Abstract

Multiple sclerosis (MS) is a serious neurological disorder due to its widespread preva-lence, chronic nature, frequent progression to disability, and tendency to affect young people. The pathogenesis of MS is based on the immunopathogenesis hypothesis. Biocompatible magnetite na-noparticles, which exhibit selective sorption activity towards cell membrane surface proteins, circu-lating immune complexes, lymphocytotoxic antibodies, and the complement system, as well as en-hance phagocytic activity and the completion index of leukocyte phagocytosis, can be effectively used for immunocorrection. The main goal of the study is to slow the progression of MS, improve the neurological status and overall condition of the patient, and reduce the dynamics of the spread of demyelinating lesions in the brain. Materials and methods: A patient diagnosed with multiple sclerosis, secondary progressive type, cerebrospinal form, at the clinical aggravation stage was included in the study. Neurological status and disability were assessed using the EDSS scale, and brain MRI with contrast enhancement was performed. The nanodevice Micromage-B was administered orally as an immunosorbent and immunomodulator. The regimen and dosage of Micromage-B were personal-ized. Assessments of general condition and neurological status were conducted every 7 days for 6 months, with contrast-enhanced brain MRI performed in the 5th month. Results: The use of Micro-mage-B in MS treatment led to an objective improvement in neurological status, with reduced stiff-ness and fatigue in the lower extremities. Gait and coordination improved, hand tremors decreased, depression and concentration disorders subsided, appetite was restored, and speech improved. Throughout the treatment period, positive dynamics in the normalization of neurological status were observed. After 6 months, the total score on the EDSS scale decreased from 210 to 45.

The most significant improvements were observed in the evaluation of the pyramidal system and coordination, with the EDSS Disability Scale score decreasing from 6.0 to 5.0. For the first time, contrast-enhanced brain MRI showed a reduction in the number of new demyelination foci by the 4th month of Micromage-B administration. The positive changes in neurological status correlated with the MRI results. The recovery of central nervous system activity in MS is likely not only due to the immuno-suppressive properties of magnetite nanoparticles but also due to the activation of remyelination mechanisms and oligodendrocyte differentiation through enzymatic methylation. Conclusion: The use of biocompatible nanodevices in the complex treatment of MS is promising. Further improve-ment and study of the regimen and method of using biocompatible magnetite nanoparticles to en-hance MS treatment effectiveness are required.

Keywords: Multiple Sclerosis; Treatment; Nanodevice; Micromagе-B; Neurological status assessment; Remyelination; MRI, Central nervous system; Antigens; Antibodies; Complete Immunosuppression; Migration; B-lymphocytes; Immunopathogenesis; Cerebellar ataxia

Introduction

Multiple sclerosis (MS) is a significant neurological issue due to its widespread prevalence, chronic nature, frequent progression to disability, and tendency to affect young people, with an average onset age of 30 years. The primary hypothesis of MS immunopathogenesis suggests that immunological tolerance is disrupted, leading to the active penetration of autoreactive cells sensitized to nervous tissue antigens through the blood-brain barrier into brain tissue. B-lymphocytes recognize myelin and signal T cells to initiate an immune attack [1-7]. T- and B-cells secrete chemicals that attract other immune cells, causing inflammation [8,9]. Plasma cells produce antibodies that attack myelin and recruit other immune cells [10,11]. T- and B-cells establish a persistent presence in the central nervous system (CNS) and continue their attack [12,13].There are two main hypotheses for MS pathogenesis: the outside-in hypothesis, which posits that immunocompetent cells activated in the periphery penetrate brain tissue, and the inside-out hypothesis, which suggests primary damage to nervous tissue leads to the expression of damage receptors from the DAMPs (Danger-associated Molecular Patterns) family, resulting in immune activation and loss of tolerance to myelin antigens [14]. Antibodies produced by plasma cells actively destroy the protective covering of nerve cells, causing inflammation. Over time, this tissue becomes scarred, disrupting conduction. Consequently, impulses from the brain fail to reach the limbs and organs, leading to a loss of body control [15]. Common initial clinical symptoms include weakness and impaired sensation in one or more limbs, decreased vision, urinary disturbances, and cerebellar ataxia.

