from the norm, both before and during the treatments resulting from the planned experiment. As the deviations were mostly small, they do not appear to be significant from the point of view of the health of the experimental animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the control group higher level of leukocyte and neutrophil content were observed in two of three examined animals. However, these deviations were normalized at least after third NaCl administration. Moreover, in one animal thrombocyte level was also higher. As with the factors described above, thrombocyte levels returned to normal after the third administration of NaCl. The remaining few deviations from the norm were very close to the limit values and were therefore not considered as significant for animals health.\u003c/p\u003e\n\u003cp\u003eIn the WJ-MSC group a slight decrease in MCH was observed in all animals throughout the experiment, but at levels close to the lower limit of normal. In one animal, there was a slight decrease in monocytes level throughout the experiment and an increase in the level of eosinophilia were observed before the 3rd and 4th administration of the active substances. The remaining very few and small deviations from the norm did not appear to be important for animal health.\u003c/p\u003e\n\u003cp\u003eIn the blood morphology of the animals in the ASC group a very slight reduction in the MCH and MCV parameters was observed at all time points examined. One of the animals showed lower eosinophil levels before and after the first cell administration, but this parameter returned to normal after NTs administration. Another animal showed deviations in leukocyte, monocyte and AST levels before and after the first cell administration. These parameters were adjusted later in the further stage of experiment. In this group, one animal died before the end of the experiment, however, there was no direct connection between the death of the animal and the procedures performed.\u003cins cite=\"mailto:Edyta%20Paczkowska\" datetime=\"2025-02-07T11:41\"\u003e\u0026nbsp;\u003c/ins\u003e\u003c/p\u003e\n\u003cp\u003eSimilar to the groups described above, slight deviations from the norm in MHC and MCV levels were observed in the sham-operated group throughout the experiment. In addition, one of the animals examined had significantly increased ALT levels. However, as the measured ALT level was high both before and during the experiment, this deviation was not due to the procedures performed.\u003c/p\u003e\n\u003cp\u003eC-REACTIVE PROTEIN LEVEL\u003c/p\u003e\n\u003cp\u003eC-reactive protein (CRP) levels were measured in plasma (Fig.4) and cerebrospinal fluid (Fig. 5) of pigs within three examined groups: 1) control group, 2) group of pigs receiving intrathecal ASC, 3) group of pigs receiving intrathecal WJ-MSC, at four time points of the experiment: a) before the start of cells/NaCl administration, b) one week after the first administration of cells/NaCl, c) one week after the second administration of cells/NaCl, d) one week after NTs administration.\u003c/p\u003e\n\u003cp\u003eCRP levels, measured before the start of the experimental procedures, did not differ significantly between the examined groups, either in plasma or CSF. One week after the first administration of cells/NaCl, an increase of CRP concentration in plasma was observed in all groups, but a statistically significant increase was measured only in the control and \u0026nbsp;WJ-MSC groups (P\u0026lt;0.05), but not in the ASC group. After the second administration of NaCl and WJ-MSC, a decrease of CRP concentration in plasma was observed, whereas in the ASC group CRP concentration remained at the same level (P\u0026lt;0.05). Administration of NTs caused a significant decrease in CRP concentration in all studied groups, compared to the CRP level measured after NaCl/cells administration. In the ASC and WJ-MSC groups, the decrease was statistically significant (P\u0026lt;0.01). In the control group the effect was slighter (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eThe observed trends of changes in CRP after NaCl/MSC/NTs administration are similar in plasma and CSF. One week after the administration of \u003cins cite=\"mailto:Emilia%20Sinderewicz\" datetime=\"2025-06-18T11:27\"\u003e\u0026nbsp;\u003c/ins\u003eWJ-MSC, ASC or NaCl, an increased level of CRP was found in the CSF, but a statistically significant difference was found only in the control group (P\u0026lt;0.05). The second administration of NaCl and WJ-MSC resulted in a decrease in CSF CRP levels, whereas a further increase in CRP level was observed after the second administration of ASC. NTs administration resulted in a final decrease of CRP levels in CSF. In all groups, this effect was statistically significant (P\u0026lt;0.05) compared to the levels measured after the 1st and 2nd cells/ NaCl administrations. The concentration of CRP was also statistically lower after NTs administration compared to the level measured before the experiments in the CSF of animals receiving WJ-MSC. The absence of the significant differences between CRP levels measured before and after ASC and WJ-MSC administration may confirm the safety of the procedure used in the experiment. In addition, the results obtained indicate the positive effect of NTs and MSC in combination with NTs on the level of CRP in the CSF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTISSUE STAINING\u003c/p\u003e\n\u003cp\u003eHE staining was used to assess the preservation of tissue and cell integrity. Analysis of the images obtained showed no deviation from the norm. Anatomical structures were preserved and the tissues contained many normal cells. Figure 6 shows examples of the structures analysed. The analysed specimens come from the brain and spinal cord of a pigs treated with WJ-MSC and neurotrophins.