Research Article

Prophylactic and Therapeutic Effects of MOG-Conjugated PLGA Nanoparticles in C57Bl/6 Mouse Model of Multiple Sclerosis

Introduction

Multiple sclerosis (MS) is a prototypic neuroinflammatory disorder of the central nervous system (CNS), and is believed to result from a lack of functional harmony in immune response of autoaggressive and regulatory cells. ^1,2 Autoreactive T cells in MS have been implicated to recognize myelin proteins including myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP). ³ In contrast, CD4⁺CD25⁺Foxp3⁺ Treg cells (Tregs) specially orchestrate the quality and quantity of adaptive immune responses against self-antigens. ^4-6 However, Treg cells were found to have functional impairments in MS patients which make them unable to prevent the onset and progression of the disease. ⁷ Accordingly, extended efforts have been made to reinduce the immune tolerance through administration of autoantigens; but the efficiency of this approach highly depends on the continuous and low-dose stimulation of regulatory cells that mediate immune tolerance.

A promising strategy to have a steady release of autoantigens for immune tolerance induction might be through coupling the autoantigens to solid biodegradable particles. Since poly (lactic-co-glycolic acid) (PLGA) particles have demonstrated suitable characteristics such as biocompatibility, biodegradability, and received approval from the Food and Drug Administration (FDA), they seem to be a pertinent carrier of the desirable drugs with sustained-release rate. ⁸ This technology has already shown practical applicability in several settings. In particular, we have found that intravenous (i.v.) injection of MOG-conjugated PLGA before and after EAE induction, significantly inhibited proliferation of splenocytes leading to a delayed incidence of the syndrome. ⁹ In other studies, PLGA-conjugated antigens depicted enhanced tolerogenic effects compared to antigens alone in nasal vaccination. ¹⁰ More specifically, i.v. administration of PLGA-encephalitogenic peptides also indicated ameliorative outcomes in experimental autoimmune encephalomyelitis (EAE). ¹¹ We, therefore, set out to discover how PLGA-MOG nanoconjugates would be able to re-establish the required immune tolerance effectively in the EAE mouse model of MS.

The current study was conducted to gain more insight into the mechanism by which MOG-conjugated PLGA particles driving immune response towards the immunoregulatory response of particularly Tregs in EAE. To do so, either prophylactic or therapeutic injection methods were comparatively examined in the study.

Material and Methods

Mice

Inbred, 6-8 weeks old female C57BL/6 mice were purchased from Pasteur Institute of Iran (Tehran, Iran). Nine groups of mice, each containing five animals, were kept under standard housing conditions in the central animal facility at Tarbiat Modares University (TMU).

Nanoparticle preparation

Using double emulsion solvent evaporation (W/O/W) approach at room temperature, the PLGA (Sigma-Aldrich, Gillingham, Dorset, UK) microspheres were prepared in sterile conditions as described previously. ¹² In summary, 1 mL of phosphate buffer saline (PBS, pH = 7.4) containing N-cetyl-N,N, N-trimethyl ammonium bromide (Merck, Kenilworth, NJ, USA) (0.2%, w/v) was suspended in 10 mL of 4% w/v PLGA solution in ethyl acetate, and then sonicated at 50 watt (W) for 1 min in an ice-bath. The water-in-Oil (W/O) emulsion was subsequently added into 20 mL of 2% w/v aqueous polyvinyl alcohol (88% hydrolyzed, 20.000-30.000, Achros) and mixed at high speed by mechanical stirrer at 4000 rpm. After evaporation of the organic solvent, the obtained microspheres were washed and then collected by centrifugation for 5 min at 4000 rpm. Next, the supernatant was collected and washed twice with PBS and passed through 500 to 30 kDa molecular weight cut-off membranes (Merck, Kenilworth, NJ, USA). After filtration, 3 fractions were collected and lyophilized. Molecular weight distribution of particles which passed through 500 kDa filter but precipitated on 300 kDa paper was defined as PLGA₅₀₀. Moreover, PLGA₁₀₀ were all particles went through 500, 300, and 100 kDa filters but could not pass from a 30 kDa filter.