Currently, immunomodulatory and immunosuppressive drugs that alter the course of MS form the cornerstone of pathogenetic treatment. Their mechanisms of action involve: 1) selective immunosuppression, 2) complete immunosuppression, 3) prevention of the migration of activated cells from lymph nodes or into brain tissue, or 4) a combination of immunoregulatory, anti-inflammatory, antioxidant, and potentially neurotrophic actions. Future directions for new MS treatments include selective local immunocorrection, remyelination and neuroprotection, enhancement of neuroplasticity and functional relocalization, evaluation of the efficacy and safety of cell therapy, and individualized therapy selection based on predictions of pathological process variants and potential treatment responses derived from the molecular and cellular biology of MS [16-22]. The development of inflammation and demyelination in MS is driven by an impaired immune response, imbalance between regulatory and effector T cells, activation of B-cell immunity, and microglia. All drugs that impact the course of MS either deplete T- or B-cells or modify the signaling pathways involved in immune response formation [23]. Recently, significant attention has been given to the role of B-cells in chronic CNS inflammation, evidenced by their involvement in autoantibody formation, antigen presentation, and continuous T-cell activation in the brain parenchyma. Anti-B-cell therapies (Rituximab, Ocrelizumab, Ofatumumab) have shown effectiveness in both remitting and progressive MS. Stimulating remyelination in MS is associated with the development of new monoclonal antibody preparations, such as anti-LINGO and human immunoglobulin M (IgM), which promote remyelination and the differentiation of oligodendrocytes and their precursors [24]. One of the key mechanisms of axonal degeneration is mitochondrial dysfunction. Some drugs, such as Dimethylfumarate, Idebenone, and Biotin, show promise in addressing this issue [25]. Additionally, the use of drugs that affect the redistribution of ion channels in demyelinated axons (Lamotrigine, Amiloride, Fampridine) can help reduce the energy deficit in axons and neurons [26].

Recently, biocompatible nanotechnological preparations have seen increasing use in medicine. Since 1998, Ukrainian clinics have been officially using nanodevices developed by Belousov’s Applied Nanotechnology Laboratory, including the Micromage-B, MCS-B, and ICNB brands (Figure 1) [27]. These nanodevices are based on biocompatible magnetite nanoparticles, whose unique physical and chemical characteristics enable a wide range of applications. They influence the quantitative and qualitative composition of bodily fluids, metabolic and biochemical processes, and the energy balance of cells. Their selective sorption activity against surface proteins of cell membranes, circulating immune complexes, lymphocytotoxic antibodies, and the complement system, as well as their ability to enhance phagocytic activity and leukocyte phagocytosis completion index [28], allow these nanodevices to be effectively used for immunocorrection. Moreover, these nano preparations impact glycolysis processes, cell membrane ion channel activity, normalize erythrocyte function, improve microcirculation, and reduce the platelet aggregation index (Figure 2) [29-31]. They also activate the system of antiradical enzymes and inhibit lipid peroxidation processes [32,33].

Figure 2. Effect of biocompatible magnetite nanoparticles on functional status of erythrocytes and hemorheology. The results of these studies lay the foundation for developing innovative techniques for the effective and safe use of biocompatible magnetite nanoparticles in treating severe autoimmune diseases, including multiple sclerosis. The primary objective of the study is to slow the progression of MS, improve the neurological status and overall condition of the patient, and reduce the spread of demyelinating foci in the brain.

Materials and Methods

Patient K. was diagnosed with multiple sclerosis, secondary progressive type, cerebrospinal form, at the clinical aggravation stage. The patient presented with pronounced spastic tetraparesis, more severe in the lower extremities, leading to walking impairment, pronounced urinary-ataxic syndrome, and sphincter and sensory disorders. MRI revealed signs of multifocal diffuse brain lesions (more than 30) of a demyelinating nature, indicating the active phase of the disease, and a diffuse atrophic process in the cerebral cortex. The average number of relapses in the year before study inclusion was 1.0, and the EDSS disability score was 6.0. The disease had progressed for 24 years since the onset of the first symptoms. For 14 years, the patient received regular treatment with vascular and metabolic drugs in combination with hormonal therapy. In the 6th year, despite active treatment, the disease progressed to a secondary progressive course, leading to the addition of the immunosuppressive drug Teriflunomide to the therapy regimen. However, despite all treatment efforts, the patient’s general condition progressively worsened, and the neurological status did not stabilize. MRI examinations over the last four years showed a continuous increase in the number of new demyelinating foci in the brain. Given the above, the therapy regimen was supplemented with the prescription of Micromage-B [34]. Before starting the study, the patient’s formal consent to the use of Micromag-B was obtained. This fact was recorded in the medical history, and the plan of treatment and subsequent study was agreed with the attending physician. Micromage-B is an oral nanodevice officially registered by the Ministry of Health of Ukraine. It is a powder form of magnetite (Fe3O4) nanoparticles designed for the prevention and treatment of various diseases and to enhance the body’s resistance to adverse environmental factors.

