Novel nano-carriers with N-formylmethionyl-leucyl-phenylalanine-modified liposomes improve effects of C16-angiopoietin 1 in acute animal model of multiple sclerosis

Abstract

Gradual loss of neuronal structure and function due to impaired blood–brain barrier (BBB) and neuroinflammation are important factors in multiple sclerosis (MS) progression. Our previous studies demonstrated that the C16 peptide and angiopoietin 1 (Ang-1) compound (C + A) could modulate inflammation and vascular protection in many models of MS. In this study, nanotechnology and a novel nanovector of the leukocyte chemotactic peptide N-formyl-methionyl-leucyl–phenylalanine (fMLP) were used to examine the effects of C + A on MS. The acute experimental autoimmune encephalomyelitis (EAE) model of MS was established in Lewis rats. The C + A compounds were conjugated to control nano-carriers and fMLP-nano-carriers and administered to animals by intravenous injection. The neuropathological changes in the brain cortex and spinal cord were examined using multiple approaches. The stimulation of vascular injection sites was examined using rabbits. The results showed that all C + A compounds (C + A alone, nano-carrier C + A, and fMLP-nano-carrier C + A) reduced neuronal inflammation, axonal demyelination, gliosis, neuronal apoptosis, vascular leakage, and BBB impairment induced by EAE. In addition, the C + A compounds had minimal side effects on liver and kidney functions. Furthermore, the fMLP-nano-carrier C + A compound had better effects compared to C + A alone and the nano-carrier C + A. This study indicated that the fMLP-nano-carrier C + A could attenuate inflammation-related pathological changes in EAE and may be a potential therapeutic strategy for the treatment of MS and EAE.

Keywords:

• Neurodegenerative diseases
• inflammation
• C16 peptide and ang-1
• EAE
• fMLP-modified nano-carriers

Introduction

As a degenerative disease of autoimmune and neuronal systems, the features of multiple sclerosis (MS) include infiltration of immune cells, demyelination, gliosis, edema, and damage to axons and neurons in the central nervous system (CNS). The diffuse CNS impairments cause massive clinical neurological defects to multiple systems, including motor and sensory systems. As such, commonly observed symptoms include limb weakness, sensory disorders, optic neuritis, ataxia, dysuria, fatigue, and cognitive deficits (Herz et al., 2010).

As a disease of the CNS caused by inflammatory demyelination, experimental allergic encephalomyelitis (EAE) shares many clinical and pathological features with MS, and is considered a model of MS (Arnon & Aharoni, 2009). A previous study has indicated that chemokines can induce inflammatory processes and demyelination in both EAE and MS (Karpus, 2020). Among the previously established rodent models of EAE, the acute model using Lewis rats has demonstrated a single peak of paralysis followed by spontaneous recovery, which is a characteristic of MS (Eng et al., 1996). This acute model can be helpful to elucidate the immune response in MS induced by inflammation and to study novel therapeutic agents. The inflammatory response in MS is complex, and suppressing the inflammation is a crucial goal when developing effective treatments (Han et al., 2013). Of the available anti-inflammatory drugs, glucocorticoids have shown central efficacy in MS (Eng et al., 1996). Unfortunately, this class of potent anti-inflammatory drugs, including methylprednisolone, gives rise to several undesirable side effects related to immunosuppression.

The fundamental events during the onset of acute EAE include extensive edema and cellular inflammation due to an impaired blood–brain barrier (BBB). Importantly, integrin αVβ3 permits endothelial cells to interact with many extracellular matrix proteins, including laminin. The synthesized peptide C16, a γ1 chain peptide of laminin-1, can selectively bind the αVβ3 and αVβ1 integrins in endothelial cells to block the interaction between leukocytes and endothelial cells, which ultimately inhibits the transmigration of inflammatory cells (Gaillard et al., 2012). A previous study has also verified that C16 had no effect on the total number of leukocytes, suggesting that C16 is not an immunosuppressant (Han S et al., 2010). As a member of the endothelial growth factor family, angiopoietin 1 (Ang-1) has important roles in the establishment and maintenance of the maturation, stabilization, and integrity of the vascular system (Fang et al., 2013). Moreover, C16 and Ang-1 can synergistically alleviate vascular leakage and inflammation and prevent the demyelination and axonal loss in the EAE rat model (Jiang et al., 2014). However, the solubility of C16 is largely affected by the pH of the solvent, which decreases its bioavailability and may limit its clinical application (Han et al., 2010).

As the first discovered chemoattractant, the chemotactic peptide N-formyl-methionyl-leucyl–phenylalanine (fMLP) can be used to enhance drug concentrations in areas with inflammation (Qin et al., 2014). This study aimed to develop a nanovector of a leukocyte liposome (LIP) modified by fMLP to deliver the C16 and Ang-1 compound (C + A) to sites of inflammation in the EAE animal model in Lewis rats. The inflammatory response, axon loss, neuronal apoptosis, demyelination of white matter, and formation of gliosis in the CNS were measured by western blotting, histology, immunohistochemistry (IHC), electron microscopy (EM), enzyme-linked immunosorbent assays (ELISA), electrophysiological recordings, and behavioral tests. The C + A compound was delivered by direct intravenous injection either alone, with the control nano-carrier (LIP), or with the fMLP-LIP nano-carrier to compare their delivery effects.

Materials and methods

Establishment of EAE model

Lewis rats (male, 9–10 weeks, 200–250 g, n = 100 total, Vital River Laboratory Animal Technology Co., Ltd, Cat number: 113) were randomly assigned into one of five groups: normal control group, vehicle-treated group, C + A group, nano-carrier C + A group, or fMLP-nano-carrier C + A group (n = 20/group). The EAE model of Lewis rats was established in all rats except the normal control group by intradermal injection of guinea pig spinal cord homogenate (GPSCH) and complete Freud adjuvant (CFA) mixture (0.2 mL, 1:1), which contained heat-killed Mycobacterium tuberculosis (0.5 mg, Difco Laboratories, Detroit, MI). The rats in the normal control group were given the same volume of CFA emulsion in 0.9% saline (1:1). The rats were checked daily for clinical signs of disease beginning on day 7 after the intradermal injection. The rats were placed in a temperature-and humidity-controlled chambers. Manual bladder emptying was performed at least three times daily when the clinic score of animal reached 4.

The severity of disease was continuously marked using the following scale: 0, no disease; 1, attenuated tail tonicity; 2, disappearance of tail tonicity; 3, imbalance of gait and mild paralysis; 4, moderate to complete paralysis of hind limb and incontinence; and 5, moribund or death. The EAE model was considered successfully established when the score exceeded 2. Experiments were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, with approval from the Animal Ethics Committee at Zhejiang University (NO. 25327) and adhered to the ARRIVE guidelines.

Cervical somatosensory and motor evoked potential (c-SEP, MEP) recordings

The latency of waveform initiation and smaller wave amplitudes in both c-SEP and MEP were recorded and measured according to our previous study (Wang et al., 2016).