Conjugation of MOG and OVA peptides to the PLGA particles

PLGA particles were pre-activated in a mixture containing 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (4 mg/mL final) and N-hydrosulfosuccinimide (Sulfo-NHS) (50 mM final) as described earlier. ¹³ The activated particles were incubated on a rotator for 2 hours at room temperature. MOG_35-55 or OVA_323-339 (Sigma-Aldrich, Gillingham, Dorset, UK) peptides were added to the PLGA (1 mg/mL) solution and then further incubated for 72 hours in order to achieve the desired antigen coupling rates. Glycine (7 mg/mL final) was then used to saturate the unbound sites on the particles and the whole solution was incubated again for 30 min at room temperature. To remove the unbound glycine, the final solution was dialyzed in a 10-14 kDa molecular weight cut-off membrane against PBS (pH = 7.2) at 4°C overnight. Given that the final solution of antigen-conjugated PLGA could not be sterilized via filtration or UV irradiation, as it may cause peptide degradation; we, therefore, conducted the whole conjugation process under aseptic conditions and then stored the lyophilized powder at 4°C.

Evaluation of coupling efficiency

Conjugation efficiency of the particles was indirectly calculated by measuring the amount of unconjugated proteins within the supernatant of the final solution. In summary, an aliquot of the solution was ultracentrifuged (Beckman TLA-100.3) (Beckman Coulter, Fullerton, CA, USA) at 70 000 rpm for 20-30 minutes. After collecting the supernatant, Micro BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to assess the free proteins according to the manufacturer’s guideline.

Administration of PLGA-conjugated peptides to mice

Each of the investigating groups consisted of five C57BL/6 mice, and the administrations’ protocol was conducted as shown in Table 1.

EAE Induction

To recapitulate MS, EAE was induced in 6-8 weeks old C57/BL6 mice, using an emulsion of MOG_35-55 peptides (1 mg/mL) and complete Freund’s adjuvant (4 mg/mL of Mycobacterium tuberculosis H37Ra (Difco, Amsterdam, Netherlands)). Moreover, mice received intraperitoneal injection of Pertussis toxin on the same day and 24 hours later. ^14,15 Thereafter, daily clinical symptoms of the EAE were examined using a standard scoring system ranging from 0 to 5 as follows: 0, no disease; 1, tail paralysis; 2, loss of tail tonicity and hindlimbs weakness; 3, hindlimbs paralysis; 4, hindlimb paralysis as well as forelimb paralysis; and 5, moribund.

Histopathological analysis of brain tissue sections

The mice were deeply euthanized by ketamine-xylazine and the brain samples were carefully removed. The brains were fixed in 10 % formalin. Then, serial tissue sections from 5 to 10 μm of thicknesses were obtained from each sample, dewaxed and stained with the hematoxylin-eosin (H&E) to assess the infiltration of mononuclear cells into the brain.

Treg cell quantification and flow-cytometry

In order to quantify Treg cells, the mice were sacrificed by cervical dislocation, and splenocytes were collected 14 days after EAE induction. Briefly, splenocytes were washed once with PBS and resuspended in 100 μL of staining buffer (PBS + 2% fetal bovine serum (FBS)). The cells were firstly stained for surface markers with anti-CD4-FITC and anti-CD25-APC (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instruction. Each sample was then fixed and permeabilized with LEUCOPERMTM (a reagent for cell permeabilizing which is supplied from Bio-Rad) (Bio-Rad, California, CA, US), based on the manufacturer’s manual. Anti-Foxp3-PE (BioLegend, San Diego, CA USA) was used for staining and the cells were evaluated by BD FACSCalibur^TM (BD Biosciences, Heidelberg, Germany).