As a nanotechnology device, Micromage-B features magnetite nanoparticles sized between 6 and 12 nm. The therapeutic effect of Micromage-B is based on the mechanism of sorption and the action of a constant magnetic field on cellular and subcellular structures induced by the magnetite nanoparticles. The nanoparticles have a sorption surface area of 800 to 1200 m²/g and an induced magnetic field strength of 300-400 kA/m. The target of Micromage-B is the microenvironment of the cell’s aqueous spaces and surface membrane proteins. Through selective sorption, the magnetite nanoparticles alter the quantitative and qualitative composition of cell surface proteins, and the constant magnetic field changes the mobility and orientation of hydrogen protons in the cell’s aqueous microenvironment (Figure 3).

Figure 3. The effect of magnetite nanoparticles on hemolysis processes by changing the polarization structure of the aqueous sector of the erythrocyte microenvironment. This leads to the activation of hydrolysis of the phosphate residue ATP, ultimately modifying the cell’s transmembrane exchange and metabolism, and altering its susceptibility. The nanodevice enhances adaptive mechanisms and the potential of cell organelles, accelerates reparative processes at the membrane and macromolecule levels, significantly increases the level of synthetic intracellular reactions. Additionally, Micromage-B nanoparticles adsorb toxic substances and circulating immune complexes, significantly enhancing the treatment efficacy of various allergic diseases, autoimmune processes (such as rheumatoid arthritis, acute and chronic polyarthritis, eczema, etc.), and acute poisoning. Micromage-B regulates the activity of antioxidant enzymes, absorbs lipid peroxidation products, and rebalances antiradical and pro-radical product levels [35,36]. The dosing regimen for Micromage-B entails 500 mg daily for the first month, 500 mg every other day for the second month, and subsequently, 500 mg once every three days. The selection of Micromage-B dosage and regimen is tailored individually, considering the patient’s rate of improvement and neurological recovery.

The study monitored changes in neurological status using a modified version of the Multiple Sclerosis Patient Assessment Scale [37,38], which evaluates the severity of motor impairments alongside other nervous system damage indicators. Disability was quantitatively assessed using Kurtzke’s online EDSS calculator [39]. Manifestations of cerebral demyelination foci were examined through contrast-enhanced MRI.The patient’s overall condition and neurological status were assessed every 7 days over a 6-month period. As per the protocol, a contrast-enhanced MRI of the brain was conducted annually, aligning with the 5th month of Micromage-B usage.

Results

The progression of neurological changes was assessed using a modified scale. One week following the administration of Micromage-B, a marked enhancement in the patient’s neurological condition was evident. The patient reported a notable reduction in lower limb stiffness and rapid fatigue. Objectively, improvements were observed in gait and coordination, reduction in hand tremors, complete alleviation of depression and concentration issues, restoration of appetite, and enhancement in speech. Positive trends in normalizing neurological status persisted throughout the entire duration of Micromage-B treatment. Table 1 illustrates the evaluation of a multiple sclerosis patient’s neurological status before and after 6 months of Micromage-B administration. Estimated scores of the neurological status before and after using the Micromage-B. The data presented in Table 1 illustrate a positive trend in normalizing neurological status following 6 months of Micromage-B usage. Initially, the total points amounted to 210, which decreased to 45 after the 6-month period, indicating a reduction of 165 points. The most significant improvement was observed in the assessment of the pyramidal system and coordination. Additionally, the EDSS disability scale score decreased from 6.0 to 5.0.

