Nanocarrier characteristics
One of the most challenging issues in pharmaceutics is defining an efficient particle size to recover SCI. Recent decades have witnessed an explosive growth in the design and delivery of nanomedicines. In this regard, FTY-nanocarriers at two distinct particle sizes were prepared and compared, and their therapeutic efficacy was investigated in an SCI rat model. We assumed that encapsulation of FTY-720 into a nanoemulsion drug delivery system probably promotes neuronal survival and functional reconstruction of this powerful immunomodulatory medicine.
DLS was performed to study particle size and Zeta potential of stable nanocarriers. DLS results showed that nanocarriers synthesized by stirring and ultrasound methods had particle sizes of 60 and 195.5 ±7.78 nm, respectively (P <0.001). Meanwhile, they showed that monodisperse nF60 and nF190 nanocarriers had the PDI value of 0.1 and 0.2, respectively (Fig. 1a and b). In other words, low energy emulsification method produced larger nanocarriers compared to high-energy methods of ultrasound. However, both methods at an optimized oil, surfactant, and co-surfactant ratio make monodisperse nanoemulsions.
ζ-potential data related to nF60 and nF190 showed a slightly negative surface charge. ζ-potential of nF60 and nF190 nanocarriers were -9.98 ± 0.15 mV and -10.58 ± 0.35 mV, respectively. Their ζ-potential had significantly difference together (p = 0.0198). Smaller nanocarriers showed less negative ζ-potential than larger nanocarriers. However, both showed ζ-potential around -10 mV.
SEM micrographs revealed that both nF60 and nF190 nanocarriers had spherical morphology with uniform size and homogeneous distribution without agglomeration (Fig. 1c and d). Environmental conditions such as temperature, ionic strength influence the curvature of apolar spontaneous nanoemulsion. It might be said that the heating produced by the ultrasound method reduces the particle size of nanocarriers with more homogeneous particles (PDI: 0.1) through decreasing viscosity and interfacial tension between oil and water phases. However, cooling following the high energy method reduces particles' velocity and enhances oil phase viscosity 18.
NSC viability and LDH release
NSCs viability was evaluated in the face to the nanocarriers and FTY-720 at 10 ng/ml concentration. Results indicated that there was no significant difference between cell viability of nanocarriers together and compared to the bulk drug (P > 0.05). However, the control group had significantly less cell viability than the nanocarriers and bulk FTY-720 (P< 0.001). Nevertheless, both nanoemulsions manifested higher cell viability as compared to the control group. It seems that FTY-720 can significantly enhance NSCs viability and may be considered as a promising drug for neurogenesis (Fig.2a).
There is just one report, belonged to our group, associated with the dependency of neuro-toxicity to drug nanocarriers' particle size. There are some other reports related to the effect of nanocarriers on different cell types, such as fibroblast. For example, mesoporous silica NPs with a particle size of 250 nm induced higher endothelial toxicity than 30 nm NPs in part through the mitophagy mechanism 19. Moreover, Tavakol et al. reported that larger curcumin nanocarriers induce higher fibroblast cell viability than smaller nanocarriers with a particle size of approximately 60 nm through the down expression of Bax and NFκB genes 20.
Earlier, we showed that small nanocarriers exhibited higher cellular viability than larger nanocarriers in neural BE(2)-M17 cells. This data is contrary to our recent results and is due to different cell types. Therefore, to neurotoxicity investigation, the cell type is critical, and we cannot refer an outcome to other cell types. Although nanocarriers with a particle size of 60 nm may be considered a candidate particle size in dopaminergic cells, larger nanocarriers of 190 nm will act as the preferred particle size for SCI recovery. Besides, it has been shown that silica NPs at the range of 200 nm exhibits significantly higher neuronal viability than small NPs (50 nm) through the calcium perturbation and apoptosis mechanisms 21. However, in accordance with our results, Prabhu et al. demonstrated that copper NPs with particle sizes of 40, 60, and 80 nm did not induce a significant impact on the cell viability of DRG neurons 22. Besides, Coelho et al. reported the anti-apoptosis mechanism of FTY-720 to enhance oligodendrocytes viability 23. This data was similar to our study in that FTY-720 had increased neural cell viability compared to the control group.