Cytokine production characterization of splenic lymphocytes

The murine spleens were removed on day 14 under sterile conditions, placed into the cell strainer separately, and homogenized. The strainer was then rinsed with 5 mL incomplete RPMI 1640 (Gibco, Gaithersburg, MD, USA) containing penicillin (100 U/mL) and streptomycin (100 μg/mL). The cell suspension was centrifuged at 500 × g for 10 min. The supernatant was discarded and the pellet was resuspended in Ammonium-Chloride-Potassium (ACK) lysing buffer. The cells were incubated at room temperature for 5 minutes, and subsequently, quenched with complete RPMI containing 10% FBS. The centrifugation was repeated at 500 × g for 10 min and the pellet was resuspended in 5 mL of complete media. A suspension of 5 × 10⁵ cells/ mL was cultivated in presence of the MOG_35-55 (10 μg/mL) and then incubated at 37°C and 5 % CO₂. After 72 h, the supernatant was collected and concentration of interferon-γ (IFN-γ), interleukin-10 (IL-10), and interleukin-17 (IL-17) were measured through commercial ELISA kits (Invitrogen, Waltham, MA, USA).

Statistical analysis

All statistical analyses were conducted by SPSS software v. 24 (SPSS Inc, Chicago, IL, USA) and data were plotted using GraphPad Prism version 6.00 (GraphPad Software Inc, La Jolla, CA, USA). Each experiment was conducted in duplicate or triplicate and one-way ANOVA with Tukey’s post-hoc test as well as independent t test were applied to compare the differences between various treated and untreated control groups. Data are presented as means ± standard error of the mean (SEM) unless otherwise stated, and two-sided P values < 0.05 were considered as statistically significant.

Results and Discussion

In our previous study, ¹⁶ we evaluated characteristics of the PLGA particles using scanning electron microscopy. PLGA500 and PLGA₁₀₀ particles had spherical shape and a smooth surface (). The PLGA₅₀₀ particles were microparticles in sizes ranging from 400 to 500 nm, while PLGA₁₀₀ particles were nano-scale particles smaller than 100 nm. The polydisperse index (PDI) of PLGA₁₀₀, PLGA₃₀₀ and PLGA₅₀₀ particles were 0.1, 0.53, and 0.85, respectively. The zeta potential of PLGA₁₀₀, PLGA₃₀₀, and PLGA₅₀₀ particles was determined to be -14.1, -5.65, and -18.0, respectively. According to the dynamic light scattering (DLS) outcomes, the average size of PLGA₁₀₀, PLGA₃₀₀ and PLGA₅₀₀ particles was determined as 151 nm, 389 nm and 521 nm. On the other hand, the particles integrity has not been significantly altered after conjugation of MOG_35-55 or OVA_323-339 with PLGA₅₀₀ particles and were still smooth on the surface with no aggregation, as observed through atomic force microscopy (AFM) (). The conjugation efficiency of 9.56% and 25.85% were measured for MOG peptides conjugated to PLGA₅₀₀ and PLGA₁₀₀, respectively. In addition, OVA binding efficacy to the PLGA₅₀₀ and PLGA₁₀₀ particles was 8.59% and 24.2%, respectively.

Close

Figure 1. Characteristics of the PLGA particles. Scanning electron microscope micrographs of PLGA500 (a) and PLGA100 (b) particles demonstrated smooth spherical morphologies before conjugation. Additionally, atomic-force microscopy ofPLGA500 particles illustrated no morphological change as well as aggregation inPLGA500 particles before (c) and after (d) conjugation. Naturally, size of the particles was increased after coupling to the MOG peptides.

Fourier transform infrared spectroscopy (FTIR) technique subsequently was used to check conjugation of the peptides to the PLGA₅₀₀. PLGA₅₀₀ particles were placed in the device before and after coupling to the peptides, and their infrared transmittance was plotted. As shown in , there were differences in the infrared spectrum of the nanoparticles before () and after () binding to the peptide, which may be due to the addition of new bands over the nanoparticles. In the plots of PLGA, the important peaks were related to those described in Table 2, before and after peptide coupling to the PLGA particles.

