Multiple sclerosis (MS) is a chronic autoimmune disorder of central nervous system which is increasing worldwide. Although immunosuppressive agents are used for the treatment of MS disease, nevertheless the lack of non-toxic and efficient therapeutic method is perceptible. Hence, this study aims to evaluate the effect of Contactin-associated protein (Caspr) antibody-, poly ethylene glycol (PEG)- and exosome combined gold nanoparticles (GNPs) in comparison to Glatiramer acetate as a selective treatment of MS disease in the experimental autoimmune encephalomyelitis (EAE) mouse model. EAE was induced in female C57BL/6 mice and 25-day treatment with anti-Caspr-, PEG- and exosome combined GNPs was evaluated. Histopathological examination of spinal cord, regulatory T cells as well as inflammatory pathway including IFN-ɣ and IL-17 and mir-326 were investigated. The results showed the severity of MS symptoms was significantly decreased in all treated groups. Histological examination of the spinal cord indicated the reduced demyelination and immune cell infiltration. Besides, regulatory T cells were significantly increased following all treatments. Remarkably, the cytokine levels of IFN-ɣ and IL-17 as well as mir-326 is altered in treated groups. Taken together, the obtained findings demonstrate that the administration of anti-Caspr-, PEG- and exosome combined GNPs can be considered a potential treatment in MS disease.
Research Article
Anti-Caspr-Conjugated Gold Nanoparticles Emergence as a Novel Approach in the Treatment of EAE Animal Model
https://doi.org/10.21203/rs.3.rs-736595/v1
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Multiple sclerosis
Experimental autoimmune encephalomyelitis
Gold nanoparticle
Contactin-associated protein
Poly ethylene glycol
Exosome
Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder of the central nervous system (CNS) which still is increasing worldwide (1). Experimental autoimmune encephalomyelitis (EAE) is commonly used as the best animal model for CNS inflammation and demyelinating diseases (2). It has recently been shown that T cell-mediated inflammation plays a dominant role in the pathogenesis of MS (3). Different environmental factors may disrupt the peripheral tolerance of myelin-specific T cells in genetically susceptible individuals (4). In general, regulatory T cells (Treg) are one of the key elements in maintaining the balance of immune responses in a variety of autoimmune disorders as well as MS disease (5). Several reports have demonstrated that pathogenic helper T (Th) lymphocytes, especially Th1 and Th17 are major players in CNS inflammation and MS development (6–8). Moreover, pro-inflammatory cytokines including interleukin 1-beta (IL-1β), IL-12, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), IL-17, and IL-22 are highly contributed to the MS progression (9). Therefore, aberrant pathogenic T cell responses, as well as impaired Treg cell populations most likely, underlies the MS immunopathogenesis.
MicroRNAs (miRNAs) are small regulatory non-coding RNAs that play an important role in the regulation of immune responses (10). Notably, dysregulation of miRNAs is strongly associated with neurodegenerative diseases (11). Based on previous studies miR-155 and mir-326 are involved in MS development through Th1/Th17 differentiation (12, 13).
Despite available immunomodulatory medications for MS such as Glatiramer acetate, discovering an effective and safe management strategy remains a subject of debate. Interestingly, electrical stimulation is an important factor in axon growth and neuron survival (14). Thus, the electrically conductive materials can be helpful in nerve regeneration through the construction of nerve guidance channels (15). Gold nanoparticles (GNPs) also known as colloidal gold, are small water-soluble particles with a diameter of 1 to 100 nm in size (16). GNPs play a critical role in the improvement of axonal myelination and neuronal repair due to electrical stimulation characteristics (17). Hence, GNPs are considered a potential non-cytotoxic tool for the treatment of inflammatory diseases as well as neurodegenerative disorders (18–20). Contactin-associated protein (Caspr) is a transmembrane protein that appears on the neuronal surface following demyelination (21). Conjugation of GNPs with anti-Caspr antibody potentially targets unsheathed neurons by which may prevent the deposition of GNPs in other tissues, especially the kidneys.