This study was approved by the Animal Care Committee of the Chinese Academy of Medical Sciences and performed in accordance with the NIH guidelines.

Preparation of liposomes

After dehydration, the DSPE-mPEG liposomes (Avanti Polar Lipids, Inc.) were rehydrated with 0.2 mL of Tris–HCl buffer (50 mmol/L, pH 7.0), followed by sonication in a water bath for five cycles (20 min per cycle) at room temperature (RT). A fMLP peptide containing a terminal cysteine (Cys–fMLP) was produced by ZiYu Biotechnology Co. Ltd. (China) according to the standards of solid phase peptide synthesis (SPPS), and the amount of fMLP–LIP used was optimized with LIP. Rhodamine-labeled C16 peptide was complexed with Cy5-labeled Ang-1 (synthesized by ZiYu Biotechnology Co. Ltd.) and the fMLP-nano-carrier C + A was made by coupling the proteins with the lipid at a molar ratio of 1:1,000. In the nano-carrier group C + A, the mixture of C + A was entrapped by LIP that was not conjugated to the fMLP peptide.

In the direct intravenous injection group, the rhodamine labeled C16 peptide was dissolved in deionized water by adding 0.3% acetic acid, was filtered with a 0.22 um disk filter, and the pH value was adjusted to 7.4 with a sterilized solution of NaOH. Finally, the solution was added to the same volume of sterilized phosphate-buffered saline (PBS), resulting in a final concentration of 1 mg/mL. The Cy5-labeled Ang-1 peptide was dissolved in deionized water to a final concentration of 1 mg/mL.

Intravenous injection

The liposome-entrapped C + A compounds with or without fMLP (400 µL) were injected into the tail vein of rats. In the vehicle and normal control groups, the same volume of solution without the C + A compound was injected. The solutions were injected immediately following the intradermal injection of the EAE inducer (GPSCH with CFA and M. tuberculosis), which was repeated daily for a period of 2 weeks.

Quantification of the drug concentration in the plasma by ELISA

The blood samples were collected at 1, 2, 4, 6, 12, 24, 48, and 72 h after the last injection of the C + A compound or vehicle. The standard curve ranges of the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A groups were made from 5, 50, 100, 500, 1000, 2500, and 5000 pg/mL concentrations. The serum concentration of C16 and Ang-1 at different time points was assessed by ELISA according to the absorbance ratio.

Detection of vascular wall stimulation in different groups

Six male adult health rabbits (2–2.5 kg, New Zealand white, purchased from Qingdao Kangda Aibo’s company, Cat number: 43513) and six lewis rats (Vital River Laboratory Animal Technology Co., Ltd, Cat number: 113) were randomly assigned into one of three groups: C + A, nano-carrier C + A, and fMLP-nano-carrier C + A (n = 2/group). The injection was performed three times (0.5 mL/injection) into both marginal ear veins (rabbits) and the tail veins (rats) at an interval of 1 h. After the last injection, the tissues at the injection site were collected under deep anesthesia with sodium pentobarbital (40 mg/kg), immerged in 10% formalin for 24 h, and stained following a standard hematoxylin and eosin (H&E) procedure. Damage to the vessel wall, as well as the presence of hemorrhage, blood clots, phlegmon and fibroplasia of the vein and its surrounding tissue were examined under a light microscope.

Tissue preparation for immunostaining and EM

Blood and urine samples were collected for biochemical examination before the animals were killed at 2 weeks post-immunization (pi). Next, under deep anesthesia with sodium pentobarbital (40 mg/kgi.p.), the rats were perfused via the left ventricle with chilled saline followed by 4% paraformaldehyde (in 0.1 M PBS, pH 7.4). The brain and entire spinal cord were collected with caution. A segment of the lumbar spinal cord (1 cm) and half of the brain were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 4 h, then dehydrated in PBS containing 30% sucrose until the tissue sank to the bottom of the container. The cortex and spinal cord were cut into 20 mm-thick coronal and transversal sections, respectively, using a Leica cryostat. The sections were mounted onto slides precoated with 0.02% poly-lysine, and were used for histological assessment and immunostaining. The other segments of spinal cord and other half of the brain were fixed with a 5% glutaraldehyde solution and cut into 50 nm sections for examination by transmission electron microscopy (TEM).

In vivo biodistribution

The fluorescence intensity of rhodamine was assessed at 6 h following the last injection, using neutral infrared imaging. Under deep anesthesia, samples from rats were collected, weighed, and homogenized in PBS. The rhodamine dye was extracted with a mixture of chloroform and methanol at a 2:1 volume ratio and the fluorescence intensity was evaluated with a spectrophotometer at 560 and 580 nm. The data were standardized to the negative control saline-treated animals. The biodistribution of drugs in the spinal cord and brain was using a slide scanner (VS120-L100, Olympus, Tokyo, Japan; 20x objective) to image tissue sections.

Histology assessment

Inflammation and neuron survival were assessed by cresyl violet (Nissl) staining, and scored in accordance with a previous study (Qin et al., 2014): 0, no cellular infiltration; 1, leukocyte infiltration only around blood vessels and meninges; 2, slight parenchyma infiltration with 1–10 leukocytes/slice; 3, moderate parenchyma infiltration with 11–100 leukocytes/slice; and 4, serious parenchyma infiltration with >100 leukocytes/slice. Neurons with smooth nuclei and cell bodies with adequate amounts of endoplasmic reticulum were counted in the anterior spinal horn. Three fields were randomly selected from each slice under bright-field to collect digital images using a Nikon TE-300 microscope.

Demyelination in the white matter tract was examined with a modified eriochrome cyanine R (ECR) staining protocol, according to a previous study (Han et al., 2010). In brief, the slides were thawed and dried in a slide warmer at 37 °C for 1–2 h, then treated with xylene at RT for 1 h and rehydrated with graded solutions of ethanol. Next, the slides were rinsed with caution in flowing tap water for 5 min, briefly in deionized water, and subsequently differentiated in 5% ferric ammonium sulfate for 5–10 min. After another brief rinse in deionized water, slides were dehydrated in graded solutions of ethanol, immersed in xylene, and sealed with a coverslip. The degree of demyelination was evaluated according to the following criteria described in a previous study (Wang et al., 2016): 0, integral white matter; 1, rare lesion of demyelination; 2, sparse demyelination; 3, affluent demyelination in perivascular or subpial areas; 4, massive demyelination in perivascular and subpial areas involving 50% of the spinal cord with leukocyte infiltration in the parenchyma; and 5, extensive demyelination in perivascular and subpial areas involving the entire spinal cord section with parenchyma infiltration by leukocytes.