Close

Figure 2. The FTIR spectrum of the PLGA500-MOG35-55 particles before (a) and after (b) conjugation to the MOG peptides. During conjugation, the decrease in wavelength peak of carboxylic group and increase of the etheric peak confirmed conjugation of PLGA to the MOG peptides. FTIR, Fourier transform infrared spectroscopy.

The increasing peaks for aliphatic, hydroxyl, or amide groups represented in of PLGA graphs compared to the PLGA₅₀₀-MOG_35-55 graph from 2900 cm-¹ to 3400 cm-¹ indicates a change in the surface of the PLGA₅₀₀ nanoparticles following conjugation to the MOG_35-55 peptides. Moreover, the peak reduction of carboxylic groups from the wavelength of 1759 cm-¹ in PLGA to 1756 cm-¹ in PLGA₅₀₀-MOG_35-55, as well as the peak increase of the etheric groups from 1174 cm-¹ in PLGA to 1187 cm-¹ in PLGA₅₀₀-MOG_35-55 imply the conjugation of MOG peptides to the PLGA₅₀₀. The surface coupling can increase the peak energy of carboxylic and reduce the peak energy of the etheric groups, leading to a decrease in the peak associated with the wavelength of the carbonyl group and an increase in the peak associated with the wavelength of the etheric group. ¹⁷

In the area of vaccination, non-toxic nanoparticles with low inflammatory activity and sustained release of antigens, have shown great promises to induce the proper immune responses. ¹⁸ The current available data are controversial regarding the inflammatory activity of PLGA nanoconjugates. To investigate the tolerogenic effects of PLGA₅₀₀-MOG_35-55, the particles were prophylactically and therapeutically administered (i.v.) to the groups of mice on day -7 and 0 of EAE induction. Results indicated that PLGA₅₀₀-MOG_35-55 injection on day 0 had no significant impact on the onset and severity of the EAE. However, minor changes were observed compared to the control group received PLGA₅₀₀-OVA_323-339 after day 20. Interestingly, prophylactic injection of the conjugates on day -7 reduced the clinical complications (). It seems that the theragnostic application of these nanoparticles could partially attenuate the relapse of the disease either. The reason behind this could be possibly due to the priming condition of the mice. The prophylactic use of PLGA₅₀₀-MOG_35-55 provides sufficient time to strengthen the existing tolerance, while in the therapeutic manner there is less opportunity to trigger a tolerogenic response before EAE induction. Afterward, we noticed that PLGA₅₀₀-MOG_35-55 and not PLGA₁₀₀-MOG_35-55 displayed a rehabilitative impact on EAE clinical scores (). Indeed, the next experiments were conducted using only PLGA₅₀₀-MOG_35-55 particles in a prophylactic manner. Cappellano et al have also indicated that vaccination with a mixture of PLGA₅₀₀-MOG_35-55 and PLGA-IL-10 successfully ameliorated EAE complications in C57BL/6 mice. ¹⁹ Compared to our analysis, they had an extra intervention of extrinsic IL-10 and consequently its tolerogenic effects. However, the two investigations were done within 40 days, and therefore, it is not possible to determine which method was more effective to induce a long-term tolerance. To predict the stability of immune tolerance, it may be advantageous to refer to the increased number of Tregs which leads to a strong tolerance in long term. ¹⁴