Polyethylene glycol (PEG) is a synthetic biocompatible compound that can be used as a membrane adhesive by induction of cell membrane fusion (22). Furthermore, PEG additives result in the stability enhancement of biopharmaceutical proteins (23). Surprisingly, PEG rapidly repairs neuronal membranes and inhibits the production of free radicals after acute spinal cord injury in the animal model (24). Moreover, it has indicated that PEGylated GNPs can reduce inflammation and microglia response and improve myelin regeneration (17). On the other hand, exosomes are extracellular vesicles (30 to 100 nm in diameter) that are released from all eukaryotic cells through different pathways (25). Besides, exosomes can be used as molecules to transport different drugs and molecules (26). Recently, it has recently been shown that nanoparticles pass through the blood-brain barrier and are transported to the brain with the help of exosomes (27).
Hence, the aim of this study was to investigate the effect of anti-Caspr-, PEG- and exosome combined GNPs in comparison to Glatiramer acetate as a selective treatment of MS disease in the experimental autoimmune encephalomyelitis (EAE) mouse model by assessing histopathological and clinical scores.
Animals
Female C57BL/6 mice (n = 36, 8–12 weeks old) were purchased from Pasteur Institute, Tehran, Iran. All mice were housed at room temperature (RT) with a relative humidity of 40% and had free access to water and pelleted diets. Experiments were conducted based on the NIH Guideline for the Care and Use of Laboratory Animals. The research proposal was approved by the ethics committee of the Iran University of Medical Sciences (code: IR. IUMS.FMD.REC.1398.431).
Synthesis of gold nanoparticle (GNP) products
Synthesis and characterization of GNPs:
GNPs of 20 nm were synthesized according to the citrate reduction of HAuCl4 described by Frens, et al. (28). Concisely, 25 mL of 1% HAuCl4 solution (Sigma Chemical Co. Philadelphia, PA) was mixed under continuous stirring. After reaching the boiling temperature, 1 mL of 1% trisodium citrate (Merck, Darmstadt, Germany) was added. A few minutes later, the color mixture first turned to dark violet then to deep pink which confirmed the formation of stable GNPs of 20 nm in size. Afterward, the mixture was boiled for an additional 5 min, then cooled at RT and stored at 4°C until use. The shape and size of synthesized GNPs were determined by transmission electron microscopy (TEM). Also, dynamic light-scattering (DLS) was used to evaluate the hydrodynamic diameters.
Synthesis of GNP- conjugated to anti- Contactin associated protein (Caspr)
The 0.5 mM EG6 -COOH and the 0.5 mM EG3 -OH solutions were mixed at a ratio of 1:10. One mL of the mixed solution was added to 9 mL of HAuCl4 colloidal suspension and stored for 25 h at RT. Then the mixed solution was centrifuged for 50 min at 14,000 RPM to separate the unbound and carboxylated GNPs and formation of a carboxylated self-assembled monolayer on GNPs. The pellet was resuspended in 0.1 M phosphate-buffered saline (PBS) (pH 7.4), which was repeated two times to remove the unbound GNPs. Then 100 mL of 0.1 M EDC was mixed with 10 mL of carboxylated GNPs, followed by adding 100 µl of 0.1 M NHS to activate the GNP solution as a coupling agent. The NHS-terminated GNPs covalently combined with the side-chain amino groups on the protein surfaces. After 10 min, 1 mL of 100 g mL–1 anti-Caspr monoclonal Ab was added to the EDC/NHS-activated GNP solution. After 24 h, the solution was centrifuged for 50 min at 14 000 RPM to remove the unbound anti-Caspr mAb. Finally, the obtained anti-Caspr mAb-conjugated GNPs (GNPs–caspr mAb) were resuspended in 5 mL of 0.1 M PBS (pH 7.4) (29).