Evaluation of BBB damage with Evans blue extravasation

At week 2 pi and under deep anesthesia with sodium pentobarbital (60 mg/kg, i.p.), the rats were infused with Evans blue dye (4 mL/kg body weight, 2% in 0.9% saline) through the right femoral vein for 5 min. After 2 h, the animals were subjected to perfusion with 300 mL saline through the left ventricle to remove any residual dye in the vascular system. To evaluate the permeability of the BBB, half of the motor cortex and spinal cord were collected and mechanically homogenized in 750 µL of N,N-dimethylformamide (Sigma, St. Louis, MO). The resulting suspension was kept in the dark at RT for 72 h and then centrifuged at 10,000 g for 25 min. The concentration of dye (represented as µg/g tissue) in the supernatant was analyzed using a spectrophotometer (Molecular Devices OptiMax, San Jose, CA) at 610 nm, and calculated with a standard curve plotted using known concentrations of the dye. The remaining tissue was cut into 20 µm sections for digital imaging.

Assessment of edema

The water content of the brain was assessed by comparing the wet weight and dry weight at 2 and 4 weeks pi. Under deep anesthesia, the rats (n = 3/timepoint/group) were decapitated and the brain tissue was weighed (wet weight), wrapped with pre-weighed aluminum foil and dried for 72 h in an oven at 60 °C. The brain was then re-weighed (dry weight). The water content of brain was measured according to the following equation (Jiang et al., 2014): brain water content (%) = [(wet weight – dry weight)/wet weight] × 100.

FG retrograde tracing

The retrograde tracer FG (Fluorochrome Inc., Denver, CO) was utilized to measure the spare extent of descending axons (n = 3/group). In brief, 3 d before sacrifice, the bilateral surface of the lumbar enlargement of the spinal cord was injected with 4% FG (0.5 µL per injection) through a glass micropipette driven by a Pneumatic PicoPump (WPI, Sarasota, FL). After sacrifice, the FG-labeled neurons with nuclei in transverse sections from the motor cortex were counted.

Cytokine quantification by ELISA

Peripheral blood samples harvested from each rat after 2 weeks of immunization (n = 3/group) were tested with the following ELISA Kit: To assess cytokine expression, plasma samples were incubated for 1 h at 37°Cin 96-well plates that were precoated with rabbit anti-interleukin (IL)-1 (1:100, BioLegend Inc., San Diego, CA), rabbit anti-IL-10 (1:100, BioLegend Inc., San Diego, CA), and rabbit anti-reactive oxygen species (ROS; 1:100, R&D Systems, Minneapolis, MN). Next, the samples were incubated with the secondary goat anti-rabbit IgG antibody (1:2000; Bio-Rad, Hercules, CA) at 37 °C for 60 min. The optical density (OD) of bound proteins was measured at 450 nm using a model 680 microplate reader (BioRad Laboratories, Corston, UK) and the results were analyzed using the GraphPad Prism version 4 software (GraphPad Prism Software, Inc., La Jolla, CA).

TEM

The TEM procedure was based on a previous study (Cai et al., 2021). In brief, samples of the spinal cord and cortex were first fixed with 2.5% glutaraldehyde, followed by immersion in 1% osmium tetroxide at 4 °C and rinsing three times with 0.1 M PBS. The samples were then dehydrated through graded ethanolsolutions for 5 min, followed by three changes of pure ethanol (10 min for each change). After two baths in 1,2-propylene oxide (PO; 15 min for each change), the samples were incubated with a mixture of PO and Epon (1:1) for 1 h, then incubated in pure Epon overnight at RT, and continuously embedded in pure Epon at 60 °C for 3 d. The Epon-embedded samples were cut into 90-nm sections with a diamond knife on an ultracut microtome (Leica EM UC7 Ultramicrotome) and harvested on 200-mesh copper grids that were stained with freshly filtered lead citrate (3%) and uranyl acetate (8%) in a Petri dish for 20 min. The grids were finally rinsed three times with deionized water and examined by TEM (Cai et al., 2021).

Immunofluorescence staining

Sample sections were incubated with many primary polyclonal rabbit antibodies at 4 °C for 24 h, including anti-CD4 (1:500; Abcam, Cambridge, MA), anti-CD45 (1:200; Abcam), anti-ionized calcium-binding adapter molecule (Iba-1; 1:1000, ProSci Incorporated, CA), anti-caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI), anti-myelin basic protein (MBP, 1:500, Abcam, Cambridge, MA), anti-nuclear factor κB (NF-κB; 1:500; R&D Systems, Minneapolis, MN), anti-cyclooxygenase-2 (COX-2; 1:1000; Neuromics, Minneapolis, MN), anti-zonulaoccludens-1 (ZO-1;1:500; Santa Cruz Biotechnology, Dallas, TX), anti-CD206 and anti-CD86 (1:1000; Neuromics), and anti-glia fibrillary acidic protein (GFAP; 1:200; Thermo Fisher, Waltham, MA). After rinsing three times with PBS, the sections were incubated with a secondary goat anti-rabbit IgG antibody (1:200; Invitrogen, Waltham, MA) at 37 °C for 1 h and mounted to slides with anti-fade mounting medium (Fluoromount-G^® anti-Fade, Southern Biotech, AL). Inactive antibodies were used as controls to verify the specificity of the antibodies of interest. Immunoreactive regions were randomly selected and analyzed using ImageJ software.

For experiments involving histological and immunohistochemical staining, five transverse sections from each animal and three fields of view from each slice were randomly examined with digital images. The histological results were analyzed for quantification by an investigator who was blinded to the experimental groups.

Western blotting

Under deep anesthesia, the animals were decapitated at 2 weeks pi (n = 3/group). The entire cerebral cortex and a 10 mm segment of the lumbar spinal cord were collected for western blotting. The primary polyclonalrabbit antibodies included anti-CD4 (1:1000; Abcam, Cambridge, MA), anti-CD45 (1:500; Abcam, Cambridge, MA), anti-Iba-1 (1:2000, ProSci Incorporated, Poway, CA), anti-NF-κB (1:800; R&D Systems, Minneapolis, MN), anti-caspase-3 (1:800; Cayman Chemical, Ann Arbor, MI), anti-MBP(1:800, AbCam, Cambridge, MA), anti-COX-2 (1:2000; Neuromics), anti-ZO-1 (1:500; Santa Cruz Biotechnology), anti-CD206 and anti-CD86 (1:2000; Neuromics), and anti-GFAP (1:800; Thermo Fisher, Waltham, MA). For the normalization of protein bands to a loading control, the membranes were rinsed with Tris-buffered saline containing Tween 20 (TBST), incubated with the internal control antibody: the primary rabbit anti-β-actin antibody (1:5000; Abcam), and re-incubated with peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000; Santa Cruz). Finally, protein bands on the film were examined by enhanced chemiluminescence (ECL). On some membranes, primary antibodies were omitted to serve as a negative control.

Statistical analysis

Data were expressed as mean ± standard deviation (SD) and were analyzed with SPSS 13.0 software (SPSS, Chicago, IL) by an individual blinded to the experimental groups. Intergroup differences were compared with two-way analysis of variance (ANOVA) and Tukey’s post-hoc test. The difference was considered statistically significant with a p value < .05. All graphs in this study were plotted using GraphPad Prism version 4.0 software (GraphPad Prism Software, Inc., San Diego, CA).

