Intranasal Administration of N-acetyl-L-cysteine Combined with Cell-Penetrating Peptide-Modified Polymer Nanomicelles as a Potential Therapeutic Approach for Amyotrophic Lateral Sclerosis

Intranasal administration is a promising route for direct drug delivery to the brain; its combination with nanocarriers enhances delivery. We have previously shown that intranasal administration combined with PEG-PCL-Tat (a nanocarrier) efficiently delivers drugs to the brain and exhibits excellent therapeutic efficacy against brain diseases. We aimed to clarify whether intranasal administration combined with PEG-PCL-Tat represents a useful drug delivery system (DDS) for amyotrophic lateral sclerosis (ALS) pharmacotherapy. We used N-acetyl-L-cysteine (NAC) as a model drug with low transferability to the spinal cord and determined the physicochemical properties of NAC/PEG-PCL-Tat. After intranasal administration of NAC/PEG-PCL-Tat, we measured the survival duration of superoxide dismutase-1 G93A mutant transgenic mice (G93A mice), widely used in ALS studies, and quantitatively analyzed the tissue distribution of NAC/PEG-PCL-Tat in ddY mice. The mean particle size and zeta potential of NAC/PEG-PCL-Tat were 294 nm and + 9.29 mV, respectively. Treatment with repeated intranasal administration of NAC/PEG-PCL-Tat considerably prolonged the median survival of G93A mice by 11.5 days compared with that of untreated G93A mice. Moreover, the highest distribution after a single administration of NAC/PEG-PCL-Tat was measured in the spinal cord. These results suggest that intranasal administration combined with PEG-PCL-Tat might represent a useful DDS for ALS therapeutics.


Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective degeneration of the upper and lower motor neurons, causing muscle atrophy, which ultimately leads to death due to respiratory failure. There are 2 types of ALS: familial ALS, which represents 10% of cases, and sporadic (idiopathic) ALS, which represents 90% of cases. It has been reported that approximately 20% of familial ALS patients are associated with mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1) [1]. Transgenic mice (G93A mice) carrying the mutant human SOD1 gene with the glycine 93 changed to alanine (SOD1 G93A ) are widely used as mouse models for mechanistic and therapeutic studies of ALS. Several hypotheses for ALS pathogenesis include the involvement of oxidative stress, mitochondrial dysfunction, excitotoxicity, neuroinflammation, and protein aggregation [2]. Riluzole, a glutamate release inhibitor, and edaravone, a free radical scavenger, were developed for ALS treatment based on these etiological hypotheses. These drugs have been approved for use in treating ALS in several countries despite their limited beneficial effects. small water-soluble compounds for delivery from the nose to the spinal cord. In this study, we aimed to clarify whether intranasal administration in combination with our nanocarrier represents a useful DDS for ALS pharmacotherapy using G93A mice, which show pathological conditions and symptoms similar to those of ALS patients [29]. Specifically, NAC was selected as the model compound, as it has low transferability to the brain and spinal cord [7]. The physicochemical properties of NAC/PEG-PCL-Tat were evaluated and the effects of daily intranasal administration of NAC/PEG-PCL-Tat on the survival duration of G93A mice were examined. In addition, we quantitatively evaluated drug distribution from the nose to the spinal cord following a single intranasal administration of NAC/PEG-PCL-Tat in healthy mice.

Synthesis of PEG-PCL-Tat Micelles
The PEG-PCL-Tat was synthesized as previously described [27,30]. Briefly, the Tat peptide (0.02 mmol) and PEG-PCL block copolymer (0.02 mmol) were dissolved in dimethylformamide. Water-soluble carbodiimide hydrochloride (0.02 mmol) and 4-dimethylaminopyridine (0.02 mmol) were then added to this solution. To form an ester bond between the Gly-COOH on the C terminus of Tat peptide and the -OH group on PEG-PCL, the solution was stirred at room temperature (25 • C) for 24 h. The reaction solution was transferred to a dialysis membrane (Molecular weight cut-off: 3.5 kDa) suitable for use with organic solvents and dialyzed against ultrapure water for 24 h under continuous stirring (on a magnetic stirrer) at 150 rpm. Subsequently, the reaction solution was freeze-dried to obtain PEG-PCL-Tat.