\u003c/p\u003e\n\u003cp\u003eStaining with potassium ferrocyanide confirmed the presence of trivalent iron, which was used to label the administered cells (Fig. 7). \u0026nbsp;As a result of this staining, iron ions took on a dark blue color. In order to assess the degree of MSCs deposition labeled with iron ions in the nervous system tissues during the analysis of images obtained as a result of potassium ferrocyanide staining, the signal strength was marked with numerical values (1 - 4; 1 means a weak signal in the form of few iron deposits, and 4 means a strong signal in the form of very numerous iron deposits). Analysis of histological slides from the animals\u0026apos; spinal cords showed the greatest presence of iron ions at the site of administration and its nearest area (end thoracic and lumbar, Fig. 7, Fig. 8B). The presence of trivalent iron was also observed in the terminal part of the spinal cord and brain, indicating the migration of labelled cells with the circulating intracellular fluid. In animals brain preparations, the highest concentrations of iron ions were found in fragments located at the periphery of the tissue on the side of the ventricles and on the side of the medulla oblongata (Fig. 7, Fig. 8A). No penetration of the administered cells through the blood-brain barrier into the tissues was observed (Fig. 7). The presence of iron ions used to label MSCs around the nervous tissue along the entire length of the CNS and the lack of penetration of the administered cells through the blood-brain barrier into the tissues. This result suggests that the presence and action of MSCs is local. Below there are sample images of the structures analysed showing staining for trivalent iron. The specimens analysed were taken from the brain and spinal cord of a pig treated with WJ-MSC and neurotrophins. Since a qualitative analysis of the obtained images was used, the results presented at Fig. 8 are intended to indicate the CNS fragments in which iron ion deposits were identified, but not to statistically assess the signal strength between the analyzed CNS sections or between experimental groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMRI\u003c/p\u003e\n\u003cp\u003eThe analysis of the MRI images after cells and NTs administration was compared with the image obtained before the start of the MSC/NTs administration procedure. In animals in the control group, no changes were observed in the MRI image after administration of the placebo compared to the image obtained before surgery. In the analysis of images gained before the start of the cell/NaCl administration procedure, no artefacts were observed in the spinal canal (Fig. 9 A). Analysis of MRI images from animals that received the MSC/NTs in the operating room and were then transported to the MRI laboratory did not reveal the presence of artefacts (Fig. 9B-E). This is probably due to the fact that the cells were suspended in a larger volume of cerebrospinal fluid and their displacement in the spinal canal under the influence of fluid circulation during transport of the animal. Analysis of MRI images obtained during cell administration using a drain under MRI guidance revealed the presence of darkening in the spinal canal, characteristic of the presence of iron (Fig.10A-D). The darkening was located approximately 10 cm above the drain insertion site, which is consistent with the recorded length of the drain in the canal.In the MRI images taken one week after the last surgical procedures, no changes were observed in the spinal canal of the pigs in any of the study groups (Fig. 9 E). During the experiment, the animal caretakers did not report any deviations from the norm that could indicate a deterioration in the health of the animals. The results obtained may indicate the safety of the method used for the intrathecal administration of the prototype medicinal product.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCell therapies, with their multifactorial mechanisms, offer promising options for treating many diseases, including neurological ones. Current treatments - such as riluzole and edaravone for ALS, dopaminergic drugs for PD, and immunomodulators for MS - mainly relieve symptoms, have limited efficacy, and often cause adverse effects [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Despite progress in neuroprotective agents and gene therapies, their long-term effectiveness remains uncertain. These limitations, especially in rare conditions like ALS, underline the need for approaches that regenerate neural tissue and support functional recovery, with stem cell therapy presenting a potential solution.\u003c/p\u003e\u003cp\u003eInitially, MSCs were expected to differentiate into neural cells and replace damaged motor neurons. However, later studies showed their main role is regulating the microenvironment by releasing neurotrophic factors and cytokines that modulate immune responses [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This supports activation of endogenous cells, stimulates neurogenesis and angiogenesis, and reduces secondary cell death through anti-inflammatory effects [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Administration of hUCB was shown to decrease reactive gliosis and slow disease progression, while SCF-activated bone marrow cells improved motor function and survival in ALS mice by lowering pro-inflammatory cytokines and increasing IGF-1 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings highlight the importance of modifying the CNS microenvironment and suggest that supplementing stem cell therapy with additional trophic factors may enhance its effectiveness in ALS treatment.\u003c/p\u003e\u003cp\u003ePreclinical animal models have shown that a number of stem cell types could be viable options for ALS therapy. These include autologous BM- and adipose-derived MSCs, Wharton's jelly-derived MSCs, granulocyte colony-stimulating factor (G-CSF)-stimulated peripheral blood stem cells (PBSCs), embryonic stem cells (ESCs), NPCs derived from fetal or adult tissues, and non-neural progenitor cells (non-NPCs) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the optimal type for human testing remains unclear [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Early clinical trials have assessed the safety and potential efficacy of various stem cell sources and delivery methods, from intravenous and intra-arterial to intrathecal, intraspinal, and intracerebral administration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Considering ALS heterogeneity and expert recommendations [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], we chose intrathecal administration of allogenic ASC and MSC, avoiding long autologous preparation times and the need for immunosuppression.