Results

C + A Compound improved clinical demonstrations in EAE rats

The clinical function scores indicated that MS symptoms appeared 7-9 d after immunization (clinical scores >2) in EAE rats of the vehicle treatment group. These rats quickly progressed to the acute stage (clinical scores 3–4) and peaked at 2 weeks pi (Figure 1(A)). The peak clinical scores in the vehicle group were significantly higher compared to the C + A group, nano-carrier C + A group, and fMLP-nano-carrier C + A group (all p < .05). The fMLP-nano-carrier C + A compound also showed a significant improvement compared to nano-carrier C + A and the C + A compound alone (p < .05, Figure 1(A)). Following the peak disease activity within the acute phase at 2 weeks pi, the clinical scores showed gradual recovery. At 4 weeks pi, the clinical scores in the fMLP-nano-carrier C + A group returned to a mean level of 2 (Figure 1(A)). Animals treated with C16 and nano-carrier C + A showed a similar disease course, but with an obvious delay. The fMLP-nano-carrier C + A compound also showed a significant improvement in functional recovery compared to the other treatment groups (p < .05, Figure 1(A)).

Figure 1. C + A, nano-carrier C + A, and fMLP-nano-carrier C + A improved the damaged electrophysiological functions, delayed the onset of motor-related symptoms, and reduced the disease severity in EAE rats. (A) The effects of treatment with different C + A compounds on clinical scores of rats. (B and C) The effects of treatment with different C + A compounds on c-SEP (B) and MEP (C). n = 10/group. *p < .05 vs. vehicle group. ^#p < .05 vs. C + A group. ^&p < .05 vs. nano-carrier C + A. C: C16 polypeptide (γ1 chain peptide of laminin-1); A: angiopoietin 1 (Ang-1); fMLP: N-formyl-methionyl-leucyl-phenylalanine; EAE: experimental autoimmune encephalomyelitis; c-SEP: cervical somatosensory evoked potentials; MEP: motor evoked potentials.

Furthermore, rats in the EAE vehicle group showed significantly longer latency of waveform which is related to the reduced speed of nerve conduction, and smaller wave amplitudes which is related to the number of surviving fibers, in both c-SEP and MEP recordings when compared to rats in the normal control group (Figure 1(B,C)). However, application of C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds significantly attenuated these changes (all p < .05, Figure 1(B,C)). Most notably, the fMLP-nano-carrier C + A compound was significantly better compared to the C + A compound regarding the latency and amplitude of the c-SEP recordings (p < .05, Figure 1(B)).

fMLP-nano-carrier C + A attenuated CNS inflammation and suppressed activated astrocytes to the largest extent

To determine the types of inflammatory cells involved in EAE, CD45, and CD4 antibodies were used to label extravasated T lymphocytes and leukocytes, respectively. The results indicated that both CD45+ and CD4+ cells were significantly higher in the CNS of rats in the EAE vehicle group (Figure 2, Supplementary Figure 1 for CD45+ cell qualification and Supplementary Figure 2 for CD4+ cell qualification) and the increase of CD45+ and CD4+ cells was significantly attenuated by C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds (p < .05, Figure 2(I–T), Supplementary Figure 1(A–J)) at both time points (2 and 4 weeks pi). In addition, fMLP-nano-carrier C + A was significantly different from C + A alone (p < .05, Figure 2(V), Supplementary Figures 1(L) and2(P)). T-lymphocyte infiltration and leukocyte extravasation were remarkably decreased in rats given C + A, nano-carrier C + A, or fMLP-nano-carrier C + A compounds, which verified the lower inflammatory scores (Figure 2(U), Supplementary Figure 1(K)). Early in EAE (2 weeks pi), a sizeable proportion of macrophages in the CNS were monocyte-derived (Figure 3(B)) as shown by the monocyte-specific marker CD14 and macrophage-specific marker CD68 double stain (Wang et al., 2021), while in later stages of EAE (4 weeks pi) the proportion of macrophages and monocytes were decreased. However, in both the early and late stages, the C + A, nano-carrier C + A, or fMLP-nano-carrier C + A compounds reduced CD14+ and CD68+ cells, with the fMLP-nano-carrier C + A compound resulting in the greatest reduction (Figure 3(K,L)).

Figure 2. Inflammation-induced infiltration in cerebral cortex and spinal cord of animals at 2 weeks after immunization with different treatments. (A–T) Representative images of staining with CD45+ (leukocyte marker) in spinal cord, posterior funiculus, anterior funiculus, and cerebral cortex. The perivascular cuffing formed by the infiltration of inflammatory cells surrounding blood vessels was shown in (H) by arrow. Scale bar =100 µm. (U) Quantitative analysis of inflammatory scores. (V) Quantification of CD45-labeled cells in different groups. a, p < .05 vs. vehicle group; b, p < .05 vs. C + A group; c, p < .05 vs. nano-carrier C + A group.

Figure 3. Effects of C + A compounds on monocytes labeled by CD14 (red) and macrophages labeled by CD68 (green) at 2 and 4 weeks post-immunization (pi). (A–J) Immunostaining with CD14 (red) and CD68 (green) in different groups at 2 and 4 weeks pi. (K and L) Quantification of CD14-labeled monocytes (K) and CD16 + CD68 double-labeled macrophages (derived from monocytes) (L). Scale bar = 100 µm. a, p < .05 vs. vehicle group; b, p < .05 vs. C + A group; c, p < .05 vs. nano-carrier C + A group; d, p < .05 vs. nano-carrier C + A group.

Furthermore, microglia-derived macrophages (labeled by Iba-1; Supplementary Figure 3(A–J,U)) and activated astrocytes (labeled by GFAP, Supplementary Figure 3(K–T,V)) in the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A groups were significantly reduced when compared to the EAE vehicle group. The fMLP-nano-carrier C + A group demonstrated the largest differences regarding these comparisons.

When compared to the normal control group, the serum levels of pro-inflammatory factors IL-1β and ROS were increased with significant differences in the EAE model (vehicle group Figure 4; p < .05), and the increases in IL-1β and ROS were attenuated by treatments with C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds (Figure 4(A,B)). In contrast, the anti-inflammatory factor IL-10 was significantly decreased in the EAE vehicle group compared to the normal control group, and this downregulation was reversed by treatment with C + A, nano-carrier C + A, or fMLP-nano-carrier C + A compounds (Figure 4(C)). When comparing the different compounds, the fMLP-nano-carrier C + A compound showed the greatest effect.

Figure 4. Effects of C + A compounds on the serum concentration of IL-1β, ROS, and IL-10 measured by ELISA. a, p < .05 vs. normal control group; b, p < .05 vs. vehicle group; c, p < .05 vs. C + A group; d, p < .05 vs. nano-carrier C + A group.