Close

Figure 3. Tolerogenic effects of different PLGA particles on the clinical scores of EAE. First, we intravenously primed three groups of C57B/L6 mice with PLGA500-MOG35-55 (red line: 7 days before EAE induction, blue line: on the day of EAE induction) and PLGA500-OVA323-339 particles (control group) in order to compare prophylactic and therapeutic effects of the vaccine. Mice prophylactically treated with PLGA500-MOG35-55 (7 days before EAE induction) indicated a delayed disease onset and significant decrease of clinical scores compared to therapeutic application of the vaccine and control group (a). Next, effect of molecular size (PLGA500 and PLGA100 particles) and tolerogenic capacity of the conjugates were evaluated by prophylactic and i.v. injection of particles to three groups of mice. In this regard, we found that prophylactic PLGA500-MOG35-55 reveals significantly higher immunosuppressive effects relative to the PLGA100-MOG35-55 and control group (b). We further hypothesized if different routes of injection also affect the mean clinical scores of EAE mice. Accordingly, PLGA100-MOG35-55, PLGA500-MOG35-55, and PLGA500-OVA323-339 were subcutaneously injected to mice, seven days before EAE induction. Our results demonstrated an early onset of EAE plus exacerbated clinical conditions for PLGA100-MOG35-55 and PLGA500-MOG35-55 treated mice compared to the control group (PLGA500-OVA323-339) (c). Finally, we aimed to confirm whether tolerance PLGA500-MOG35-55 particles was antigen-specific. Thus, PLGA500-MOG35-55, PLGA500-PLP139-151, or PLGA500-OVA323-339 particles were i.v. administered to three groups of mice seven days before the EAE induction. Our results demonstrated a specific break and reinduction of immune tolerance against MOG peptides in PLGA500-MOG35-55-treated mice compare to PLGA500-PLP139-151 and PLGA500-OVA323-339 groups (d). One-way ANOVA and Tukey’s post hoc were conducted to compare efficacy of the treatments between different groups. Data are presented as the mean ± SEM and the number of mice in each group was 4 to 6. P value less than 0.05 is summarized with *.

As shown in , prophylactic and subcutaneous priming of mice with both PLGA₁₀₀-MOG_35-55 and PLGA₅₀₀-MOG_35-55 particles on day -7, exacerbated clinical conditions of the groups. This procedure also caused an early onset of the disease in the EAE mice. Thus, we found intravenous route more effective than the subcutaneous injection procedure to induce the immune tolerance. No anaphylactic reaction was observed after either s.c. or i.v. administration but the adjuvant effect of nanoparticles in subcutaneous route resulted in the exacerbation of the disease with no amelioration. On the other hand, it is worthy to note (it is worth noting) that intravenous vaccination has the potential capability to induce T cell anergy, as demonstrated by splenocyte proliferation inhibition in our previous study. ¹⁶ Moreover, tolerance specificity of EAE induction was also evaluated when a group of mice was intravenously primed with PLGA₅₀₀-PLP_139-151 on day -7. Then, EAE was induced and this group demonstrated no significant improvement of clinical scores relative to the control group that received PLGA₅₀₀-OVA_323-339 (). During MS and also EAE pathogenesis, neuroinflammation permanently attracts several clones of lymphocytes that react to different epitopes of autoantigens. This phenomenon is called “epitope spreading” and points to the fact that the appropriate cure of the disease must establish the required immune tolerance against all reactive antigens. ^20-22 As a solution, exertion of homogenized CNS tissue could possibly be more clinically applicable to prevent EAE in this case. However, the present study successfully broke and then reinduced the specific immune tolerance against only MOG_35-55, when treatment with PLGA₅₀₀-PLP_131-159 nanoparticles did not affect initiation and severity of MOG-induced EAE. By contrast, Getts et al used the two epitopes PLP_139-151 and PLP_178-191 to demonstrate that although the induced tolerance was antigen-specific, the second epitope prevented the epitope spreading. ¹¹ The proximity of two epitopes in the site of lesions could possibly be the reason for their outcome. However, it needs further experimental testing in the future.

In tissue level, others have previously reported infiltration of lymphocytes into the brain parenchyma during EAE pathogenesis. ²³ We, therefore, have performed H&E staining on the brain sections for each sample and found that the degree of mononuclear cell migration was significantly (markedly) reduced following the treatment with PLGA₅₀₀-MOG_35-55 () compared to the PLGA₅₀₀-OVA_323-339-treated group and naïve mice ().