PEGylation of GNPs
To prepare an aqueous polyethylene glycol (PEG) solution (MW 1500, 30% w/v in PBS), 30 g of PEG powder (Sigma-Aldrich, Philadelphia, PA) was dissolved in 100 mL PBS. In order to PEGylation, 20 µL of PEG solution was added to each 1 mL GNP solution which was mixed on a magnetic stirrer overnight. To concentrate the PEGylated GNPs and remove any free ligand, the mixture was centrifuged at 14,000g for 30 min and then the pellet is resuspended in PBS.
Isolation and characterization of exosomes:
Exosomes were purified from the mouse serum using a Total Exosome Isolation kit (EXOCIB, Iran) according to the manufacturer’s protocol. Briefly, serum was centrifuged at 3000 RPM for 20 min to remove cellular debris. The supernatant was transferred to a new conical tube and the heated reagent-A was added as a 1:5 ratio. Following the incubation overnight at 4°C, the mixture was centrifugated at 3000 RPM for 40 min and the supernatant was discarded. The pellet of the exosomes resuspend with reagent-B and the total protein concentration was measured for exosome quantification using the Bradford method (30). The quality of isolated exosomes was assessed by western blot using a primary antibody (mouse anti-CD63 antibody, concentration of 100µg/mL, (Abcam, USA) and secondary HRP-conjugated anti-mouse IgG, concentration of 200µg/mL (Santa cruz, USA). Also, TEM and DLS were done to confirm the presence of isolated exosomes (Fig. 1). Finally, 4µg (≈ 8 x 109) exosomes were incubated with 10mL of 20nm GNPs for 3h at 37°C and then centrifuged and washed.
Induction and evaluation of EAE
The EAE was induced in female C57BL/6 mice by the “Salari Institute of Cognitive and Behavioral Disorders” as previously described (31). Following a ketamine/xylazine (Merck, Germany) anesthesia, all animals were immunized according to the previous protocol described by Aghaie T, et al. (17). After 48 h of the immunization, all animals have received intraperitoneal (IP) pertussis toxin (Sigma-Aldrich, P7208). Clinical signs and bodyweight of mice were assessed daily by a blind observer. Symptoms were scored according to Table 1.
| Score | Clinical signs |
|---|---|
| 0 | No symptoms |
| 1 | Partial loss of tail tonicity |
| 2 | Complete loss of tail tonicity |
| 3 | Flaccid tail and abnormal gait |
| 4 | Hind leg paralysis |
| 5 | Hind legs paralysis with hind body paresis |
| 6 | Hind and foreleg paralysis |
| 7 | Death |
Study Design:
Mice were randomly divided into six groups and six animals were included in each group. The healthy control group (Healthy Control) is not EAE induced and received only PBS. The EAE control group (EAE Control) was EAE induced and received PBS. The third group (GNPs-Caspr mAb) is EAE induced and treated GNPs conjugated with anti-Caspr antibody and polysorbate 80. The EAE induced and treatment GNPs coated with PEG and polysorbate 80, considered a fourth group (GNPs-PEG). The EAE induced and treatment with GNPs, exosome, and polysorbate 80 (GNPs-Exosome). The EAE induced and treatment with the Glatiramer acetate (GA) drugs (GA group). Keeping in mind that polysorbate 80 (Merck, Germany) was used for the component transportation across the blood–brain barrier (BBB). In this study, 100µl of PEGylated-, Casper- conjugated and exosome-labeled GNPs with a concentration of 200µg/mL was used in all mice. All the animals received treatments through IP injection 1 day in between from day 3 to day 24 post-immunization (in total 12 injections). GA treatment (150µg/mouse by subcutaneous injection) started at day 3 after EAE induction. Daily injection of GA continued until the end of experiment. 25 days after EAE induction, the mice were anesthetized and spleens were removed and single-cell suspension of splenocytes were obtained. In brief, the spleen was perfused and the cells were passed through the cell strainers of 70 and 40µm. The cells were centrifuged at 300g for 10min, and the supernatant was discarded. To lysis the red blood cells, the splenocytes were incubated with 10mL ACK 1X (ammonium-chloride-potassium) lysing buffer for 5min at 4°C. The mixture was centrifuged at 300g for 5min and the supernatant was removed. After washing once with RPMI-1640 medium, the reminding cell suspensions were the spleen mononuclear cells. Then, all animals were sacrificed according to the ethics protocol and the spinal cord was removed for further histopathological studies.