\u003c/p\u003e\u003cp\u003eA number of clinical trials have selected MSCs as the cell source for ALS therapy. The safety of stem cell therapy in ALS was confirmed for BM-MSC, WJ-MSC, ASC or NSC [\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Phase I trials confirmed the safety of intraspinal NSI-566RSC NSC transplants via lumbar or cervical injections, with some improvement in ALSFRS-R scores, especially at higher doses [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Mazzini et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] also demonstrated safe intraparenchymal administration of human NSCs, showing transient slowing of ALS progression without adverse effects. Multiple intrathecal (IT) or intravenous BM-MSC administrations were safe, reduced neuroinflammatory biomarkers, but efficacy remained uncertain due to small patient numbers [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Phase II trials with repeated IT BM-MSC injections showed clinical improvement related to dosing intervals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Repeated IT WJ-MSCs increased median survival with no serious adverse effects [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Intrathecal autologous ASC injections were well tolerated, with minor CSF changes and nerve root thickening; some subjective symptom improvement was reported, but ALSFRS-R scores did not improve [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese studies highlight the potential of stem cells in ALS therapy but show that further optimization is needed due to lower-than-expected efficacy. Intrathecal and intramuscular autologous BM-MSCs improved disease progression by increasing neurotrophic factors and reducing inflammatory CSF biomarkers in rapidly progressing patients [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Despite promising results, some trials lacked statistical significance. Preclinical research explored combining MSCs with neurotrophin-releasing polyelectrolyte nanoparticles in pigs to enhance efficacy. The administration route also influences therapy outcomes. Considering effectiveness and minimal invasiveness, the intrathecal route was selected for cell and neurotrophin delivery.\u003c/p\u003e\u003cp\u003eThe first indicator of the procedure\u0026rsquo;s potential safety was the assessment of animals\u0026rsquo; general health and well-being before, during, and after the procedures. Caretakers reported no behavioral deviations suggesting health deterioration. Safety evaluation also included potential side effects and systemic health parameters, such as blood counts. Analysis showed no significant changes in these parameters at any time point in either the control or experimental groups, and no trends affecting animal health were observed. Thus, the absence of significant differences in the measured markers before and after administration of cells/NTs/NaCl supports the safety of the method. Similar results have been documented in clinical trial of intrathecal administration of WJ-MSCs in ALS patients [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Among 34 patients, only one adverse event manifested as a headache without any other signs and abnormalities at neurological examination. No serious adverse effects were noted also after intrathecal injections of autologous BM-MSC [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Only minor adverse drug reaction was noted, manifested by pyrexia, pain and headache. Similarly, no adverse effects were observed in studies evaluating influence of MSCs administration via lumbar puncture [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the link between ALS and neuroinflammation, CRP levels in plasma and cerebrospinal fluid were measured. CSF CRP increased after the first NaCl/cell administration, likely due to procedural stress. This effect disappeared after the second administration in the control and WJ-MSC groups but increased again with ASC. Similar CRP dynamics across groups suggest stress from procedures, not cell administration, caused the changes. Notably, NTs administration reduced CSF CRP in all groups, also lowering plasma CRP compared to post-NaCl/cell levels. Our findings are in accordance with phase III of the clinical trial findings also revealed significant improvements in cerebrospinal biomarkers of neuroinflammation, neurodegeneration, and neurotrophic factor support in patients who received three intrathecal MSC-NTF treatments. The treatment was well tolerated but did not reach statistical significance [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, it was documented, that injections of MSCs and Lin- cells, isolated from bone marrow, induce the secretion of neurotrophic factor and diminish the inflammation in ALS patients [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our results, indicating the positive effect of NTs and WJ-MSC in combination with NTs on the level of neuroinflammation in the CSF, suggest that enhancing the neuroprotective effect by slow releasing NTs from biocompatible polyelectrolyte nanoparticles may improve the effectiveness of the adiuvant therapy of ALS.\u003c/p\u003e\u003cp\u003eSuccessful stem cell therapy requires reliable outcome measures, but no in vivo assay universally confirms transplantation, incorporation, or neurotrophic factor secretion. Clinical trials track cell fate to ensure safety and prevent migration to undesirable regions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To avoid interference with cell biology, we used iron as a contrast agent. Superparamagnetic iron oxide particles (SPIOs) are widely studied [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] due to fast labelling, long half-life, and high MRI resolution [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. SPIOs do not affect stem cell survival, proliferation, or differentiation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. SPIO-labelled cells produce hypointense areas on T2/T2*-weighted MRI, indicating transplanted cell location.\u003c/p\u003e\u003cp\u003eMRI during spinal cord administration showed hypointense areas above the drain site, confirming procedure effectiveness. SPIO-labelled cells, however, cannot be distinguished from hemorrhage or clots [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Absence of artefacts in animals transported to MRI suggests signals were not due to hemorrhage. Lack of hypointense signal in animals without a drain likely resulted from cell suspension dispersal in cerebrospinal fluid. Future strategies may use positive contrast agents, such as gadolinium (Gd-DTPA), fluorine (^19F), or manganese complexes, for clearer localization [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eComparing MRI before, during, and one week after procedures showed no spinal canal changes, consistent with previous studies, supporting the safety of intrathecal administration of the prototype medicinal product.