RETRACTED: Fabrication, Optimization, and Evaluation of Rotigotine-Loaded Chitosan Nanoparticles for Nose-To-Brain Delivery

: The objective of the present study was to develop, optimize, and evaluate rotigotine-loaded chitosan nanoparticles (RNPs) for nose-to-brain delivery. Rotigotine-loaded chitosan nanoparticles were prepared by the ionic gelation method and optimized for various parameters such as the effect of chitosan, sodium tripolyphosphate, rotigotine concentration on particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency. The prepared nanoparticles were characterized using photon correlation spectroscopy, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, fourier-transform infrared spectroscopy, and X-ray diffraction. The developed RNPs showed a small hydrodynamic particle size (75.37 ± 3.37 nm), small PDI (0.368 ± 0.02), satisfactory zeta potential (25.53 ± 0.45 mV), and very high entrapment efficiency (96.08 ± 0.01). The 24-h in vitro release and ex vivo nasal permeation of rotigotine from the nanoparticles were 49.45 ± 2.09% and 92.15 ± 4.74% while rotigotine solution showed corresponding values of 95.96 ± 1.79%and 58.22 ± 1.75%, respectively. The overall improvement ratio for flux and permeability coefficient were found to be 4.88 and 2.67 when compared with rotigotine solution. A histopathological study showed that the nanoparticulate formulation produced no toxicity or structural damage to nasal mucosa. Our results indicated that rotigotine-loaded chitosan nanoparticles provide an efficient carrier for nose-to-brain delivery.


Introduction
Parkinson's disease (PD) is a neurodegenerative disorder, primarily associated with degeneration of the neurons in the substantia nigra, resulting in decreased nigrostriatal availability of the neurotransmitter dopamine.The major symptoms are characterized by resting tremor, muscular rigidity, and bradykinesia [1].Oral levodopa (LD) represents the most clinically useful drug in the treatment of PD [2].Unfortunately, the clinical response to oral LD is variable and unreliable as absorption of oral LD has been found to be erratic.In addition, marked fluctuations in plasma concentrations of LD following oral LD treatment has been observed.The responses, i.e., marked changes in motor performance as well as drug-induced dyskinesia, have posed major challenges in developing an efficacious treatment for PD.More recently, there has been more studies that have focused on developing newer therapeutic strategies that can lead to continuous dopaminergic stimulation (CDS).One of the main aims of developing this therapeutic approach is to lower the incidence and severity of motor functions and impairment of voluntary movements that are associated with treatments of PD, which would be able to provide good long-term safety and tolerability [3].The CDS concept favors long-acting dopamine receptor agonists to prevent or treat motor fluctuations.However, most dopamine agonists present low bioavailability when they are orally administered as free drugs and they must be administered daily in several doses, since their effect lasts only a short time.
Rotigotine is a non-ergoline dopamine D1/D2/D3 agonist with demonstrated effectiveness in treating PD [4].However, its use has been hindered by its low oral bioavailability (1%), extensive first-pass effect, and relatively short plasma half-life (5-7 h) [5].Various formulations of rotigotine have been investigated, these include the rotigotine transdermal, once-daily delivery system (Neupro ® ), which provided 24-h continuous drug levels intended to improve efficacy and reduce motor complications compared with other currently available products [5,6].However, this product was withdrawn from the US market in April 2008 because of drug crystallization on the patches, leading to poor absorption through the skin and a resultant broad variation in efficacy [7].Given the problems associated with earlier tried approaches, there is a need for an improved drug delivery system for rotigotine, which would increase the bioavailability of rotigotine and provide site-specific deposition in the brain.However, transport of rotigotine to the brain remains a challenge due to the blood brain barrier (BBB).Researchers have studied several approaches to overcome the BBB in an effort to deliver drugs efficiently to the brain for various therapeutic and diagnostic applications.
Alternative drug delivery approaches that can improve efficacy and reduce toxicity of PD drugs have been developed in the last decade.One such approach is the intranasal (IN) delivery method, which is considered to be a promising alternative method to rapidly deliver PD drugs to the brain compared to the to the intravenous route, as this approach can circumvent the BBB [8].Using the IN route, it would be possible to deliver drugs directly to the brain along both the olfactory and trigeminal nerves [9].Some of the benefits of administering a drug via the IN route is that this route offers a large surface area for absorption, which can allow the patient to achieve desired levels of the drug in the brain rapidly as well as avoid first pass metabolism [10].All these benefits can improve the bioavailability of the therapeutic drug.In addition, this method of delivery also shows marked improvement in patient comfort and compliance when compared to the parenteral route.However, this method has some limitations.This is related to the rapid clearance of any drugs or formulation introduced through the nasal route by the mucociliary system in the nose.This action often limits the bioavailability of drugs delivered using the IN route.Hence, it is important to develop a newer method that can allow the drug to remain in the nasal cavity for a longer period, for instance, by using mucoadhesives or nanoparticles, which could facilitate in augmenting the effectiveness of IN delivery.Furthermore, retaining the drug for a longer period in the nasal cavity using the nanoparticulate systems can enhance the permeability across the epithelial membrane.Over the last decade, polymeric NPs have attracted increasing attention as a drug delivery system due to their biocompatibility, biodegradability, and prolonged half-life in the circulation after intravenous (i.v) administration [11][12][13][14].More specifically, nanoparticles have been shown to enhance interaction with mucus barriers and protect drugs from biological and/or chemical degradation that may occur during intranasal delivery [10].To date very little research has been undertaken to develop ways of delivering rotigotine directly to brain bypassing the BBB.One study showed rotigotine-loaded PLGA microparticles (Ro-MP) provide high and stable plasma and striatal drug levels for up to 14 days, in 6-OHDA-lesioned rats; intramuscular Ro-MP enhanced the effect of pulsatile L-DOPA treatment in reducing dyskinesias in these rats [15].Bi et al. [16] have reported intranasal delivery of rotigotine to the brain with lactoferrin-modified PEG-PLGA nanoparticles for Parkinson's disease treatment.No previous work appears to have been undertaken on the delivery of rotigotine to the brain using intranasal administration of chitosan nanoparticles.In this research we have develop, optimized, characterized, and evaluated rotigotine-loaded chitosan nanoparticles for intranasal administration to enable the clinical potential of rotigotine to be explored for the treatment of PD.