Detection of regulatory T cells by flow cytometry analysis
The treatment effect on Treg cells was determined by flow cytometry technique using mouse regulatory T cell staining kit (eBioscience, San Diego, CA). In order to detect the cell surface markers, 1 × 106 spleen cells from all treatment groups were stained with the following fluorochrome labeled monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (clone RM4) and phycoerythrin (PE)-conjugated anti-mouse CD25 (clone PC61.5) antibodies at 4°C for 30 min. Cells were fixed and permeabilized with fixation/permeabilization buffer for the evaluation of FoxP3. Then the cells were incubated in this buffer containing (APC)-conjugated anti-mouse/Rat Foxp3 (clone FJK16s) antibody at 4°C for 30 min. Ultimately, all samples were washed and suspended in kit-provided staining buffer and then analyzed in a Mindray (BriCyte E6, China) with associated FlowJO 10 software. A minimum of 50000 events in the lymphocyte gate was collected per sample.
Histopathology
The spinal cord was isolated and horizontal paraffin-embedded sections (5µm thick) of the spinal cords were stained by H&E and Luxol Fast Blue Staining for evaluating the cell infiltration and myelination, respectively. Slides were assessed in a blinded manner (by analysis of three fields) and histological scores were determined as in our previous study.
RNA Extraction, Quantitative real-time PCR (qPCR), and MicroRNA Quantification
The spleen cells were isolated from EAE mice treated with GNPs. Large and small (total) RNA was extracted from splenocytes using GeneAllR RiboEx Total RNA extraction kit (GeneAll Biotechnology, Korea) according to the manufacturer’s instructions. The quantity and quality of RNA were evaluated by a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). The cDNA synthesis was performed from large RNA in the thermal cycle using miScript Reverse Transcription kit (Parstoos, Iran) according to manufacturer’s protocols for the treatment effect on mRNA expression of IL-17 and IFN-ɣ cytokines were determined by qPCR. HGPRT was considered as a housekeeping gene. The specific forward and reverse primers for the evaluation of IL-17 and IFN-ɣ genes expression were designed by NCBI primer blast (Table 2). The specific forward and reverse primers of miR-326-3p were purchased from Pars Genome, Tehran, Iran. Then the qPCR was carried out using the miScript SYBR Green PCR Kit (Pars Genome, Iran) and Qiagen Real-Time PCR system (Applied Biosystem). The RNU6 gene was used as an internal control for normalization. The threshold cycles (CT) were normalized and the relative expression levels were calculated using the 2-ΔΔct method.
| Gene Name | Sequence | Product length (bp) | |
|---|---|---|---|
| Interleukin- 17A (IL-17a) | Forward | 5’-CAGACTACCTCAACCGTTCC-3’ | 120 |
| Reverse | 5’-CCTCCGCATTGACACAGC-3’ | ||
| Interferon- gamma (IFN-γ) | Forward | 5’-CACACCTGATTACTACCTTCTTC-3’ | 180 |
| Reverse | 5’-AGCAGCGACTCCTTTTCC-3’ | ||
| Hypoxanthine guanine phosphoribosyl transferase (HGPRT) | Forward | 5’-TCCCAGCGTCGTGATTAG-3’ | 138 |
| Reverse | 5’-CGAGCAAGTCTTTCAGTCC-3’ | ||
Statistical analysis
Statistical analysis was performed using Prism (GraphPad) Software (version 8.3.0). Data are presented as Mean ± SEM and p values < 0.05 were statistically considered as the significance level. The normal distribution of data was examined by the Kolmogorov-Smirnov test. ANOVA test was used to compare the groups and analysis of the data. The Friedman test is used to evaluate the clinical score and body weight of all groups.