In addition, the expression of COX-2 and NF-κB, which are vital transcription factors involved in inflammatory signaling pathways, was significantly increased in EAE rats (p < .05, Supplementary Figure 4). The increased expression of COX-2 and NF-κB was significantly reduced by C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds (Supplementary Figure 4(U,V)). Moreover, western blotting also confirmed the immunostaining results of NF-κB, COX-2, CD45, CD4, Iba-1, and GFAP (Figure 5(A–F)). The fMLP-nano-carrier C + A compound demonstrated the largest effect concerningCD45, CD4, and COX-2 compared to the C + A and nano-carrier C + A compounds.

Figure 5. Effects of C + A compounds on the expression of NF-kB (a), COX-2 (B), CD45 (C), CD4 (D), Iba-1 (E) and GFAP (F) by western blot. a, p < .05 vs. normal control group; b, p < .05 vs. vehicle group; c, p < .05 vs. C + A group; d, p < .05 vs. nano-carrier C + A group.

C + A compounds alleviated demyelination and axon damage induced by EAE

The immunohistochemical labeling with ECR and MBP staining for myelination at both 2 and 4 weeks pi indicated that the EAE rats in the vehicle control group had large areas of demyelination (Supplementary Figures 5(B,G) and 6(B,G,L,Q)) when compared with rats in the normal control group (Supplementary Figures 5(A,F) and 6(A,F,K,P)). Treatment with C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds visibly decreased the areas of demyelination (Figure 6(V,X)). Furthermore, the fMLP-nano-carrier C + A treatment group exhibited greater areas of myelination (Figure 6(E,J,O,T)) compared to the C + A and nano-carrier C + A groups (Figure 6(V,X) p < .05). Moreover, the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A groups all had improved demyelination scores at both 2 and 4 weeks pi (Figure 6(U,W)), and the greatest improvement was found in the fMLP-nano-carrier C + A group (Figure 6(U,W)).

Figure 6. Effects of C + A compounds on demyelination at 2 and 4 weeks post-immunization. (A–T) Representative images for staining of MBP in spinal cord and motor cortex of different groups. (U and W) Quantification analysis of demyelination score. a, p < .05 vs. vehicle group; b, p < .05 vs. C + A group; c, p < .05 vs. nano-carrier C + A group, and (V, X) MBP + area in different groups. a, p < .05 vs. normal control group; b, p < .05 vs. vehicle group; c, p < .05 vs. C + A group; d, p < .05 vs. nano-carrier C + A group. Scale bar = 100 µm.

The TEM results indicated that the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds prevented demyelination induced by EAE. Compact and dense myelin without signs of axonal damage was observed in TEM samples from the normal control group (Figure 7(C) at low magnification and (D) at high magnification), while EAE vehicle-treated animals presented with a loosened, wobbly, and unfastened myelin sheath, indicative of demyelination (Figure 7(G) at low magnification and (H) at high magnification). The treated groups also had compressed, solid myelin (Figure 7(K,O,S) at low magnification and (L,P,T) at high magnification). The myelin sheaths showed a more compact structure compared to the EAE-vehicle group, but still had signs of unwrapping when compared to normal healthy control animals. Of all the treated groups, the greatest improvement was found in the fMLP-nano-carrier C + A group.

Figure 7. Representative images of electron micrographs showing that C + A compounds reduced demyelination, axonal loss, and neuronal apoptosis. A–C,E–G,I–K,M–O,Q–S, Scale bar =2 μm. D, H, L, P, T), Scale bar =1 μm.

The western blotting results demonstrated similar changes of active MBP in each group as compared to immunofluorescence staining (Figure 8(C,D)).

Figure 8. Effects of C + A compounds on the expression of MBP (A,B), caspase-3 (C,D), and ZO-1 (E,F). left panels show western blots of MBP, capsase-3, and ZO-1 in different groups; right panels show quantitative analysis of the expression of capsase-3, MBP, and ZO-1 in different groups. a, p < .05 vs. normal control group; b, p < .05 vs. vehicle group; c, p < .05 vs. C + A group; d, p < .05 vs. nano-carrier C + A group.

C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds reduced neuronal apoptosis in EAE rats

Under TEM, apoptotic cells had a dark, shrunken appearance with a condensed nucleus, and crescent margination of chromatin against the nuclear envelope (Figure 7(E)) when compared to the normal cells that showed well-defined nuclei with dispersed chromatin (Figure 7(A)). In the rescued animals, the ultrastructural morphology of the nucleus more closely resembled normal control samples (Figure 7(I,M,Q)). To further examine the effect of C + A on neuronal apoptosis, active caspase-3, an enzyme involved in the execution of apoptosis, was examined. Western blotting results indicated that the enhanced activity of caspase-3 in EAE rats was significantly attenuated in the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A groups, which was similar to the results obtained by immunofluorescence staining (Figure 8(A,B)). In addition, the immunostaining results indicated that caspase-3 was upregulated in the population of large multipolar motor neurons in the anterior horn of the spinal cord and in the pyramidal motor neurons of the precentral gyrus in EAE vehicle treated rats (Figure 9(B,G)) compared to rats in the normal control group (Figure 9(A,F)). The upregulation of caspase-3 was visibly reversed in the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A groups (Figure 9(C–E,H–J). Nissel staining also showed visible neuron loss in the spinal cord (Figure 9(L); Supplementary Figure 6 (B) and brain cortex in rats from the EAE vehicle group (Figure 9(Q); Supplementary Figure 6G) when compared with rats from the normal control group (Figure 9(K,P); Supplementary Figure 6 (K,A,F). Compared to the EAE vehicle group, there were significantly more neurons in the anterior horn of the spinal cord and the motor cortex in rats from the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A groups (Figure 9(M–O,R–T,V); Supplementary Figure 6(C-E,H-J,K). The fMLP-nano-carrier C + A compound showed more prominent changes than the C + A and nano-carrier C + A compounds (Figure 9(U,V); Supplementary Figure 6 (K). Furthermore, these results were consistent with the preservation of FG-labeled brain neurons (Supplementary Figure 7).

Figure 9. C + A compounds decreased apoptosis induced by EAE and increased neuronal survival in EAE rats at 2 weeks post-immunization. (A–J) Representative images of caspase-3 staining of spinal cord and cerebral cortex in different groups. (K–T) Representative images of Nissel staining of spinal cord and cerebral cortex in different groups. (U,V) Quantitative analysis of caspase-3 + cells (U) and survival neurons (V) in spinal cord and brain from different groups. a, p < .05 vs. normal control group; b, p < .05 vs. vehicle group; c, p < .05 vs. C + A group; d, p < 005 vs. nano-carrier C + A group. Scale bar = 100 µm.