Close

Figure 4. Histopathological analysis of mononuclear cells infiltrated into the brain tissues. For this purpose, brains of mice that prophylactically received PLGA500-MOG35-55 (a) or PLGA500-OVA323-339 (b) peptides, along with a healthy sample (c) were stained with hematoxylin-eosin (H&E) dyes. Arrows indicate the infiltrated cells into the different regions of the brain (hematoxylin-eosin, original magnification ×200).

To determine the potential effect of MGO-conjugated PLGA treatment on Treg frequency, three groups of mice were subcutaneously treated with PLGA₅₀₀-MOG_35-55 or PLGA₅₀₀-OVA_323-339 particles on day -7 of EAE induction. Another group was also designed as the healthy control group with no EAE induction and treatment. On day +14, the splenocytes were isolated and stimulated by MOG_35-55 for 72 h to assess quantity of Treg cells. For analysis of CD4⁺CD25⁺Foxp3⁺ cells, lymphocytic population of isotype control was selected on forward scatter/side scatter (FSC/SSC) and then gating was applied for CD4⁺ cells. Afterward, quadrants were drawn to find percentage of CD4⁺CD25⁺FoxP3⁺ subpopulation (). According to the , PLGA₅₀₀-MOG_35-55-treated mice had significantly (P< 0.0001) higher count of CD4⁺CD25⁺Foxp3⁺ cells compared to those which received PLGA₅₀₀-OVA_323-339 particles (1.15±0.1 and 0.70±0.01, respectively).

Close

Figure 5. Flow cytometric analysis of CD4 + CD25 + FoxP3 + T cells (Tregs). Gating strategy of the splenic Tregs (a). Effect of prophylactic and i.v injection ofPLGA500-MOG35-55 and PLGA500-OVA323-339 particles on percentage of splenic Treg cells compared to a naïve mice (b). Quantification of increase in percentage of Treg cells in PLGA500-MOG35-55 compared to PLGA500-OVA323-339-treated mice (c). Independent t-test was conducted to compare PLGA500-MOG35-55, PLGA500-OVA323-339-treated. Naive group were healthy mice without any injection. Data are presented as the mean ± SEM. P value less than 0.01 is summarized with **.

In order to determine the immune response balance of splenocytes, the concentration of IFN-γ, IL-17, and IL-10 was measured in the supernatant of the activated cell culture. As shown in the , IFN-γ and IL-17 produced by the splenocytes of PLGA₅₀₀-MOG_35-55-treated mice (IFN-γ: 156±9,IL-17: 51.6±2.2 pg/mL) were significantly lower than that in PLGA₅₀₀-OVA_323-339-treated mice (IFN-γ: 1175±55, IL-17: 226.5±6.5 pg/mL) (both p values < 0.0001). In contrast, PLGA₅₀₀-MOG_35-55-treated group produced a significantly higher amount of IL-10 in culture, compared to PLGA₅₀₀-OVA_323-339-treated mice (567.2±22.8,196.25±12.75 pg/mL, respectively with P < 0.0001) (). Cappellano et al also reported that the PLGA₅₀₀-MOG_35-55 vaccine affects the balance of cytokines, by repressing either IFN-γ or IL-17 and promoting IL-10 production, which is in consensus with our results. ¹⁹ However, uptake of PLGA particles was also shown to induce proinflammatory responses in murine macrophages and human dendritic cells. ^24,25 Moreover, we have already demonstrated that PLGA₅₀₀-MOG_35-55 treatment reduces proliferation capacity of activated splenocytes in EAE mouse without obvious cell toxicity, ¹⁶ as stated by Cappellano et alin MTT assay. ¹⁹

Close

Figure 6. Balance of splenic cytokines in naïve mice as well as EAE-induced mice vaccinated with PLGA500-MOG35-55 or PLGA500-OVA323-339 particles. The mean concentration of IFN-γ (a), IL-17 (b), and IL-10 (c) was evaluated in the supernatant of splenic lymphocytes cultured for 72 h in presence of MOG peptides. Independent t-test were conducted to compare PLGA500-MOG35-55 -, PLGA500-OVA323-339-treated, and naïve mice together. The number of mice in each group was 5 (P≤0.00001). P value less than 0.0001 is summarized with ****.