Characterization of GNPs:
TEM and DLS are respectively used to determine the shape and size distribution of synthesized GNPs (Fig. 2). As Fig. 1b shows, the mean size of over 90% of GNPs was ~ 20 nm in diameter.
Improved clinical Scores and reduced Body Weight in treated mice
Body-weight and clinical signs of all mice were monitored, daily. The obtained results have shown an improvement in the behavioral and neurological symptoms of mice that are treated with GNPs. Also, a significant weight loss was observed over the 25 days period in the EAE control group. The treatment with GNPs increases the body-weight of mice in all treatment groups (Fig. 3). Clinical manifestations of EAE were observed about 10 days after immunization in the untreated group (EAE ctrl) and between days 14 and 15 in the treated groups (Fig. 4). Moreover, the severity of clinical signs had significantly decreased in the treated mice in comparison to the EAE control group (P ≤ 0.05). Treatment with GNPs-Caspr mAb, GNPs-PEG, and GNPs-exosome reduced the incidence of disease. However, there was no significant difference in clinical scores between the GNP-treated groups (Table 3).
|
Groups |
Incidence (sick/total) |
Day of onset |
Peak Day Score |
Maximum mean clinical score |
Cumulative disease index CDI |
|---|---|---|---|---|---|
|
EAE Control |
6/6 100% |
10 |
19 |
2.95 ± 1.28 |
242 |
|
Glatiramer Acetate |
5/6 83% |
14 |
18 |
0.55 ± 0.50 |
56 |
|
GNPs + Caspr |
4/6 66% |
15 |
19 |
0.37 ± 0.47 |
19 |
|
GNPs + PEG |
5/6 83% |
15 |
17 |
0.41 ± 0.60 |
35 |
|
GNPs + Exosome |
5/6 83% |
14 |
18 |
0.50 ± 0.61 |
43 |
GNPs effects on demyelination/inflammation in the spinal cord of EAE mice
The histopathological analysis of the spinal cord from all treated mice was carried out to evaluate the underlying reasons for the improved clinical manifestations. The infiltration of mononuclear cells, as well as the myelin loss, were characterized in order to evaluate inflammation and demyelination, respectively. The semi-quantitative results of the histopathological features demonstrated a remarkable infiltration of inflammatory cells into the spinal cord and local demyelination in the EAE control group. Furthermore, declined demyelination occurred due to the reduced inflammatory cell infiltration to the spinal cord of all GNPs-treated mice. Interestingly, histopathological evaluation of mice showed that the treatments with GNPs modulate the severity of EAE (Fig. 4).
MS is viewed as a more frequent chronic immune-mediated demyelinating disease of the CNS worldwide. Despite the notable progress in the treatment of MS, it still remains a major neurological disorder at a global level. Thus, the discovery of a novel effective therapeutic method in order to the management of disease through reduction MS complications is perceptible. This study provides new insight into the potential role of anti-Caspr, PEG- and exosome combined GNPs in the treatment of EAE that is considered a prototype of MS disorder.
According to recent researches, it is reasonable to perceive that GNPs have an anti-inflammatory effect which can be considered as a therapeutic agent in neuro-immunological disorders (32). In general, previous studies have shown that GNPs play an important neuroprotective role through the improvement of clinical symptoms in mice with autoimmune disorders including Parkinson's disease, Alzheimer's disease, and EAE (17, 33–35). However, targeted therapy emerges as the best strategy regarding the treatment of MS disease and animal model of EAE. Based on the MS pathogenesis, GNPs combined with an anti-Caspr mAb can be considered a novel approach to target neuronal damage and unsheathed myelin. This study highlights three different approaches in the treatment of the EAE animal model. Interestingly, this study has demonstrated that the treatment with GNP- conjugated with anti-Caspr antibody improved the clinical symptoms in EAE mice (P ≤ 0.05). Recently, in-vivo studies have reported that PEG treatment has a major influence on the reduction and improvement of clinical signs in EAE animal models (36). Moreover, Aghaie, T. et al have depicted that EAE treatment by PEG significantly decreases the severity of MS symptoms (17). Hence, PEGylation of GNPs could open a new horizon in the management and limitation of MS disorder.