C + A compounds reduced vascular leakage and improved BBB permeability induced by EAE

As shown in Figure 10, vascular leakage and BBB permeability were increased in EAE vehicle treated rats when compared to rats in the normal control group (p < 0.05). However, the increased blood vessel leakage and BBB permeability induced by EAE were significantly attenuated by the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds (all p < 0.05). Furthermore, the fMLP-nano-carrier C + A compound had significantly more improvement compared to the C + A and nano-carrier C + A compounds (Figure 10(F–G)). The TEM results indicated that the EAE model animals exhibited edema with enlarged perivascular spaces (Figure 7(F)), while normal control blood vessels appeared with healthy perivascular spaces (smaller than 2 µm, Figure 7(B)). Perivascular microcavities were identified in blood vessels of treated MS model animals, which also showed enlarged perivascular spaces (Figure 7(J,N,R)). However, all these perivascular spaces were much smaller compared to the spaces seen in the vehicle group. Furthermore, the tight junctions (immunofluorescence labeling of ZO-1, arrow in Supplementary Figure 8) between endothelial cells (ECs) were absent in EAE rats of the vehicle group (Supplementary Figure 8(B,G)) but were present in EAE rats treated with C + A, nano-carrier C + A, or fMLP-nano-carrier C + A compounds. The tight junctions in the fMLP-nano-carrier C + A group had better structure compared to the C + A, and nano-carrier C + A groups (Supplementary Figure 8(K)). These results were comparable to the ZO-1 western blotting results in each group (Figure 8(E,F)).

Figure 10. C + A compounds reduced the permeability of BBB and vascular leakage. (A–E) Representative staining of Evans blue in different groups. Scale bar =100 µm. (F) Quantification of EB. (G) Quantification of brain water content (%). a, p < .05 vs. normal control group; b, p < .05 vs. vehicle group; c, p < .05 vs. C + A group; d, p < .05 vs. nano-carrier C + A group.

fMLP-nano-carrier C + A compound better suppressed the M1 phenotype and promoted the M2 phenotype of microglia compared to C + A and nano-carrier C + A

Immunofluorescence staining of the spinal cord and brain tissues indicated that the C + A, nano-carrier C + A, and fMLP-nano-carrier C + A compounds significantly upregulated the expression of CD206 (marker for M2 phenotype), which was down-regulated in the EAE vehicle group. However, the compounds inhibited the expression of CD86 (M1 phenotype marker), which was up-regulated in the EAE vehicle group (Figure 11(A–O)). The western blotting results indicated that the fMLP-nano-carrier C + A compound was better at downregulating CD86 and upregulating CD206 compared to the C + A and nano-carrier C + A compounds (Figure 11(Q–T)).

Figure 11. Immunofluorescence staining (A–P) and western blotting (Q–T) showing the effects of C + A compounds on CD206 (blue) and CD86 (green) expression in microglia. a, p < .05 vs. normal control group; b, p < .05 vs. vehicle group; c, p < .05 vs. C + A group; d, p < .05 vs. nano-carrier C + A group.

Nano-carrier C + A and fMLP-nano-carrier C + A penetrated parenchyma of CNS better than C + A alone

The biodistribution analysis indicated that the C + A compound could pass though the BBB (and blood–spinal cord barrier) to enter the parenchyma of the cortex and spinal cord (Figure 12(A–L)). However, the C + A compound delivered by the nano-carrier (Figure 12(B,E,H,K)) or the fMLP-nano-carrier (Figure 12(C,F,I,L)) was able to diffuse in a larger area in the brain compared to C + A alone (Figure 12(A,D,G,J)). The concentration of the C + A compound in the blood reached a peak at 6 h post-injection and was significantly enhanced by the nano-carrier or fMLP-nano-carrier, and the greatest effect was seen with the fMLP-nano-carrier (Table 1). Moreover, at 12 h post-injection, the C + A mixture was mainly distributed in the spinal cord and brain (target organs), as well as the kidney (excretory organ). The concentration of the compound in these organs was enhanced by the nano-carrier, and even further enhanced by the fMLP-nano-carrier (Table 2).

Figure 12. fMLP-nano-carrier had the greatest Ability to pass through the blood-brain/spinal cord barrier in the CNS: labeled C16 (rhodamine, red) and Ang-1 (Cy5, blue).

Table 1. Blood concentrations of drugs at different time points (ng/mL, mean ± SD).

			Compound
	C + A		Nano-carrier C + A		fMLP-nano-carrier C + A
Time (h)	C16	Ang1	C16	Ang1	C16	Ang1
0.5	26.47 ± 5	22.18 ± 2.9	40.08 ± 3.37*	22.47 ± 1.1	122.55 ± 6.02*^#	79.69 ± 3.28*^#
1	170 ± 5.54	26.43 ± 1.09	433.32 ± 35.36*	28.8 ± 3.5	480.48 ± 38.8*^#	93.16 ± 3.8*^#
2	175.14 ± 8.11	33.005 ± 1.36	528.9 ± 41.14*	94.08 ± 8.5*	843.28 ± 66.12	102.64 ± 4.18*^#
4	418.15 ± 9.9	37.74 ± 1.55	715.22 ± 53.15*	152.04 ± 36.19*	850.51 ± 61.72	191.85 ± 7.81*^#
6	505.99 ± 9.93	61.97 ± 2.53	997.99 ± 71.04*	160.83 ± 26.54*	1677.82 ± 111.38*^#	215.95 ± 26*^#
12	122.37 ± 7.15	58.03 ± 2.37	725.72 ± 53.83*	67.97 ± 7.8*	499.58 ± 9.53*^#	69.7 ± 8.78*^#
24	90.6 ± 3.17	20.5 ± 3.94	420.55 ± 45.3*	33.25 ± 1.37*	493.93 ± 9.31*^#	35.25 ± 2.67*^#
48	13.97 ± 1.7	7.97 ± 1.35	29.94 ± 6.93*	6.76 ± 2.4	314.57 ± 27.8*^#	6.93 ± 25.7

*p < .05 vs. C + A group. ^#p < 0.01 vs. nano-carrier C + A group.

Table 2. In vivo distribution of C + A compounds at 12 h post-injection.

Organ	C + A (ng/mL)	Nano-carrier C + A (ng/mL)	fMLP-nano-carrier C + A (ng/mL)
Kidney	720.31 ± 33.58	856.74 ± 38.55	751.53 ± 266.62
Brain	1352.63 ± 60.55	1676.08 ± 93.64*	1975.38 ± 88.19*^#
Spinal cord	1722.64 ± 48.82	1886.51 ± 46.64*	2289.62 ± 42.59*

*p < .05 vs. C + A group. ^#p < .01 vs. nano-carrier C + A group.