Conclusion

In this study, improved clinical conditions of EAE mice were obtained in consequence of PLGA-based vaccinations, without subsequent complications reported for other treatments. Strategies exerting such particles are simple and inexpensive techniques to induce an autoantigen-specific immune tolerance. Using tolerogenic nanoparticles alongside with the immunosuppressive modules could open up new horizons towards MS treatment.

Above all, efficacy and safety of this strategy for other autoantigens of MS need to be taken for granted. Accordingly, more clinical trials for each vaccine are required to guarantee the procedure.

Ethical Issues

The current study was conducted with approval from the Animal Ethics Committee of Tarbiat Modaress University (TMU), Tehran, Iran (thesis number: 3768).

Conflict of Interest

The authors declare that they have no competing interests.

Acknowledgments

We are thankful to the Department of Microbiology and Immunology facilities at Islamic Azad University-Tehran Medical Branch.

References

1. Hsieh CS, Zheng Y, Liang Y, Fontenot JD, Rudensky AY. An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat Immunol 2006; 7(4):401-10. doi: 10.1038/ni1318 [Crossref]
2. Chang CC, Ciubotariu R, Manavalan JS, Yuan J, Colovai AI, Piazza F. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat Immunol 2002; 3(3):237-43. doi: 10.1038/ni760 [Crossref]
3. Johnson D, Hafler DA, Fallis RJ, Lees MB, Brady RO, Quarles RH. Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein, and myelin basic protein in multiple sclerosis. J Neuroimmunol 1986; 13(1):99-108. doi: 10.1016/0165-5728(86)90053-6 [Crossref]
4. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25) Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995; 155(3):1151-64.
5. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008; 133(5):775-87. doi: 10.1016/j.cell.2008.05.009 [Crossref]
6. Hoffmann P, Eder R, Boeld TJ, Doser K, Piseshka B, Andreesen R. Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion. Blood 2006; 108(13):4260-7. doi: 10.1182/blood-2006-06-027409 [Crossref]
7. Azimi M, Ghabaee M, Naser Moghadasi A, Noorbakhsh F, Izad M. Immunomodulatory function of Treg-derived exosomes is impaired in patients with relapsing-remitting multiple sclerosis. Immunol Res 2018; 66(4):513-20. doi: 10.1007/s12026-018-9008-5 [Crossref]
8. Houchin ML, Topp EM. Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms. J Pharm Sci 2008; 97(7):2395-404. doi: 10.1002/jps.21176 [Crossref]
9. Gholamzad M, Ebtekar M, Shafiee Ardestani M. Intravenous injection of myelin oligodendrocyte glycoprotein-coated PLGA microparticles have tolerogenic effects in experimental autoimmune encephalomyelitis. Iran J Allergy Asthma Immunol 2017; 16(3):271-81.
10. Keijzer C, Slütter B, van der Zee R, Jiskoot W, van Eden W, Broere F. PLGA, PLGA-TMC and TMC-TPP nanoparticles differentially modulate the outcome of nasal vaccination by inducing tolerance or enhancing humoral immunity. PLoS One 2011; 6(11):e26684. doi: 10.1371/journal.pone.0026684 [Crossref]
11. Getts DR, Martin AJ, McCarthy DP, Terry RL, Hunter ZN, Yap WT. Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis. Nat Biotechnol 2012; 30(12):1217-24. doi: 10.1038/nbt.2434 [Crossref]