On the other hand, exosomes are extracellular double-membrane microvesicles that contain different biomolecules particularly microRNAs with immunomodulatory effects (37). Approximately 30% of exosomes isolated from serum were originated from neuronal cells (38). Therefore, a combination of the exosome and GNPs could improve the application of neurodegenerative disease treatment.
Surprisingly, the results of this study showed that treatment of EAE mice with Caspr-, PEG- and exosome combined GNPs reduced the inflammation and help the nerve cell repairment which subsequently results in the improvement of clinical symptoms. Besides, the obtained results have indicated that there is no significant difference between the three treatment groups (P ≤ 0.05). Remarkably, no significant difference was observed between the effects of three different treatments in comparison to the glatiramer acetate as a selective treatment for MS. Furthermore, all treatments result in a significant reduction in weight loss among EAE mice (P ≤ 0.05). Recently, disruption in T cell polarization, as well as Treg cell reduction, have been shown in MS disease. In this sense, the development of regulatory T cells may be useful to inhibit the autoreactive T cells activation and modulation of autoimmune disorders. The results show a significant increase in the number of Treg lymphocytes in the spleen of EAE mice treated with anti-Caspr-, PEG- and exosome combined GNPs. Besides, regulatory T cells reduce inflammation; In addition, previous studies have shown that the Th1/Th17-mediated inflammatory pathways play an important role in the pathogenesis of MS disease. It is reasonable to presume that proinflammatory cytokines including INF-γ and IL-17 significantly higher in MS patients compared to normal subjects. The obtained results indicated that anti-Caspr-, PEG- and exosome combined GNPs reduce the proinflammatory cytokine production.
On the other hand, Du Ch, et al. have demonstrated mir-326 promotes the Th17 differentiation that results in the severity of MS disease (39). Moreover, the expression level of mir-326 in active MS plaques is significantly upregulated in comparison to healthy individuals (40). The present study also showed that the level of mir-326 in the EAE animal model was significantly higher than in the healthy control group (P ≤ 0.05). Interestingly, the treatment of mice using anti-Caspr mAb conjugated GNPs significantly reduced mir-326 expression. Also, administration of PEG- and exosome combined GNPs remarkably influence mir-326 expression in EAE treated mice.
Taken together, administration of anti-Caspr-, PEG- and exosome combined GNPs can be considered a potential treatment in MS disease due to induction of Treg development and reduction in proinflammatory cytokines especially IL-17 and IFN-γ.
Acknowledgment:
This work was supported by a grant from the Iran University of Medical Sciences, Tehran, Iran. We would like to thank Dr. Salari who are our colleague from Salari Institute of Cognitive and Behavioral Disorders for their kind assistance.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
We declare that there is no conflict of interest in publication of this contribution.
Author contributions:
Shirin Taghizadeh1, Morteza Motallebnezhad1, Tayebe Aghaie1, Maryam Azimi2, Azin Aghamajidi1, Ali-Akbar Salari3, Mahmoud Bozorgmehr4, Mohammad Ali Assarezadegan1,2, Mir-Hadi Jazayeri1,2*
S.T.; Designed and performed experiments, analyzed data and co-wrote the paper
M.M., T. A., M. B; Performed experiments
M.A; Designed experiments
A.A; Co-wrote the paper
AA. S.; Provided essential mouse strains and performed EAE induction
MA. A; Supervised the research
MH. J; Designed experiments and supervised the research
- Filippi M, Bar-Or A, Piehl F, Preziosa P, Solari A, Vukusic S, et al. Multiple sclerosis. Nat Rev Dis Primers. 2018;4(1):43.
- Burrows DJ, McGown A, Jain SA, De Felice M, Ramesh TM, Sharrack B, et al. Animal models of multiple sclerosis: From rodents to zebrafish. Mult Scler. 2019;25(3):306-24.
- Zhang XM, Liu CY, Shao ZH. Advances in the role of helper T cells in autoimmune diseases. Chin Med J (Engl). 2020;133(8):968-74.