Physicochemical Characterization of the NAC Containing PEG-PCL-Tat Micelles Solution
The mean particle size, polydispersity index (PDI), and zeta potential of the PEG-PCL-Tat, 0.2NAC/PEG-PCL-Tat, and NAC/PEG-PCL-Tat solutions were measured after diluting them to an appropriate concentration range via the Zetasizer Ultra (Malvern Instruments, Worcestershire, UK).

Animals
All efforts were made to minimize the number of animals used and their distress. Animal experiments were carried out after obtaining approval from the Nihon University Animal Care and Use Committee (Approval no.: AP19PHA026-1 and AP20PHA006-1) (Tokyo, Japan). Animals were housed under controlled conditions (temperature 23 ± 1 • C, relative humidity 50 ± 10%, and 12 h light/dark cycles [light on 8:00 a.m. to 8:00 p.m.]) and with ad libitum access to feed and water.

G93A Mice
Transgenic mice of the B6SJL-TgN (SOD1-G93A) 1Gur strain were used as an ALS mouse model, purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The hemizygous G93A mice were obtained by mating male G93A mice with wild-type (WT) females. Mice were genotyped by polymerase chain reaction (PCR) using genomic DNA isolated from blood as previously described [31]. Only male mice weighing 25-30 g were used in this study to control for possible gender differences.

ddY Mice
Four-week-old ddY mice (male) were obtained from Japan SLC, Inc. (Shizuoka, Japan) for the pharmacokinetic investigation of the intranasal administration. Mice weighing 25-35 g were used for the experiments after being pre-fed as previously reported [28].

Intranasal Administration
Intranasal administration was performed as previously described [32,33]. In brief, the nasal area of mice under isoflurane (4% induction, 2% maintenance) inhalation anesthesia was covered with an openable inhalation mask (SN-487-70-09, Shinano Seisakusho, Tokyo, Japan). After opening the silicone cap of the mask, mice were administered a total volume of 20 µL dosing solution at 30-s intervals over 10 min, via 1-µL doses alternatively into each naris (1 µL/30 s). The dosing solution was administered by timing the droplet from the tip of the microtip to be brought close to the nasal cavity and synchronized with the respiration of the mouse, thereby allowing for spontaneous aspiration. Intranasal administration of 0.2NAC/PEG-PCL-Tat or NAC/PEG-PCL-Tat solutions was performed in a total volume of 20 µL per mouse, equivalent to receiving 0.2 mg or 1.0 mg of NAC, respectively.

Drug Administration and Survival Analyses
For survival analyses, a total of 108 male G93A mice were randomly assigned into six groups as shown in Table 1. Starting at 105 days of age, which is shortly after the onset of ALS [34], G93A mice were treated with NAC or NAC complex (NAC/PEG-PCL-Tat) on weekdays (5 d a week). In cases when the 105th day fell on a holiday or national holiday in Japan, treatments were started on day 106 or 107. The treatment was continued until the endpoint. The endpoint was defined as the date on which a mouse could not right itself within 30 s after being placed on its side [35]. At that point, the G93A mice were euthanized with CO 2 . The median survival duration is the time period taken for 50% of G93A mice to die. The mean survival time is the average amount of days that G93A mice reached the endpoint. In the group that received intranasal administration (Groups 3-6) of Table 1, some mice did not show spontaneous respiration during administration or until awakening from anesthesia. Accordingly, we excluded these mice from the assessment of survival duration.

Collection of CSF, Plasma, and Tissues Following an Intranasal Administration to ddY Mice
CSF and tissue samples were obtained at 60 min after an intranasal administration of [ 14 C]-NAC or [ 14 C]-NAC/PEG-PCL-Tat solution to ddY mice. CSF was slowly withdrawn (approximately 20 µL) from the cisterna magna through a needle (30 G) connected to a cannula. Plasma samples (approximately 0.5 mL) were obtained through blood centrifugation (15 min, 2100× g), collected from the heart. Tissues (trigeminal nerve, brain, spinal cord [including the thoracic spinal cord and lumbar spinal cord], lungs, liver, and kidneys) were obtained after systemic perfusion with phosphate-buffered saline (pH 7.4) using a peristaltic pump until the circulating fluid was decolored. Subsequently, each tissue sample was weighed (wet weight) and prepared as previously described [33].