12. Saini V, Jain V, Sudheesh MS, Jaganathan KS, Murthy PK, Kohli DV. Comparison of humoral and cell-mediated immune responses to cationic PLGA microspheres containing recombinant hepatitis B antigen. Int J Pharm 2011; 408(1-2):50-7. doi: 10.1016/j.ijpharm.2011.01.045 [Crossref]
13. Gaumet M, Vargas A, Gurny R, Delie F. Nanoparticles for drug delivery: the need for precision in reporting particle size parameters. Eur J Pharm Biopharm 2008; 69(1):1-9. doi: 10.1016/j.ejpb.2007.08.001 [Crossref]
14. Getts DR, Turley DM, Smith CE, Harp CT, McCarthy D, Feeney EM. Tolerance induced by apoptotic antigen-coupled leukocytes is induced by PD-L1+ and IL-10-producing splenic macrophages and maintained by T regulatory cells. J Immunol 2011; 187(5):2405-17. doi: 10.4049/jimmunol.1004175 [Crossref]
15. Ghaffarinia A, Parvaneh S, Jalili C, Riazi-Rad F, Yaslianifard S, Pakravan N. Immunomodulatory effect of chymotrypsin in CNS is sex-independent: evidence of anti-inflammatory role for IL-17 in EAE. Iran J Allergy Asthma Immunol 2016; 15(2):145-55.
16. Gholamzad M, Ebtekar M, Shafiee Ardestani M. Intravenous injection of myelin oligodendrocyte glycoprotein-coated PLGA microparticles have tolerogenic effects in experimental autoimmune encephalomyelitis. Iran J Allergy Asthma Immunol 2017; 16(3):271-81.
17. Bellisola G, Sorio C. Infrared spectroscopy and microscopy in cancer research and diagnosis. Am J Cancer Res 2012; 2(1):1-21.
18. Leleux J, Roy K. Micro and nanoparticle-based delivery systems for vaccine immunotherapy: an immunological and materials perspective. Adv Healthc Mater 2013; 2(1):72-94. doi: 10.1002/adhm.201200268 [Crossref]
19. Cappellano G, Woldetsadik AD, Orilieri E, Shivakumar Y, Rizzi M, Carniato F. Subcutaneous inverse vaccination with PLGA particles loaded with a MOG peptide and IL-10 decreases the severity of experimental autoimmune encephalomyelitis. Vaccine 2014; 32(43):5681-9. doi: 10.1016/j.vaccine.2014.08.016 [Crossref]
20. Waldner H, Whitters MJ, Sobel RA, Collins M, Kuchroo VK. Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptor. Proc Natl Acad Sci U S A 2000; 97(7):3412-7. doi: 10.1073/pnas.97.7.3412 [Crossref]
21. Bettelli E, Pagany M, Weiner HL, Linington C, Sobel RA, Kuchroo VK. Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop spontaneous autoimmune optic neuritis. J Exp Med 2003; 197(9):1073-81. doi: 10.1084/jem.20021603 [Crossref]
22. Liu GY, Fairchild PJ, Smith RM, Prowle JR, Kioussis D, Wraith DC. Low avidity recognition of self-antigen by T cells permits escape from central tolerance. Immunity 1995; 3(4):407-15. doi: 10.1016/1074-7613(95)90170-1 [Crossref]
23. Stromnes IM, Cerretti LM, Liggitt D, Harris RA, Goverman JM. Differential regulation of central nervous system autoimmunity by T(H)1 and T(H)17 cells. Nat Med 2008; 14(3):337-42. doi: 10.1038/nm1715 [Crossref]
24. Nicolete R, dos Santos DF, Faccioli LH. The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response. Int Immunopharmacol 2011; 11(10):1557-63. doi: 10.1016/j.intimp.2011.05.014 [Crossref]
25. Look M, Saltzman WM, Craft J, Fahmy TM. The nanomaterial-dependent modulation of dendritic cells and its potential influence on therapeutic immunosuppression in lupus. Biomaterials 2014; 35(3):1089-95. doi: 10.1016/j.biomaterials.2013.10.046 [Crossref]