To further study the effect of nanocarriers and bulk FTY-720 on NSCs, LDH release as a marker of necrosis and cell membrane damage was investigated. Results showed that smaller nanocarriers exhibit more serious cell membrane damage to NSCs and produce higher LDH release than the larger nanocarrier (P< 0.01). At the same time, there was no significant difference between other groups (P> 0.05). In other words, it seems that larger nanocarriers induced minor NSCs membrane damage, while there was no significant difference between cell membrane damage of smaller nanocarriers and bulk FTY-720 (P> 0.05) (Fig.2b). To sum things up, it seems that although both nanocarriers induced significantly high cell viability in NSCs, smaller nanocarriers caused higher NSCs membrane damage compared to the larger nanocarrier with 190 nm particle size. Prabhu et al. disclosed that small and large copper NPs of 40 and 80 nm did not significantly induce LDH release from DRG neurons, and notably, these NPs significantly induced higher LDH release compared to the control group 22. Moreover, Gillespie et al. reported that although both fine and ultrafine particles induced apoptosis in the neural cell, ultrafine particles induced more apoptosis of neural cells than fine particles 24. This finding was in good agreement with our results that larger nanocarriers induced less cell membrane damage of NSCs.
BBB score
Nanocarriers and FTY-720 were directly injected into the lesion to investigate their effects on the SCI model. In vivo SCI models were exerted on Wistar male rats employing the weight drop contusion model. Blunt injury models, including weight drop, represent human injuries and can efficiently study secondary damages 3. Herein we choose local administration to transport the medicine across the blood-spinal cord barrier directly to the lesion area. Local delivery significantly decreases systemic administration side-effects and effectively eliminates the risk of potential exposure and toxicities within the non-targeted organs 14.
To assess motor function recovery, BBB open-field locomotor rating scales were carried out in contusion rat models received local FTY- nanocarriers and bulk drug during six weeks post-injury (Fig. 2c). In the first four weeks, no significant difference was detected among different groups. However, from week four, nanocarriers and bulk FTY-720 started to show a gradual rise in BBB score compared to the control group. Finally, bulk FTY720 and nanocarriers induced an improved motor hind limb function compared to the control group (P< 0.001). There was no significant difference between the motor neuron recovery of nanocarriers and free FTY-720 (P> o.o5). These findings were in accordance with MTT assay data. It seems that NSC viability has a direct impact on the potential of nanocarriers to enhance motor neuron recovery. To further investigate the effect of nanocarriers on SCI rats, bladder reflux, body and muscle weights were evaluated.
Return of bladder reflex
Impaired bladder function is another incapacitating consequence of SCI in humans and animals. The degree of SCI severity is correlated with bladder dysfunction 9. In other words, the return of bladder reflex was considered as a sign of improvement and recovery. As shown in Fig. 3a, nF190 nanocarriers exhibited a noticeable faster regain of spontaneous bladder function compared to nF60 (P<0.01), free drug (P< 0.05), and the control group (P<0.001). There was no significant difference between the return of bladder reflux in nF60 and free drug (P> 0.05). Based on these results, although larger nanocarriers could enhance motor neuron recovery at the score of nF60 and free drug, they positively impacted bladder reflux, which is very important in SCI patients. Kangmin et al. demonstrated that daily IP injections of FTY-720 for four weeks following a contusion model in a Long-Evans hooded rat model significantly enhanced functional outcomes and bladder recovery 9. Moreover, it has been shown that FTY-720 regulates detrusor muscle tone and preserves the integrity of vessels by acting on endothelial cells 25.
Bodyweight changes
Bodyweight changes were recorded weekly as an indicator of general health and recovery 9. The weight of animals was recorded each week until the sacrifice (Fig. 3b). There was a marked decline in all groups' body weight in the first week after surgery. All animals' weight was increased upon the time, as expected. However, this increment trend was more significant in nanocarrier treated groups (P< 0.001). By the time, the body weight of rats treated by nanocarriers was significantly enhanced compared to the control group and free drug on four weeks. nF190 induced significantly higher body weight than the nF60 on 42 days post-treatment (p< 0.001).
Meanwhile, nF60 significantly influenced higher bodyweight than the free drug (P< 0.001). Based on these findings, it seems that although there was no significant difference on motor neuron recovery of nanocarriers and free drugs, gained bodyweight and bladder reflux as the markers of health and recovery in rats have been enhanced using nF190 and nF60. Eventually, the return of bladder reflex in SCI rats has positively impacted bodyweight and general recovery.