Determination of [ 14 C] Radioactivity
The radioactivity of [ 14 C] in plasma, CSF, or tissues was measured using a liquid scintillation counter (Tri-Carb 4810TR, PerkinElmer Inc., Waltham, MA, USA), as per our previous report [33]. The percentage of injected dose per gram of tissue (%ID/g tissue) or per milliliter of plasma or CSF (%ID/mL plasma or CSF) was calculated for each sample. A maximum drug concentration (C max ) was determined as the highest value among the calculated values of %ID/mL plasma. The area under the plasma concentration-time curve (AUC 0-90 ) was calculated by the linear trapezoidal method using the [ 14 C]-NAC radioactivity in the plasma to the last time point (t = 90 min).

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Data were expressed as the mean ± standard error (SE) or standard deviation (SD). Student's t-test was used for comparisons between two groups. The significance of differences between multiple groups was analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Comparisons of survival duration analyzed by the Kaplan-Meier method were performed using the log-rank test. The differences were considered to be significant at p < 0.05.

Physicochemical Characterization of NAC/PEG-PCL-Tat Micelles
The physicochemical characterization of PEG-PCL-Tat, 0.2NAC/PEG-PCL-Tat, and NAC/PEG-PCL-Tat is demonstrated in Table 2. The mean particle size and PDI values of 0.2NAC/PEG-PCL-Tat and NAC/PEG-PCL-Tat showed no significant difference compared with those of PEG-PCL-Tat. In contrast, the zeta potential of NAC/PEG-PCL-Tat was significantly decreased compared with that of PEG-PCL-Tat and 0.2NAC/PEG-PCL-Tat (p = 0.00001 and p = 0.00009, respectively). The results revealed that the zeta potential decreases in a NAC concentration-dependent manner. It has been reported that nanoparticles with zeta potentials of −10 mV to +10 mV are generally regarded as neutrally charged, while nanoparticles with an absolute value of zeta potentials exceeding ± 10 mV are positively or negatively charged [36]. PEG-PCL-Tat and 0.2 NAC/PEG-PCL-Tat exhibited positive charges, while NAC/PEG-PCL-Tat exhibited neutral charges. Table 2. Particle diameter, PDI, and zeta potential of PEG-PCL-Tat, 0.2NAC/PEG-PCL-Tat, and NAC/PEG-PCL-Tat.

Effect of NAC on Survival Duration of G93A Mice
We  Figure 1A). These results revealed that intranasal administration of NAC with PEG-PCL-Tat as a nanocarrier increased the survival duration of G93A mice in a concentration-dependent manner.
The mean survival time of G93A mice is shown in Figure 1B. Similar to the result illustrated in Figure 1A, the mean survival time of untreated mice, NAC-IP-treated mice, PEG-PCL-Tat-treated mice, and 0.2NAC/PEG-PCL-Tat-treated mice was 128.5, 126.8, 127.8, and 129.9 d, respectively. There were no significant differences between groups. In contrast, NAC/PEG-PCL-Tat (1.0 mg) treatment significantly extended the mean survival duration of G93A mice when compared with that of untreated mice (135.6 d vs. 128.5 d, p = 0.0042; Figure 1B). There was no difference between the mean survival time of NAC-IN (1.0 mg)treated mice and that of untreated mice (133.9 d vs. 128.5 d). NAC/PEG-PCL-Tat-treated mice were the only group that markedly extended mean survival time compared with that observed in untreated mice, thus indicating that intranasal administration combined with PEG-PCL-Tat is the most efficacious for the NAC therapy.  Figure 1A). These results revealed that intranasal administration of NAC with PEG-PCL-Tat as a nanocarrier increased the survival duration of G93A mice in a concentration-dependent manner. duration of G93A mice when compared with that of untreated mice (135.6 d vs. 128.5 d, p = 0.0042; Figure 1B). There was no difference between the mean survival time of NAC-IN (1.0 mg)-treated mice and that of untreated mice (133.9 d vs. 128.5 d). NAC/PEG-PCL-Tat-treated mice were the only group that markedly extended mean survival time compared with that observed in untreated mice, thus indicating that intranasal administration combined with PEG-PCL-Tat is the most efficacious for the NAC therapy.   6 or 7). The significant differences in mean plasma concentrations at the same time or AUC 0-90 between the two groups were analyzed using a t-test.  Figure 3B). In CSF, the [ 14 C]-NAC distribution after an intranasal administration of [ 14 C]-NAC/PEG-PCL-Tat was 1.4-fold higher than that measured in the [ 14 C]-NAC without PEG-PCL-Tat group. For the spinal cord, the distribution of [ 14 C]-NAC/PEG-PCL-Tat was increased compared with that of [ 14 C]-NAC 60 min after the intranasal administration and was the highest among the other tissue (0.46 ± 0.09% ID/g tissue). In the [ 14 C]-NAC/PEG-PCL-Tat group, the [ 14 C]-NAC amount distributed within the spinal cord was 1.5-and 2.1-fold higher than that measured in the brain and CSF, respectively ( Figure 3B). However, there were no significant differences in the amount of [ 14 C]-NAC distributed in any tissue or CSF of the central regions. Similarly, there was no significant difference in the amount of [ 14 C]-NAC distributed in the peripheral tissues analyzed ( Figure 3C). Thus, no significant differences were observed between the two groups in any of the tissues in the single intranasal administration.