\u003c/p\u003e\u003cp\u003eBesides promising therapeutic outcomes, a key consideration in cell therapy is their fate and potential consequences. Human DNA was detected in peripheral organs but not in the brain after intravenous hUCB administration [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], prompting further studies on IV or CNS delivery. In SOD1G93A mice, few IV hUCB-HSCs reached the spinal cord, while most localized in the spleen, modulating immune response and increasing Th2 cytokines [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. hUCB-MNCs injected into CSF remained in the subarachnoid space or lateral ventricles, likely secreting neurotrophic and anti-inflammatory factors [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study the biodistribution of mesenchymal stem/stromal cells, the preservation of tissue and cell integrity in CNS was assessed. Similarly to our previous studies, anatomical structures of the tissues and phenotype of the cells were preserved with no deviation from the norm. Moreover, the presence of the iron ions, used for MSCs labelling, were detected surrounding the nervous tissue of the spinal cord and the brain. The highest concentrations of iron ions were located at the periphery of the tissue on the side of the ventricles and on the side of the medulla oblongata. However, no penetration of the administered cells through the blood-brain barrier into the tissues was observed, which indicates that the presence and action of MSCs is local. These results seem to confirm the final location of cell migration and the safety of the method used. Furthermore, these findings substantiate the efficacy of cell intrathecal administration as a therapeutic modality for diseases of the nervous system.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eETHICAL APPROVAL\u003c/p\u003e\n\u003cp\u003eAll methods were conducted in accordance with relevant guidelines and regulations. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by the Local Ethics Committee in Olsztyn (approval no. 36/2021) and were performed in compliance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003eHuman mesenchymal stem/stromal cells isolated from Wharton\u0026rsquo;s Jelly of the umbilical cord (WJ-MSC) were obtained under approval of the Bioethics Committee (Resolution no. 27/2015) at the University of Warmia and Mazury in Olsztyn, Poland.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdipose tissue was collected during the liposuction procedure of Plastic Surgery Department at Orlowski\u0026rsquo;s Clinical Hospital in Warsaw. The study protocol was approved by the Institutional Review Board (IRB) at the Centre of Postgraduate Medical Education (No. 62/PB/2016) on September 14, 2016. All procedures involving human tissue were performed in accordance with relevant guidelines and regulations. Written informed consent was obtained from all donors (or their legal guardians) prior to sample collection.PROCEEDING OF THE IN VIVO EXPERIMENT\u003c/p\u003e\n\u003cp\u003eIn order to investigate the safety and tolerance of intrathecal administration of mesenchymal stem/stromal cells (MSC) and biocompatible nanoparticles made of polyelectrolytes releasing neurotrophins (NTs), an \u003cem\u003ein vivo\u003c/em\u003e experiment on the animal model was conducted. During the experiment, the tested products were administered intrathecally, in the lumbar section of the spinal canal of pigs, and the number of adverse events, biochemical parameters as well as biodistribution of transplanted cells were analysed. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by Local Ethics Committee in Olsztyn (approval 36/2021) and were performed in accordance with the ARRIVE guidelines. The experiment was conducted on the 12 castrated male pigs with an average weight of approximately 30 kg. The animals were housed in the animal facility of the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn. The light cycle was 12 hours on and 12 hours off. The rooms were equipped with a continuous and emergency ventilation system with the capacity ensuring 15 to 20 air changes per hour. The noise level did not exceed 60 dB. The air temperature was maintained at 21 Celsius degrees, and the humidity was maintained at a level of 50-60%. The animals were provided with access to a clean place to lie down, drinking water, hay and straw. Following their transportation from the breeder to the animal facility, the boars were subjected to the required adaptation period before the start of the \u003cem\u003ein vivo\u003c/em\u003e experiment. The animals were earmarked, divided into four research groups (n=3) and placed in the aforementioned pens (3 animals per pen).\u003c/p\u003e\n\u003cp\u003eIn the experiment human\u0026nbsp;mesenchymal stem/stromal cells isolated from Wharton\u0026apos;s jelly of the umbilical cord (WJ-MSC; Bioethics Committee Resolution No. 27/2015), human mesenchymal stem/stromal cells isolated from adipose tissue (ASC; Bioethical Committee at the Centre of Postgraduate Medical Education (No. 63/PB/2013) and neurotrophin-releasing nanoparticles (NTs) were used. The tested cells were administered inthratecally twice, at a dose of 5 million, with a seven-day interval between each administration. Seven days following the second administration of cells, the animals were administered 250 \u0026micro;l of PEGylated NT3\u0026ndash;BDNF nanoparticles, containing 13.2 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e of each neurotrophins. The third group of animals, which served as the control group, was given 0.9% NaCl twice and NTs once, at seven-day intervals. The fourth group was the sham-operated group. This group underwent a spinal canal puncture only, four times at seven-day intervals.\u003c/p\u003e\n\u003cp\u003eThe experimental setup is shown below \u0026nbsp;(Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrior to each surgical procedure, the animals were premedicated and anaesthetised in the animal facility. Xylazine (Vetaxyl) was administered intramuscularly at a dosage of 3 mg/kg, ketamine intramuscularly at a dosage of 5-8 mg/kg, and atropine (Atropinum sulfuricum WZF 1 mg/mL) subcutaneously at dosage of 0.05 mg/kg. Subsequently, butorphanol (Torbugesic) was administered \u0026nbsp;intravenously at a dosage of 0.3 mg/kg, propofol intravenously at a dosage of 2-4 mg/kg and the animals were intubated. After that, the animals were transported to the Emil Behring\u0026rsquo;s Experimental Medicine Centre of the Faculty of Medicine, University of Warmia and Mazury in Olsztyn, in a specially adapted car. Every time, the animals were under the supervision of a veterinarian, with access to life-sustaining/life-saving equipment.