Nano-carrier C + A and fMLP-nano-carrier C + A did not stimulate the vascular wall or cause functional impairment on metabolic organs

The purpose of the vascular wall studies was to assess the stimulation on blood vessels following the different treatments. Since rabbit marginal ear veins are large, the tests are easier to perform and allows for more accurate observations compared to using rat veins. Importantly, many previous studies concerning drug stimulation tests have used rabbit marginal ear veins. In this study, vascular wall stimulation was tested by injecting the C + A compounds into the marginal ear veins of New Zealand rabbits and tail vein of Lewis rats. The results showed that the C + A compound could induce hemorrhages and blood clots in the marginal ear vein as well as regional infiltration surrounding the vessel (Figure 13(A,B)), while the C + A compound delivered by nano-carrier or fMLP-nano-carrier did not induce obvious hemorrhaging or infiltration (Figure 13(D,E)). Additionally, stimulation tests were performed in the tail vein of rats, and the results were consistent with the results from the rabbit marginal ear vein tests (Figure 13(C,F)). Moreover, the functions of the liver (Table 3) and kidneys (Table 4) were not impaired by the C + A compound alone or when transported by the nano-carrier or fMLP-nano-carrier, except for a mild albumin and albumin/globulin ratio decrease, which might be due to the poor body condition of EAE animals (Table 3). In addition, ketone bodies, blood cells, and protein were detected in the urine of rats in the vehicle group, but not in rats from the -nano-carrier C + A and fMLP-nano-carrier C + A groups (Table 4).

Figure 13. The effects of different C + A compounds on vascular injection stimulation. (A–C) C16 + Ang-1 group (A and B marginal ear veins of rabbits; C tail vein of rats); (D) nano-carrier C + A group; (E and F) fMLP-nano-carrier C + A group (E) in marginal ear veins of rabbits and (F) tail vein of rats). Scale bar = 100 µm.

Table 3. Effects of different C + A compounds on hepatic function.

Items (normal range)	Normal control	Vehicle	C + A	Nano-carrier C + A	fMLP-nano-carrier C + A
AST (151.3–221.5 u/L)	N	N	N	N	N
ALT (70.42–73.45 ul/L)	N	N	N	N	N
ALP (72.1–74.09 u/L)	N	N	N	N	N
TP (51.9–59.04 g/L)	N	N	N	N	N
ALB (39.97–41.36 g/L)	N	↓ (28.9 ± 1.11 g/L)^*&	↓ (29.7 ± 1.98 g/L)^*#	↓ (29 ± 1.74 g/L)^*#	↓ (20.7 ± 1.03 g/L)^*#&
A/G (1.95–2.45)	N	↓ (0.89 ± 0.13)^*&	↓ (0.94 ± 0.08)^*#	↓ (0.96 ± 0.12)^*#	↓ (0.97 ± 0.08)^*#&
TBIL (0.9–1.3 µM)	N	↑ (2 ± 0.04 µM)^*&	N	N	N
DBIL (0.28–0.4 µM)	N	↑ (0.5 ± 0.01 µM)^*&	N	N	N
ALP (374–404 IU/L)	N	N	N	N	N

*p < .05 vs. normal control group. ^#p < .05 vs. vehicle. ^&p < .05 vs.C + A group.

Table 4. Effects of different C + A compounds on renal function.

Items (normal range)	Normal control	Vehicle	C + A	Nano-carrier C + A	fMLP-nano-carrier C + A
BUN (3–8 mM)	N	↑(16.89 mM)*	N	N	N
BUA (59–120 µM)	N	N	N	N	N
Cr (86–120 µM)	N	N	N	N	N
Ketone bodies	–	+	+	+	+
Blood	–	++++	+	+	+
Protein	–	+++	+	+	+

*p < .05 vs. normal control group. 

Discussion

In this study, the effects of different delivery strategies of C + A on MS were investigated using an EAE model in rats. The results indicated that using a fMLP-nano-carrier to deliver C + A had advantages over other delivery strategies to inhibit the neuropathological changes induced by EAE.

MS is believed to be initiated by the infiltration of myelin-specific T cells and subsequent recruitment of inflammatory leukocytes to the CNS. In the pathogenesis of EAE, leukocytes also produce cytokines, promote the breakdown of the BBB, and facilitate parenchymal CNS inflammation (Ma et al., 2010). Generation of CD4+ T cells reactive to myelin proteins is a pathological hallmark of MS and exacerbates the inflammatory micro-environment. During the early phase of EAE, infiltrating monocytes and monocyte-derived macrophages contribute to T lymphocyte recruitment, especially CD4+ T cells, into the CNS, resulting in neuronal demyelination (Wang et al., 2021). The prerequisite for leukocytes to enter the CNS is the disruption and inflammation of the BBB, which can be initiated by ROS (Pierson et al., 2018). Several agents that aim to suppress the infiltration of leukocytes and the activation of macrophages/microglia in the CNS have been developed as potential therapies for MS (Cai et al., 2018). The effects of the compounds of C16 and Ang-1 on CNS inflammatory-related diseases have been confirmed using a variety of animal models (Han et al., 2010; Aarts et al., 2017; Chen et al., 2019; Nakazato et al., 2020; Fu et al., 2021). Both at the peak (2 weeks pi) and later stage (4 weeks pi) of disease course, the C + A, nano-carrier C + A, or fMLP-nano-carrier C + A compounds reduced the number of CD14 + monocytes, CD68+ macrophages, CD45+ leukocytes, and CD4+ T lymphocytes, with the largest differences seen in the fMLP-nano-carrier C + A treatment group.

In acute EAE models (vehicle group), the CNS demonstrated high leukocyte infiltration that occurred rapidly throughout the brain and spinal cord. At the same time, activated microglia produced a proinflammatory milieu, stripped off myelin from neuronal axons, and attracted activated T-lymphocytes that augmented the destruction of myelin. This study demonstrated that the C + A compounds significantly suppressed the widespread leukocyte infiltration in perivascular and parenchymal areas and reduced the activation of microglia induced by EAE. Activated microglial cells include two phenotypes: neurotoxic M1-like (labeled by CD86) and neuroprotective M2-like (labeled by CD206) cells. M1-like microglia can establish a microenvironment that is detrimental to neurons by producing inflammatory ROS, while M2-like microglia can establish a beneficial microenvironment for neurons by secreting neurotrophic factors and anti-inflammatory mediators (Fu et al., 2020). The results of this study indicated that the C + A compounds could activate the M2 phenotype and suppress the M1 phenotype of microglial cells.

Following inflammation in the CNS, astrocytes can be activated through hypertrophy and/or proliferation, resulting in reactive astrogliosis. Generally, activated astrocytes prefer to accumulate within and at the margins of demyelinated regions, produce proinflammatory cytokines and chemokines, present antigens to T-lymphocytes, and participate in the inflammatory response during the pathological development of MS and EAE (Gaillard et al., 2012). As a transcription factor mediating CNS inflammation, NF-κB is positively involved in the transcriptional regulation of multiple genes, such as those encoding pro-inflammatory cytokines (like IL-1β) and proinflammatory enzymes (like COX-2), which are critical regulators of the inflammatory process and promote chronic inflammation (Jiang et al., 2014). Chronic inflammation induced by the reaction of astrocytes could produce glial scars at the site of lesions, which may further suppress the regeneration of axons. Data presented herein revealed that the C + A compounds could decrease the GFAP + reactive astroglia, and suppress the production of ROS, NF-κB, IL-1β, and COX-2 in serum or tissue, while upregulating anti-inflammatory IL-10 in serum. Importantly, reduced inflammatory cell infiltration and activation may be related to a more rapid recovery of locomotor function.