Discussion
This study aimed to establish a non-invasive and efficient DDS for brain and spinal cord delivery of therapeutics. We selected NAC as a model compound with low transferability to the brain and spinal cord, and investigated the effect of NAC by intranasal de-

Discussion
This study aimed to establish a non-invasive and efficient DDS for brain and spinal cord delivery of therapeutics. We selected NAC as a model compound with low transferability to the brain and spinal cord, and investigated the effect of NAC by intranasal delivery, which has been shown to bypass the BBB and blood-cerebrospinal fluid barrier. Furthermore, we combined NAC with PEG-PCL-Tat, which enhances nasal mucosal permeability and brain distribution. We assessed the delivery effectiveness using survival duration of G93A mice. In addition, we quantitatively investigated the distribution of NAC in the brain and spinal cord of healthy mice.
NAC/PEG-PCL-Tat was prepared by mixing an equal volume of NAC and PEG-PCL-Tat micelles in a weight ratio of 1:4. NAC is a water-soluble compound and thus cannot be encapsulated in the micelle, PEG-PCL-Tat. Previous studies showed that positively charged Tat peptides are able to form complexes with negatively charged plasmid DNA and siRNA via electrostatic interaction [27,37]. NAC at near neutral pH is negatively charged [38]. In this study, NAC was dissolved in HEPES buffer at a pH of 7.4, suggesting that electrostatic interactions occurred between positively charged Tat peptide and the negatively charged NAC. Therefore, part of the NAC has been loaded in the surface of PEG-PCL-Tat, which may have caused a NAC concentration-dependent decrease in zeta potential due to charge neutralization ( Table 2). Particle sizes of nanocarriers utilized in nose-to-brain delivery have been reported in the 50-500 nm range [39]. In addition, the PDI value of NAC/PEG-PCL-Tat was slightly above 0.5. However, we considered it to be close to the usually recommended value (<0.5) [40]. We have previously reported that PEG-modified neutral liposomes, among PEG-modified positively, neutrally, and negatively charged liposomes, showed higher distribution in a wide area of the brain and spinal cord via the olfactory and trigeminal nerve pathways [28]. Therefore, NAC/PEG-PCL-Tat is expected to enhance the nose-to-brain delivery due to its physicochemical characterization.
Next, we examined the effect of intranasally administered NAC/PEG-PCL-Tat on the survival duration of G93A mice. The intraperitoneal administration of NAC alone as well as intranasal administration of NAC and PEG-PCL-Tat alone were also examined. No significant differences were observed between the survival duration of NAC-IP-or NAC-IN-treated mice when compared with the untreated mice ( Figure 1). These results suggest that changing the administration route of NAC to an intranasal route was not sufficient to provide a therapeutic effect. Similarly, no significant difference was observed in the PEG-PCL-Tat-treated mice compared with the untreated mice, indicating that the PEG-PCL-Tat alone did not provide a therapeutic effect. However, the intranasal administration of NAC with PEG-PCL-Tat as a nanocarrier increased the survival duration of G93A mice in a concentration dependent manner, with the median and mean survival of the NAC/PEG-PCL-Tat-treated mice being markedly increased by 11.5 d (9.1%) and 7.1 d (5.5%), respectively, when compared with those of the untreated mice ( Figure 1). A previous study suggested that NAC administered in drinking water to G93A mice 4-5 weeks of age (before onset phase) prolonged their mean survival time by 8.6 d (6.7%) [41]. Therefore, this study revealed that the therapeutic effect of intranasal administration of NAC/PEG-PCL-Tat is comparable to that of preventative treatment even after ALS onset. In addition, despite riluzole and edaravone currently being approved by the Food and Drug Administration (FDA) and in Japan for the treatment of ALS, it has been reported that neither riluzole administered in drinking water, nor intraperitoneal administration of edaravone to G93A mice after ALS onset prolonged their survival [42,43]. These results suggested that the intranasal administration of NAC/PEG-PCL-Tat shows a therapeutic effect that outperforms the in vivo results from studies investigating riluzole administered in drinking water and the intraperitoneal administration of edaravone to G93A mice. As no valid early diagnostic methods have yet been established for sporadic ALS, most drug treatments are initiated after the onset of motor dysfunction or other symptoms. Our results suggest that intranasal administration of PEG-PCL-Tat could be a very useful drug delivery strategy for the treatment of ALS, even after disease onset.