\u003c/p\u003e\n\u003cp\u003eOn day 0, a baseline,\u0026nbsp;magnetic resonance imaging (MRI) of the spine of each pig was performed. The examinations were conducted using in T1 and T2 sequences, without contrast administration. During the study, the pigs were anaesthetised with sevoflurane 1-2.5% by inhalation. Thereafter, the pigs were then transported to the operating room. The animals were administered intrathecally in the lumbar spine with 0.5 mL of 0.9% NaCl (control group, 1), ASC and WJ-MSC (experimental groups, 2 and 3, respectively; 5 million cells suspended in 0.5 mL of sterile PBS), or only the injection was performed (sham operated group, 4).\u0026nbsp;The procedure was conducted under intraoperative\u0026nbsp;X-ray control. As during MRI, pigs were maintained under anaesthesia with sevoflurane 1-2.5% by inhalation. After surgery, the pigs underwent a repeat MRI imaging. After completion of the procedure, the pigs were transported to the animal facility under general anaesthesia and in appropriate conditions, where they were awakened under the veterinary supervision and received the necessary post-operative care. The animals were kept under veterinary supervision for 24 hours after the procedure. Biochemical tests were performed on the blood samples, including morphology,\u0026nbsp;alanine aminotransferase (ALT), aspartate aminotransferase (AST) and C-reactive protein (CRP)\u0026nbsp;levels. Additionally, cerebrospinal fluid was collected.\u003c/p\u003e\n\u003cp\u003eThe procedure was repeated after 7 days. Postoperative MRI imaging was performed. After 14 days from the beginning of the experiment, animals in the experimental and control groups received PEGylated NT3\u0026ndash;BDNF nanoparticles, containing 13.2 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e of each neurotrophins suspended in 0.5 mL of sterile PBS, instead of the cells/0.9% NaCl. In the sham-operated group, the injection was repeated. \u0026nbsp;Blood samples for biochemical tests \u0026nbsp;and cerebrospinal fluid were taken each time. MRI of the spine was performed after the procedure. The animals showed no central nervous system damage, pain symptoms or other abnormalities, after any of the treatments, which may confirm the safety of the method used. After a further 7 days (21 days from the start of the experiment), MRI of the spines was repeated. Finally, the animals were euthanised with propofol and tissues were collected for assessment of cell biodistribution. Brain, spinal cord and fragments of the liver, spleen and lung located near large blood vessels were obtained. The tissues were fixed and frozen until histochemical analyses. The corpses were transferred to the corpse repository at the Department of Pathological Anatomy of the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn and then transferred to the rendering plant.\u003c/p\u003e\n\u003cp\u003eCELL-BASED\u0026nbsp;PRODUCT PREPARATION\u003c/p\u003e\n\u003cp\u003eWJ-MSC, produced in Laboratory for Regenerative Medicine, Department of Neurosurgery, University of Warmia and Mazury in Olsztyn and ASC obtained from the \u0026nbsp;Mossakowski Medical Research Institute, Polish Academy of Sciences, were thawed and seeded into flasks containing DMEM medium (Macopharma #BC0110060) supplemented with the antibiotic penicillin-streptomycin (Sigma Aldrich #P0781) 2% v/v, Human Platelet Lysate Virally inactivated MultiPL\u0026apos;100i (Macopharma #BC0190032) 5 % v/v and heparin (Sigma-Aldrich #H3149-250KU) 0.1% v/v. Approximately 18 hours before administration, the cells were labelled with Molday ION Rhodamine B iron preparation (BioPAL #CL-50Q02-6A-50) for magnetic resonance imaging. The preparation was added to the bottles in the amount of 10 \u0026micro;L/mL of medium. On the day of administration, the cells were detached using trypsin (Sigma-Aldrich #T4049), washed and prepared for administration of 5 million cells suspended in 0.5 mL of sterile PBS for each pig.\u003c/p\u003e\n\u003cp\u003eNTs PREPARATION\u003c/p\u003e\n\u003cp\u003eIn our earlier work, we studied the PEG layers\u0026rsquo; self-assembled arrangement (Dąbkowska et al. 2023b) in a serum-free and complex serum environment with 0.1, 1, or 5 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e concentrations of both neurotrophins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main objective of this research was to produce and characterize PEGylated NT3\u0026ndash;BDNF nanoparticles \u0026nbsp; with 13.2 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e of each BDNF and NT3, with a particular focus on their physicochemical behavior over time in 0.15 M phosphate-buffered saline (PBS) at pH 7.4, and 37\u0026deg;C. To \u0026nbsp;assess the stability of the obtaining nanoparticles over 28 days, we determined \u0026nbsp;a range of physicochemical properties using transmission electron microscopy (TEM) and multiangle dynamic and electrophoretic light scattering (MADLS/ELS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. \u0026nbsp;BDNF and NT3\u003c/p\u003e\n\u003cp\u003eUnfiltered stock solutions (typically 250 mg/L) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R\u0026amp;D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248-BDB-250/CF; R\u0026amp;D Systems), were prepared by dissolving lyophilized of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 \u0026plusmn; 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at -20 \u0026deg;C. In the text, BDNF and NT3 are collectively referred to as neurotrophins (NTs). Before each measurement, the stock solution was diluted to the desired bulk concentration, 10 mg L\u003csup\u003e-1,\u0026nbsp;\u003c/sup\u003ein Ringer\u0026rsquo;s solution (pH 7.0 \u0026plusmn; 0.6, 0.16 M) (Fresenius Kabi, Polska). The exact concentrations of these solutions were determined by the commercially available enzyme-linked immunosorbent assay (ELISA) (described in 2.3.4). The temperature of all materials remained constant at 298 \u0026plusmn; 0.1 K.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCarrier-free recombinant human BDNF (rhBDNF) and carrier-free recombinant human NT3 (rhNT3), collectively referred to as neurotrophins (NTs), were prepared as unfiltered stock solutions with a typical concentration of 250 mg/L. The lyophilized NTs of known concentrations were dissolved in phosphate-buffered saline (PBS), with a pH of 7.4 \u0026plusmn; 0.2, 0.15 M (Biomed, Lublin, Poland) and stored at -20\u0026deg;C for a maximum of three months. Before each measurement, the stock solution was diluted to a bulk concentration of 13.2 mg/L in PBS solution. The exact concentrations of the solutions were determined using a commercially available enzyme-linked immunosorbent (ELISA) assay (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R\u0026amp;D Systems). The temperature of the experiments was kept at a constant value equal to 298 \u0026plusmn; 0.1 K.