- Jayaraman S, Prabhakar BS. Immune Tolerance in Autoimmune Central Nervous System Disorders. Neuroimmune Diseases: From Cells to the Living Brain. 2019:143-66.
- Sambucci M, Gargano F, Guerrera G, Battistini L, Borsellino G. One, No One, and One Hundred Thousand: T Regulatory Cells' Multiple Identities in Neuroimmunity. Front Immunol. 2019;10:2947.
- Arellano G, Acuna E, Reyes LI, Ottum PA, De Sarno P, Villarroel L, et al. Th1 and Th17 Cells and Associated Cytokines Discriminate among Clinically Isolated Syndrome and Multiple Sclerosis Phenotypes. Front Immunol. 2017;8:753.
- Milovanovic J, Arsenijevic A, Stojanovic B, Kanjevac T, Arsenijevic D, Radosavljevic G, et al. Interleukin-17 in Chronic Inflammatory Neurological Diseases. Front Immunol. 2020;11:947.
- Lassmann H, Ransohoff RM. The CD4-Th1 model for multiple sclerosis: a critical [correction of crucial] re-appraisal. Trends Immunol. 2004;25(3):132-7.
- Wang K, Song F, Fernandez-Escobar A, Luo G, Wang JH, Sun Y. The Properties of Cytokines in Multiple Sclerosis: Pros and Cons. Am J Med Sci. 2018;356(6):552-60.
- Mehta A, Baltimore D. MicroRNAs as regulatory elements in immune system logic. Nat Rev Immunol. 2016;16(5):279-94.
- Juzwik CA, S SD, Zhang Y, Paradis-Isler N, Sylvester A, Amar-Zifkin A, et al. microRNA dysregulation in neurodegenerative diseases: A systematic review. Prog Neurobiol. 2019;182:101664.
- Chen C, Zhou Y, Wang J, Yan Y, Peng L, Qiu W. Dysregulated MicroRNA Involvement in Multiple Sclerosis by Induction of T Helper 17 Cell Differentiation. Front Immunol. 2018;9:1256.
- Azimi M, Ghabaee M, Naser Moghadasi A, Izad M. Altered Expression of miR-326 in T Cell-derived Exosomes of Patients with Relapsing-remitting Multiple Sclerosis. Iran J Allergy Asthma Immunol. 2019;18(1):108-13.
- Gordon T. Electrical Stimulation to Enhance Axon Regeneration After Peripheral Nerve Injuries in Animal Models and Humans. Neurotherapeutics. 2016;13(2):295-310.
- Saberi A, Jabbari F, Zarrintaj P, Saeb MR, Mozafari M. Electrically Conductive Materials: Opportunities and Challenges in Tissue Engineering. Biomolecules. 2019;9(9).
- Herizchi R, Abbasi E, Milani M, Akbarzadeh A. Current methods for synthesis of gold nanoparticles. Artif Cells Nanomed Biotechnol. 2016;44(2):596-602.
- Aghaie T, Jazayeri MH, Manian M, Khani L, Erfani M, Rezayi M, et al. Gold nanoparticle and polyethylene glycol in neural regeneration in the treatment of neurodegenerative diseases. J Cell Biochem. 2019;120(3):2749-55.
- Abbas M. Potential Role of Nanoparticles in Treating the Accumulation of Amyloid-Beta Peptide in Alzheimer's Patients. Polymers (Basel). 2021;13(7).
- Hu K, Chen X, Chen W, Zhang L, Li J, Ye J, et al. Neuroprotective effect of gold nanoparticles composites in Parkinson's disease model. Nanomedicine. 2018;14(4):1123-36.
- Souza PS, Zaccaron RP, Vasconcellos FTF, De Paula CBV, Cunha EBB, de Noronha L, et al. Neuroinflammatory Regulation of Gold Nanoparticles Conjugated to Ethylene Dicysteine Diethyl Ester in Experimental Autoimmune Encephalomyelitis. ACS Biomater Sci Eng. 2021;7(3):1242-51.