Next, we analyzed the pharmacokinetics of a single intranasal administration of [ 14 C]-NAC/PEG-PCL-Tat to ddY mice, as the survival duration of the NAC/PEG-PCL-Tat-treated mice was considerably extended compared with that of the untreated mice. The plasma time-dependent concentration indicates that the interaction between NAC and PEG-PCL-Tat is maintained at early time points after intranasal administration, whereas this interaction is lost after 30 min (Figure 2A).
Furthermore, we quantitatively analyzed drug distribution in the brain, medulla oblongata, spinal cord, and CSF after a single intranasal administration of [ 14  ]-NAC/PEG-PCL-Tat had already reached the brain and medulla oblongata via the trigeminal nerve and olfactory bulb 60 min after administration. This is supported by our previous report that PEG-PCL-Tat is transferred to the brain via the olfactory and trigeminal nerve pathways because, after intranasal administration of Alexa-dextran/PEG-PCL-Tat, the fluorescence of Alexadextran was observed in the olfactory bulb at 15 min and then in the olfactory bulb and brainstem at 60 min [23]. The highest improvement in the relative distribution ratio of the [ 14 C]-NAC/PEG-PCL-Tat group when compared with that in the [ 14 C]-NAC group was in the CSF ( Figure 3B). The [ 14 C]-NAC in CSF is thought to flow into the brain via the glymphatic system [44], a circulatory mechanism between CSF and interstitial fluid in perivascular space (PVS). It has been previously shown that the PVS is involved in some portion of drug diffusion through the brain, as fluorescence was observed in perivascular and the PVS of cerebral arteries after intranasal administration of fluorescently labeled dextran [13]. Conversely, concerning the pathway from the brain to the spinal cord, it is unclear how diffusion in the brain affects distribution to the spinal cord. However, it has been reported that PVS was observed around arterioles and venules as well as throughout the spinal cord white and grey matter, while the tracer injected into the cisterna magna appeared to be spreading from the PVS into the surrounding parenchyma [45]. Thus, the [ 14 C]-NAC in the CSF is speculated to flow similarly into the spinal cord via the glymphatic system. We showed that the increased distribution to the brain, medulla oblongata, and spinal cord in the [ 14 C]-NAC/PEG-PCL-Tat group was accompanied by an increased distribution to the CSF when compared with that observed in the [ 14 C]-NAC group ( Figure 3B). Although no significant difference was observed between the two groups in all regions after a single administration, the [ 14 C]-NAC/PEG-PCL-Tat group tended to exhibit increased [ 14 C]-NAC distribution in each tissue when compared with that measured in the [ 14 C]-NAC group, thereby suggesting that repeated administration might have extended survival.

Conclusions
In this study, we demonstrated that intranasal administration of NAC, which has low transferability from the blood to the brain and spinal cord, extensively extended the survival of G93A mice (an ALS animal model) when combined with a PEG-PCL-Tat nanocarrier. After a single intranasal administration to healthy ddY mice, the [ 14 C]-NAC distribution to the brain and spinal cord of [ 14 C]-NAC/PEG-PCL-Tat group slightly increased compared with that observed in [ 14 C]-NAC without PEG-PCL-Tat group. We showed that intranasal administration combined with PEG-PCL-Tat is a useful DDS not only for the treatment of brain diseases such as brain tumors and cerebral ischemia-reperfusion injury but also for ALS with the primary pathophysiology of the spinal cord. Future optimizations of the nanocarrier are expected to provide wider applicability.  Institutional Review Board Statement: The animal study protocol was approved by the Nihon University Animal Care and Use Committee (Tokyo, Japan) (protocol code #AP19PHA026-1 (18 March 2020) and #AP20PHA006-1 (5 June 2020)).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.