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. \u0026nbsp; \u0026nbsp;PEG\u003c/p\u003e\n\u003cp\u003ePoly(ethylene glycol) (PEG), a 4 kDa molar mass (1546569, GMP grade, Sigma Aldrich), was used without further purification to simultaneously encapsulate NT3-BDNF nanoparticles. The working solution of PEG was prepared by aseptically dissolving 2 g of PEG-4000 in 10 mL of Ringer\u0026apos;s solution. The mixture was then gently rotated in a tube for 15 minutes at room temperature. Subsequently, the solution was filtered through a 0.22 \u0026micro;m filter (Millipore), creating 200 mg mL\u003csup\u003e-1\u003c/sup\u003e PEG-4000 working stock that was further used in the preparation of PEGylated NT3-BDNF nanoparticles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of PEGylated NT3-BDNF nanoparticles\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePEGylated BDNF-NT3 nanoparticles were prepared at the Pomeranian Medical University in Szczecin. BDNF and NT3 were adsorbed onto the PEG surface through electrostatic interactions. Initially, the hydrodynamic diameter and electrophoretic mobility of PEG molecules in the working solution were determined. Subsequently, NT3 was adsorbed by combining 20 000 mg L\u003csup\u003e-1\u003c/sup\u003e PEG in the working solution (as described in 2.1.2) with a 250 mg\u003cbr\u003eL\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eNT3 suspension (prepared as described in 2.1.1) at a ratio of 17:1. The mixture was incubated at room temperature for 900 seconds, resulting in a 13.2 mg L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003efinal concentration of the NT3 protein in the PEG-NT3 suspension. The hydrodynamic diameter and electrophoretic mobility of the PEG-NT3 nanoparticles were measured, and the corresponding zeta potential was calculated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThen, 250 mg L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBDNF (prepared as described in Section 2.1.1) was added to the PEG-NT3 suspension at a ratio of 17:1 (PEG-NT3: BDNF). The mixture was incubated at room temperature for another 900 seconds, resulting in a final concentration of 13.2 mg L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBDNF protein in the PEG-NT3-BDNF mixture. After 1 500 seconds of incubation, spontaneous self-assembly of PEG-NT3-BDNF complexes occurred, referred to as \u0026ldquo;PEGylated NTs-based nanoparticles\u0026rdquo; or \u0026ldquo;PEGylated NT3-BDNF\u0026quot;. NTs were mixed with PEG in aqueous solution without further sonication or extensive agitation. PEG chains were conjugated to nanoparticle surfaces via amide or carboxyl bonds, depending on the type of core charge of the amino acids in the protein complex NT3-BDNF and the formation of PEG amino groups and protein amino surface groups. After each component was added, samples were taken to assess the protein concentration using an ELISA (described in Section 2.4). The nanoparticles in PBS\u0026apos;s solution were stored at -20 \u0026deg;C for up to two months without any detectable protein loss.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe preparation of nanoparticles for \u003cem\u003ein vivo\u003c/em\u003e experiments was carried out within a controlled laminar flow hood to ensure sterility of the entire system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical characterization of PEGylated NTs nanoparticles\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. \u0026nbsp; \u0026nbsp;Multiangle dynamic light scattering (MADLS)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA multifaceted approach was used to determine the concentration of PEGylated BDNF-NT3 nanoparticles. The particle size distribution was measured, the time-averaged intensity was scattered by a molecular scatterer, and the sample was subjected to multiangle dynamic light scattering using a Malvern ZetaSizer Ultra instrument (Malvern Instruments, Malvern, UK) and ZS XPLORER 3.2.0 software. MADLS and DLS (dynamic light scattering) are well-established techniques for determining the hydrodynamic size distribution of molecules or NPs dispersed in solution. The MADLS technique is amenable to studying nanomaterials\u0026apos; dispersion/aggregation states. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe particle size distributions were obtained from the measured diffusion coefficients. The diffusion coefficient of the NPs was determined via DLS using a Zetasizer Nano ZS Malvern instrument at a final concentration of BDNF and NT3 of 13.2 mg L\u003csup\u003e-1\u003c/sup\u003e, as described previously ( Dąbkowska et al. 2018, Dąbkowska et al. 2021). The data analysis was performed in automatic mode at 25 \u0026deg;C. The measured size is presented as the average value of 20 runs, with triplicate measurements within each run, described in detail elsewhere (Dąbkowska et al. 2021, Wasilewska et al. 2009). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. \u0026nbsp; \u0026nbsp;Electrophoretic light scattering (ELS)\u003c/p\u003e\n\u003cp\u003eThe zeta potential and polydispersity index of the PEGylated NT3-BDNF nanoparticles were determined by laser Doppler velocimetry (LDV) at 25 \u0026deg;C with a Malvern ZetaSizer Ultra Particle Analyzer through diffusion coefficient (D) and electrophoretic mobility (\u0026mu;e) measurements. The LDV method introduced by Adamczyk et al. is based on measuring \u0026zeta;-potential/microphoretic mobility changes during the adsorption of tested proteins/particles on a model colloid particle (Adamczyk et al. 2011). The electrophoretic mobility was recalculated to the \u0026zeta;-potential using the Henry equation, which is valid for higher ionic strengths in which the polarization of the electric double layer is relevant (the double-layer thickness decreases than the protein dimension).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. \u0026nbsp; \u0026nbsp;\u003c/strong\u003eTransmission electron microscopy (TEM)\u003c/p\u003e\n\u003cp\u003eA JEOL JSM-7500F electron microscope working in transmission mode (TEM) was used to evaluate the morphology and size distribution of the PEGylated NT3-BDNF nanoparticle suspension (in 0.15 M PBS solution). Stock suspensions of nanoparticles (NPs) containing 13.2 mg/L BDNF and NT3 proteins were dispersed on a copper grid covered with carbon film to prepare the samples for microscopic imaging. After the PBS solution had evaporated, dark and bright field images of the NPs were taken. The micrographs were analyzed using MultiScan 6.08 software (a computer scanning system). All the images represent direct detection from the sample surfaces, with no coating or contrast applied. The size of the PEGylated NT3-BDNF nanoparticles was determined using ImageJ software by gathering the number and coordinates of a minimum of 300 nanoparticles. The counting of PEGylated NT3-BDNF nanoparticle dimensions involved a manual process that involved comparing the initial image and a modified version obtained through digital image filters. Specifically, the alteration of the picture background was instrumental in this method. By applying these filters, we assessed the accuracy of the particle analysis using the software mentioned above. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4. \u0026nbsp;Enzyme-linked immunosorbent assay (ELISA)\u003c/p\u003e\n\u003cp\u003eThe concentration of the neurotrophins was measured with immunoenzymatic test ELISA (cat no. DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY248, DY267, R\u0026amp;D Systems, Minneapolis, MN, USA). The test involves the use of immobilized biotinylated antibodies specific to fragments of the investigated protein. After the studied material was applied to the antibody-coated surface, streptavidin-conjugated horseradish peroxidase was added, and the reaction substrate was incubated with the samples at \u0026lambda; = 540 nm and \u0026lambda; = 450 nm. The absorbance was read with a Varioskan LUX Plate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of each protein was determined in relation to an appropriate, freshly prepared standard curve (part of the kit).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eANALYSIS OF BLOOD COUNTS AND \u0026nbsp;BIOCHEMICAL PARAMETERS\u003c/p\u003e\n\u003cp\u003eThe analysis of selected biochemical parameters and blood counts on smears from the experimental animals was performed in the accredited veterinary laboratory on samples taken at four time points: before the first administration of cells/NaCl (1), one week after the first administration of cells/NaCl (2), one week after the second administration of cells/NaCl (3), one week after the administration of NTs/NaCl (4). Levels of liver enzymes (AST, ALT) and blood morphological parameters (MCV, MCH, MCHC, hematocrit, hemoglobin content, number per volume unit and the percentage of erythrocytes, platelets, leukocytes, monocytes, neutrophils, basophils, eosinophils) were analysed.\u003c/p\u003e\n\u003cp\u003eANALYSIS OF CRP LEVEL IN PLASMA AND CELEBROSPINAL FLUID\u003c/p\u003e\n\u003cp\u003eC-reactive protein (CRP) levels were measured in the plasma and cerebrospinal fluid (CSF) of pigs in three research groups: 1) after intrathecal administration of 0.9% NaCl (control group), 2) after intrathecal administration of 5 million ASC, 3) after intrathecal administration of 5 million WJ-MSC. The CRP levels were measured: (1) before the start of the procedure, (2) one week after the first administration of cells/NaCl, (3) one week after the second administration of cells/NaCl, (4) one week after the administration of NTs. A Pig CRP ELISA kit was used (Abcam, #ab205089) was used to determine CRP levels.\u003c/p\u003e\n\u003cp\u003eANALYSIS OF MSC BIODISTRIBUTION IN THE NERVOUS TISSUE\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the biodistribution of mesenchymal stem/stromal cells in the tissues of the experimental animals, the collected tissues were subjected to histochemical analyses. The tissues, fixed in 4% buffered paraformaldehyde and permeated with 30% sucrose, were frozen and cut into 10 \u0026mu;m sections using a cryostat. The brain was divided into 4 sections, according to the diagram below (Fig. 2):\u003c/p\u003e\n\u003cp\u003eIn anticipation of cell migration from the CSF, the preparations were sectioned to obtain samples from the surfaces closest to the areas where CSF circulates. Section I was cut from the side of the cerebral ventricle, section II from the lateral side, section III from the side of the medulla oblongata and section IV from the top. The spinal cord from the lower thoracic sections (at T11) to the cauda equina was dissected together with the nerve roots and divided into sections according to the course of the spinal nerves. The sections were embedded in freezing medium and sectioned in the transverse plane in a caudal direction.\u003c/p\u003e\n\u003cp\u003eThe \u0026nbsp;sections obtained were subjected to topographic staining with haematoxylin and eosin (HE) and trivalent iron staining with 2% potassium ferrocyanide. The preparations were analysed using an Olympus BX51 microscope.\u003c/p\u003e\n\u003cp\u003eMRI\u003c/p\u003e\n\u003cp\u003eAll animals participating in the study underwent magnetic resonance imaging. Imaging was performed before the start of the surgical procedures, each time after the administration of the cell-based product, NTs or placebo, and one week after the last procedure (before the animals were sacrificed). MRI imaging was performed using Siemens Magnetom Prisma Fit 3T. The scan protocol for spine analysis included the following sequences: T2-TSE-COR, T2-TSE-SAG, T2-FL2D-SAG-HEMO, T2-TSE-TRA, T2-SPC-SAG-ISO-1.0MM.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study evaluated the safety of MSCs combined with neurotrophin-releasing polyelectrolyte nanoparticles as a preclinical ALS therapy. The procedure caused no side effects, and neurotrophins reduced inflammatory markers in cerebrospinal fluid and plasma, suggesting improved efficacy by modulating the CNS microenvironment. Intrathecal administration was safe, and biodistribution analysis showed cell movement within the CNS, around the spinal cord and brain, without crossing the blood-brain barrier. These findings indicate that cell therapy with neurotrophins is a promising alternative to traditional or genetically modified approaches, using readily available autologous or allogeneic cells. Further clinical trials are needed to assess efficacy in ALS patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKJW, AS, and BM conceived the study concept and design; MD, JSCh, DM, ID, MCh, and MS conducted the experiments; PH and ID managed the pig herd, performed animal procedures, and assessed disease symptoms; KJW, MM, AS, and EP analyzed the data; and ES and KJW wrote the manuscript. All authors read, edited, and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Medical Research Agency, Poland (No: 2020/ABM/01/00014-00).\u003c/p\u003e\n\u003cp\u003eThe clinical procedures and histopathological analyses were conducted at the Regenerative Medicine Laboratory of the Faculty of Medicine, Medical College of the University of Warmia and Mazury in Olsztyn.