As targets of the C + A compound, the expression of Tie2 and integrin αVβ3 was significantly upregulated in vascular ECs (Jiang et al., 2014). Previous studies indicated that Ang-1 may be involved in maintaining EC integrity of blood vessels, promoting survival of ECs, and preventing vascular leakage through the Ang-1-Tie2 system (López-Vales and David, 2019). However, it can also prevent inflammatory cells from adhering together through the inhibition of adhesion factors, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Liang et al., 2015). The major machinery of C16 functions in a competitive manner to disturb the binding of αVβ3 integrin and inflammatory cells to the endothelium, and ultimately attenuate the transmigration of leukocytes (Jiang et al., 2014). Although Ang-1 and C16 have some overlapping functions, together the C + A compound has been shown to prevent edema in the CNS and improve the function of tight junctions between ECs in the CNS of EAE animals. In the vehicle group of EAE rats, there was reduced ECR staining and MBP immunoreactivity, and the myelin sheath with abundant loose, fused, and fragmented spires, suggesting extensive demyelination. The CNS is susceptible to axonal injury, which can result in defective conduction and functional loss. In addition, electrophysiological recordings also indicated one reduced amplitude and delayed latency of c-SEP responses (to sensory stimuli) and c-MEP responses (for motor function), suggesting focal demyelination and axonal damage.

Myelin integrity and neuronal survival can be attributed to an improved micro-environment. In the C + A group, the activity of caspase-3, one critical enzyme involved in apoptosis in mammals, was reduced. Together with the findings from electron micrographs showing the alleviation of apoptotic signs in nuclei, these results suggested improved neuronal survival through reduced inflammation, improved micro-environment, and alleviated injury secondary to the induction of the immune response. Accordingly, treatment with the C + A compound delayed the appearance of motor-related symptoms, attenuated the clinical scores of EAE at its peak, reduced axon demyelination, and increased neuronal survival. These changes were demonstrated by the reduced amplitude and decreased latency of the waveforms in the electrophysiological testing.

The acute EAE model of Lewis rats in-primed by GPSCH is a self-recovery model, in which the clinical scores of the animals will gradually decrease following the acute phase peak (at 2 weeks pi). In this study, the clinical scores of each group (aside from the normal control group) all exhibited a downward trend following the peak stage. Although animals in the three treatment groups displayed a similar disease course as the vehicle control group, the C + A, nano-carrier C + A, or fMLP-nano-carrier C + A compounds all clearly suppressed the clinical scores when compared with the vehicle group at the same time point. Clinical scores, inflammatory markers, demyelination, and neuronal death at 4 weeks pi showed that although the peptides were administered for 14 d, the neuroprotective effects were sustained until the later stage. This observation suggests that these peptides may have long lasting effects.

In general, therapeutic efficacy and systemic reactions can be improved by increasing focal drug concentrations at the target diseased area/organ. Therefore, developing a novel system for target-delivery of drugs to a specific area/organ is an important endeavor and of great interest to the entire pharmaceutical community (Liang et al., 2015). Nanoliposomes have been used as a carrier to prepare drugs that are difficult to dissolve and to improve drug solubility and biocompatibility, thus improving the effect of treatment (Hu et al., 2020). One previous study proposed that the chemoattractant fMLP is involved in inflammation reactions to defend against microbial infections in a host (Hu et al., 2020). As the first discovered leukocyte chemotaxis peptide, which is composed of formyl methionine, leucine, and phenylalanine, fMLP can bind to formyl peptide receptors expressed on the surface of target cells such as macrophages and can be well-tolerated when administered intravenously (Cui et al., 2002). A previous study has suggested that fMLP might directly lead to an accumulation of neutrophils in the blood and drug delivery efficacy can be enhanced by the use of a novel fMLP-modified liposome (fMLP-LIP) (Qin et al., 2014). In this study, although C + A, nano-carrier C + A, and fMLP-nano-carrier C + A all demonstrated therapeutic effects, the fMLP-nano-carrier C + A achieved the best results, suggesting that the fMLP-nano-carrier could breakthrough certain limitations on drug applications of the C + A compound regarding the treatment of MS and EAE.

Regarding biosafety, the data from this study revealed that the fMLP-nano-carrier C + A compound had no evident impairment on the liver or kidneys, two important organs for metabolism and clearance. The weakness and moribund conditions at the peak of EAE could lead to the exhaustion of important organs. Regarding hepatic function, albumin and the albumin/globulin ratio decreased while total bilirubin and direct bilirubin increased in the vehicle group, and these changes might be due to the poor body condition of EAE animals. In contrast, there was no remarkable functional impairment of the liver by C + A compounds except a mild albumin and albumin/globulin ratio decrease, which might be due to the improvement of the whole-body condition of C + A-treated EAE rats. Concerning renal function, the C + A compounds alleviated the presence of protein, blood cells, and ketone body in the urine. These data suggested that potential side effects caused by the C + A compounds were relatively small.

In summary, the results of this study indicated that the C + A compounds attenuated pathological inflammation-related neurological changes induced by EAE and the fMLP-nano-carrier C + A demonstrated the most pronounced effects. In addition, the C + A compounds had minimum adverse effects on the liver and kidneys. These results suggested that the fMLP-nano-carrier C + A has potential as a therapeutic strategy for MS.

Author contributions

SH and WJ conceived and designed the study; XXF, HHC, QH, and HJ carried out the experiments; XXF and HHC performed data analysis and interpreted the data; SH and HYC wrote the initial draft. All authors have reviewed and approved the final manuscript.

Institutional review board statement

Experiments were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, with approval from the Animal Ethics Committee at Zhejiang University (NO.25327) and have adhered to the ARRIVE guidelines. Since the animals need to be taken tissue at 2 and 4 weeks two time point, both fresh tissue used for WB and fixed tissue for histological assay were needed, and some animals might dead during experiment, 20 animals were arranged in each group. The rats were placed in a temperature- and humidity-controlled chambers. Manual bladder emptying was performed at least three times daily when the clinic score of animal reached 4. Animals were sacrificed under deep anesthesia with sodium pentobarbital (40 mg/kg i.p.). This study took 106 Lewis rats (100 for observing the effects of different drugs delivery on MS treatment and 6 for detecting of vascular wall stimulation in different groups).Six male adult health rabbits were taken also for detecting of vascular wall stimulation.

Acknowledgments

The authors thank Medjaden Inc. for helping in editing and proofreading the manuscript.

Disclosure statement

The authors declared that there was no conflict of interest.

Data availability statement

The supporting data for the results of this study can be obtained by contacting the corresponding author under reasonable request.

Additional information

Funding

This study received support from the National Natural Science Foundation of China (81971069) and Science and Technology Planning Project of Jinhua City, PR China (2021-3-149).