A contrast-enhanced MRI of the brain conducted after 4 months of Micromage-B usage revealed a decrease in the number of new demyelination foci in the brain for the first time. The favorable progression of neurological status correlated with the brain MRI findings. Upon analyzing the collected data, particular attention should be given to the observed positive clinical effects attributed to immunosorption and the active contribution of Micromage-B’s magnetite nanoparticles to neurological status restoration. This effect may be attributed to remyelination processes and oligodendrocyte differentiation. Oligodendrocytes, a type of neuroglial cell, form the myelin sheath around neurons in the central nervous system (CNS). The molecular mechanisms underlying cell differentiation and specialization remain complex and poorly understood, presenting a challenging area in cell and developmental biology. The development and maturation of various cell types continue to pose significant research challenges. However, it is now understood that one mechanism underlying oligodendrocyte maturation involves enzymatic methylation, specifically the addition of a methyl group (-CH3) to the N6 nitrogen atom in adenosine’s nitrogenous base, referred to as m6A-methylation. Despite its seemingly minor nature, this modification can significantly impact subsequent stages of protein biosynthesis. The role of m6A-methylation has been demonstrated in numerous processes associated with oligodendrocyte maturation [40]. The body’s universal methyl group donor is S-adenosylmethionine, generated through the interaction between the amino acid methionine and ATP. Given that Micromag-B activates glycolysis, it significantly increases the production of the macroergic compound ATP [41,42], and promotes the formation of the reduced form of the coenzyme NADPH2, facilitating the conversion of oxidized glutathione to its reduced form [43]. These conditions are conducive to initiating enzymatic methylation processes, which likely enhance the mechanism of action of magnetite nanoparticles (Micromag-B). This, in turn, supports the differentiation of oligodendrocytes and the remyelination process. Additionally, it should be noted that these nanoparticles polarize the aqueous environment of the cellular microenvironment [44], leading to the activation of ATP hydrolysis, energy release, and the formation of ADP. For a detailed understanding of how biocompatible magnetite nanoparticles (Micromag-B) influence remyelination processes, refer to Figure 4.

Given the positive neurological status dynamics, it was deemed appropriate to continue Micromage-B nano preparation administration at the prescribed dosage. Therapy was augmented with a regimen of rehabilitative exercises aimed at expediting the restoration of physical, cognitive, and psychosocial functions in patients with MS. Considering the positive progression in neurological status, it was decided to continue the administration of the nano preparation Micromage-B at the prescribed dosage. Additionally, the therapy was augmented with a comprehensive rehabilitation exercise program designed to expedite the recovery of physical, cognitive, and psychosocial functions in a patient with MS

Conclusion

The study’s results broadened the clinical effectiveness of biocompatible magnetic nanoparticles in treating severe autoimmune diseases [45-48]. The use of the Micromage-B nano preparation in treat-ing multiple sclerosis (MS) demonstrated a significant positive clinical effect. Positive trends in nor-malizing the neurological state were observed throughout the application period of Micromage-B. After six months of treatment, the overall score decreased from 210 to 45. The most notable improvement was seen in the assessment of the pyramidal system and coordination. The EDSS Disabil-ity Scale score decreased from 6.0 to 5.0. For the first time, contrast-enhanced MRI of the brain rec-orded a reduction in the number of new demyelination foci by the fourth month of Micromage-B administration. The normalized neurological condition correlated with positive brain MRI results. The restoration of central nervous system activity in MS is attributed not only to the immunosup-pressive properties of magnetite nanoparticles but also likely to the activation of remyelination mech-anisms and oligodendrocyte differentiation through enzymatic methylation.

The design and method of using biocompatible magnetite nanoparticles to enhance MS treatment efficiency require further refinement and research. The use of biocompatible nanodevices in the comprehensive treatment of MS is a promising innovation [49-51]. Micromage-B is a biologically active form of a nanodevice officially approved by the Ministry of Health of Ukraine and authorized for clinical use since 1998. To date, no side effects or contraindications to its use have been identified. Given the key properties of bio-compatible magnetite nanoparticles, their clinical applications continue to expand. Recent studies have deepened our understanding of the mechanisms regulating cellular metabolism, microcircula-tion, eryptosis, as well as the mechanisms of action of antibacterial agents and other processes. To substantiate the necessity of further utilizing magnetite nanoparticles in larger clinical trials, it is cru-cial first to identify relevant biochemical markers. These markers should not only objectively reflect the severity of multiple sclerosis from a pathogenetic perspective but also provide a high-probability prognosis of disease outcomes.