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSabatelli, M., Conte, A. \u0026amp; Zollino, M. 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In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e, 2290\u0026ndash;2293 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHauger, O. et al. MR evaluation of the glomerular homing of magnetically labeled mesenchymal stem cells in a rat model of nephropathy. \u003cem\u003eRadiology\u003c/em\u003e \u003cb\u003e238\u003c/b\u003e, 200\u0026ndash;210 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHauger, O. et al. MR evaluation of the glomerular homing of magnetically labeled mesenchymal stem cells in a rat model of nephropathy. \u003cem\u003eRadiology\u003c/em\u003e \u003cb\u003e238\u003c/b\u003e, 200\u0026ndash;210 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarkides, H. et al. Ex vivo MRI cell tracking of autologous mesenchymal stromal cells in an ovine osteochondral defect model. \u003cem\u003eStem Cell. Res. Ther.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 25 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, T. et al. Mesoporous silica-coated hollow manganese oxide nanoparticles as positive T1 contrast agents for labeling and MRI tracking of adipose-derived mesenchymal stem cells. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cb\u003e133\u003c/b\u003e, 2955\u0026ndash;2961 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEnde, N., Weinstein, F., Chen, R. \u0026amp; Ende, M. Human umbilical cord blood effect on SOD mice (amyotrophic lateral sclerosis). \u003cem\u003eLife Sci.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e, 53\u0026ndash;59 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHabisch, H. J. et al. Intrathecal application of neuroectodermally converted stem cells into a mouse model of ALS: limited intraparenchymal migration and survival narrows therapeutic effects. \u003cem\u003eJ. Neural Transm (Vienna)\u003c/em\u003e. \u003cb\u003e114\u003c/b\u003e, 1395\u0026ndash;1406 (2007).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cell therapy, stem cells/mesenchymal stromal cells, neurotrophin-3, brain-derived neurotrophic factor, preclinical studies, porcine animal model","lastPublishedDoi":"10.21203/rs.3.rs-7641255/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7641255/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a multifactorial disease that complicates the identification of unique therapeutic targets. Stem cells and neurotrophins hold therapeutic promise due to their neuroprotective and anti-inflammatory roles. This study preclinically evaluated the safety of mesenchymal stem cells (MSCs) and neurotrophin-releasing polyelectrolyte nanoparticles (NTs) as potential adjuvant therapies in a porcine model. Four groups of castrated male pigs were used. Group I (control) received saline and pegylated NT3-BDNF nanoparticles. Group II received adipose-derived stem cells (ASCs), Group III Wharton’s jelly-derived MSCs (WJ-MSCs), each followed by NT3-BDNF nanoparticles, while Group IV underwent only spinal puncture. Treatments were administered intrathecally. Safety was assessed using MRI, hematological and biochemical parameters, and cerebrospinal fluid analysis. Cell localization was studied with iron-label staining, and tissue integrity was evaluated histologically. Biochemical tests revealed no significant blood parameter changes. C-reactive protein (CRP) levels decreased after NTs and NT–MSC combinations, indicating an anti-inflammatory effect. Biodistribution analysis showed MSC migration via cerebrospinal fluid and accumulation around the spinal cord and brain. MRI and behavioral monitoring confirmed the absence of adverse effects. These findings demonstrate that MSC therapy combined with neurotrophin-releasing nanoparticles is safe and feasible as adjunct therapy for ALS.\u003c/p\u003e","manuscriptTitle":"Safety and biodistribution of mesenchymal stromal/stem cells and biocompatible neurotrophin-releasing polyelectrolyte nanoparticles as a preclinical study in amyotrophic lateral sclerosis (ALS) cell therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-12 14:20:18","doi":"10.21203/rs.3.rs-7641255/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-10T10:26:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T15:13:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T05:39:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-07T13:20:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56764461760209976812412917328359646697","date":"2025-10-01T20:44:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170883198447272143305246543451960783884","date":"2025-10-01T14:36:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T13:45:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T11:20:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55187915331199982625337169973846897541","date":"2025-09-30T10:32:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138144231894735859335229841506780000412","date":"2025-09-30T07:15:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283748061629446437905980126032887960398","date":"2025-09-29T15:23:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236586099347206630338908976419447591914","date":"2025-09-29T12:31:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272258128269144127809165971065616195684","date":"2025-09-29T12:29:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-29T12:16:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-29T12:06:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-23T04:50:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-21T16:46:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-21T16:41:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d42e40fb-895b-43f7-84ef-91745e4b9f69","owner":[],"postedDate":"October 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56041195,"name":"Biological sciences/Biotechnology"},{"id":56041196,"name":"Health sciences/Neurology"},{"id":56041197,"name":"Biological sciences/Neuroscience"},{"id":56041198,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-03-02T16:01:18+00:00","versionOfRecord":{"articleIdentity":"rs-7641255","link":"https://doi.org/10.1038/s41598-026-40196-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-27 15:57:23","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-10-12 14:20:18","video":"","vorDoi":"10.1038/s41598-026-40196-0","vorDoiUrl":"https://doi.org/10.1038/s41598-026-40196-0","workflowStages":[]},"version":"v1","identity":"rs-7641255","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7641255","identity":"rs-7641255","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}