- Zou Y, Zhang WF, Liu HY, Li X, Zhang X, Ma XF, et al. Structure and function of the contactin-associated protein family in myelinated axons and their relationship with nerve diseases. Neural Regen Res. 2017;12(9):1551-8.
- Lu X, Perera TH, Aria AB, Callahan LAS. Polyethylene glycol in spinal cord injury repair: a critical review. J Exp Pharmacol. 2018;10:37-49.
- Wang Y, Quinsaat JEQ, Ono T, Maeki M, Tokeshi M, Isono T, et al. Enhanced dispersion stability of gold nanoparticles by the physisorption of cyclic poly(ethylene glycol). Nat Commun. 2020;11(1):6089.
- Luo J, Borgens R, Shi R. Polyethylene glycol immediately repairs neuronal membranes and inhibits free radical production after acute spinal cord injury. J Neurochem. 2002;83(2):471-80.
- Zhang Y, Liu Y, Liu H, Tang WH. Exosomes: biogenesis, biologic function and clinical potential. Cell Biosci. 2019;9:19.
- Ha D, Yang N, Nadithe V. Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges. Acta Pharm Sin B. 2016;6(4):287-96.
- Saint-Pol J, Gosselet F, Duban-Deweer S, Pottiez G, Karamanos Y. Targeting and Crossing the Blood-Brain Barrier with Extracellular Vesicles. Cells. 2020;9(4).
- Frens G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature physical science. 1973;241(105):20.
- Jazayeri MH, Amani H, Pourfatollah AA, Avan A, Ferns GA, Pazoki-Toroudi H. Enhanced detection sensitivity of prostate-specific antigen via PSA-conjugated gold nanoparticles based on localized surface plasmon resonance: GNP-coated anti-PSA/LSPR as a novel approach for the identification of prostate anomalies. Cancer Gene Ther. 2016;23(10):365-9.
- Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54.
- Majidi-Zolbanin J, Doosti MH, Kosari-Nasab M, Salari AA. Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis. Neuroscience. 2015;294:69-81.
- Kevadiya BD, Ottemann BM, Thomas MB, Mukadam I, Nigam S, McMillan J, et al. Neurotheranostics as personalized medicines. Adv Drug Deliv Rev. 2019;148:252-89.
- Xue J, Liu T, Liu Y, Jiang Y, Seshadri VDD, Mohan SK, et al. Neuroprotective effect of biosynthesised gold nanoparticles synthesised from root extract of Paeonia moutan against Parkinson disease - In vitro &In vivo model. J Photochem Photobiol B. 2019;200:111635.
- Dos Santos Tramontin N, da Silva S, Arruda R, Ugioni KS, Canteiro PB, de Bem Silveira G, et al. Gold Nanoparticles Treatment Reverses Brain Damage in Alzheimer's Disease Model. Mol Neurobiol. 2020;57(2):926-36.
- Nosratabadi R, Rastin M, Sankian M, Haghmorad D, Mahmoudi M. Hyperforin-loaded gold nanoparticle alleviates experimental autoimmune encephalomyelitis by suppressing Th1 and Th17 cells and upregulating regulatory T cells. Nanomedicine. 2016;12(7):1961-71.
- Shi R, Borgens RB. Acute repair of crushed guinea pig spinal cord by polyethylene glycol. J Neurophysiol. 1999;81(5):2406-14.
- Pang H, Luo S, Xiao Y, Xia Y, Li X, Huang G, et al. Emerging Roles of Exosomes in T1DM. Front Immunol. 2020;11:593348.
- Doyle LM, Wang MZ. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells. 2019;8(7).
- Du C, Liu C, Kang J, Zhao G, Ye Z, Huang S, et al. MicroRNA miR-326 regulates T H-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nature immunology. 2009;10(12):1252.
- Junker A, Krumbholz M, Eisele S, Mohan H, Augstein F, Bittner R, et al. MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47. Brain. 2009;132(12):3342-52.