Gastrocnemius and soleus muscle mass
Skeletal muscle as an endocrine organ 26 is associated with inflammation. Therefore, inflammation is leading to muscle atrophy and reduced satellite cells 27, 28. Muscle atrophy leads to metabolic disorders in SCI patients 29. In this study, gastrocnemius and soleus muscle were dissected and weighed after perfusion on the 42nd day. Results showed no significant difference between the gastrocnemius weights of the groups (P= 0.0982). There is some difference between the two types of gastrocnemius and soleus muscles in which gastrocnemius is predominantly glycolytic muscle while soleus is more largely slow‐twitch muscle 30. At the same time, soleus muscle mass was higher in nF190 (p<0.01) and nF60 (p<0.05) compared to the control group. nF190 showed significantly higher soleus weight compared to the free drug (p<0.05) (Fig.3c). Based on these findings, it might be said that larger nanocarriers can diminish the soleus muscle atrophy. Ormond et al. showed greater soleus muscle weight is correlated with better hind limb functional recovery 31. Waterson et al. showed a regulatory effect of FTY720 on detrusor muscle tone in a rabbit model 32. Graham et al. showed that SCI induces the up-regulation of IL‐6, TNFα, and p53 in soleus muscle while IL‐6 and TNFα return to baseline in a short period 30. However, the return of inflammatory cytokine to baseline happens in rodent 33, and they cannot undergo senescence and may not translate to humans. In other words, in human may inflammatory cytokines following SCI promotes muscle to cellular senescence. Since FTY-720 reduces the up-regulation of pro-inflammatory cytokines such as IL-17A, IL-1, IL-6, and TNFα 29, 34, 35, therefore, nF190 has diminished soleus muscular atrophy in SCI rat.
Histological evaluation
Histological evaluation was performed using H&E staining. As demonstrated in Figure 4, a scar was made in the spinal cord adjacent to T9 spin in this model. In the control group, the cavity was significantly larger and more extended outside than other groups on 42nd-day post-treatment. At the same time, the cavity had a smaller size in nF190. Since a severe model was induced in the spinal cord, the astroglial scar could not completely recover in all groups, and fibroconnective tissue is observed in the cavity of all groups. However, nanocarriers showed less cavity size compared to others. It seems that nanocarriers help the astroglial scare to be reconstructed.
Analyzing functional recovery of SCI rats treated with free FTY-720, nF60, and nF190 indicated that nF190 has remarkably improved outcomes faster regain of bladder reflux, a gain of body weight, and soleus muscles compared to the free FTY-720, and in some parts more than smaller nanocarriers. Kong et al. discovered that PLGA microfibers containing FTY-720 and NSCs reduce the glial scar cavity size and suppress astrocyte differentiation and induce NSCs differentiation into neurons oligodendrocyte, which is critical for axonal reconstruction. In vivo results of that study pointed out the remarkable efficacy of FTY-720 loaded into electrospun fibers on motor recovery in a spinal cord transection rat model 11. Moreover, Norimatsu et al. demonstrated that FTY-720 has a more extensive activity of FTY-720 than being S1P1 receptor antagonist. They declared that permanent internalization of the S1P1 receptor in astrocytes through functional antagonism is probably a primary function for FTY-720 efficacy 12. Although FTY-720 declined the migration of lymphocytes to the site of SCI, it cannot diminish the infiltrated granulocytes and glial cells 2. Cytokines and other biomolecules released by these cells are in part responsible for the neurotoxicity of SCI. However, microglial scavenger debris and inhibitory biomolecules in SCI 36 help recover injured neurons through axon regeneration signaling 37. Therefore, it seems that SCI recovery following SCI is in part through T cell attenuating.
In conclusion, the high energy method using the ultrasonicator and the low energy method using the magnetic stirrer was employed to synthesize nanoemulsions of 60 and 190 nm particle size, respectively. Particle size was analyzed using SEM and DLS. Nanocarriers were biocompatible and exhibited higher cell viability compared to the control group. Local delivery of FTY-720 nanoemulsion after contusion model in an SCI rat model significantly improved hind limb motor function recovery. Collectively, our data demonstrated that nF190 provides us with neuroprotective and neuroregenerative properties. This synthetic nanocarrier not only could impede further damages but also helps to improve motor dysfunction and recovery. Nanocarriers at the particle size of 190 nm through enhanced cellular uptake positively impact bladder reflex, bodyweight, and muscular weight. Eventually, smaller nanocarriers through enhanced cell membrane damage than the nF190 exhibit less beneficiary effect than nF60 in SCI rats. We suggest that the nanocarriers are studied in a moderate SCI model to significantly shown the impact of particle size on motor neuron recovery.