Materials and Methods
Rotigotine was purchased from Shanghai PI Chemicals Ltd., Shanghai, China and Sigma-Aldrich, St. Louis, MO, USA.Chitosan, Sodium Tripolyphosphate (TPP) and Porcine mucin were purchased from Sigma-Aldrich, St. Louis, MO, USA.Glacial acetic acid, methanol, and ethanol was purchased from Biotek Abadi Sdn, Bhd, Selangor, Malaysia.All other reagents used were of analytical grade.

Preparation and Optimization of Rotigotine Nanoparticles
An ionic gelation method was employed for the preparation of rotigotine-loaded chitosan nanoparticles (RNPs).For this purpose, chitosan was dissolved in acidified media at various concentrations in the range ranges 0.05-0.2%w/v.Cross linker tri-polyphosphate was prepared at the same concentration range as that of the chitosan solution.The rotigotine solution was added to the chitosan solution with constant stirring prior to addition of tri-polyphosphate solution.Nanoparticles were obtained by the addition of crosslinker solution to polymer solution with constant stirring at room temperature for 30 min.The drug-free polymeric NPs were obtained using the same procedure.The resultant NPs were concentrated by ultracentrifugation at 4 • C for 40 min.The supernatant was analyzed to determine the entrapment efficiency and the pellets were washed with distilled water and freeze dried for further characterization.The production variables of stirring speed, concentration of polymer, concentration of drug, and concentration of crosslinker were varied systematically to investigate the particle size, size distribution, zeta potential, and entrapment efficiency of the resulting NPs.

Characterization of Rotigotine-Loaded Chitosan Nanoparticles
2.2.1.Nanoparticle Size, Size Distribution, and Zeta Potential Hydrodynamic particle size, size distribution, and zeta potential were measured using photon correlation spectroscopy (PCS) (Zetasizer Nano-ZS-90, Malvern Instruments, Worcestershire, UK).The polydispersity index (PDI) was used to indicate the size distribution within the sample.The zeta potential which reflect charges present on the surface of nanoparticles, is responsible for the stability of formulation and interaction with cellular membranes.

Transmission Electron Microscopy (TEM)
The size and morphology of the nanoparticles were determined using TEM (Hitachi HT7700 TEM, Tokyo, Japan) at an acceleration voltage of 200 kV and viewed at a magnification of 50,000×.Rotigotine-loaded chitosan nanoparticles were diluted 10 times with distilled water and a drop of diluted sample was placed on a formvar-coated copper grid, and after complete drying, the sample was focused on a layer of photographic film grid and images were captured.

Field Emission Scanning Electron Microscope (FESEM)
The shape and morphology of nanoparticles were determined using FESEM (Hitachi, Tokyo, Japan) using the gold sputter technique.Freeze-dried RNPs were dusted onto a double-sided tape on an aluminum stub.The stubs containing the sample were coated with gold.Photomicrographs were taken at the accelerated voltage of 20 KV and chamber pressure of 0.6 mmHg.

Atomic Force Microscopy (AFM) Studies
Atomic force microscopy was used to characterize the morphology of placebo and RNPs.The AFM images were obtained using an AFM (AFM5000II, Hitachi, Tokyo, Japan) instrument in non-contact mode and using a 910-ACTA cantilever with 330 KHz resonant frequency.For surface morphology characterization, solution of NPs was drop-casted on to a quartz substrate, then, after overnight evaporation by heating at 353 K, the sample was placed on a substrate holder of the Park System and images were collected.The scan speed was between 0.5 and 1 Hz using the adaptive scan mode function set in the software.

X-ray Diffraction (XRD) and Fourier-Transform Infrared Spectroscopy (FTIR) Studies
The crystallinity of rotigotine, chitosan, physical mixture, placebo nanoparticles, and RNPs was measured using X-ray diffraction PANalytical X'pert PRO (Almelo, The Netherlands) using CuK α radiation.X-ray diffraction was carried out at 2θ of 4 • to 60 • to determine the crystal lattice arrangements and information on degree of crystallinity.The FTIR spectra was observed for rotigotine, chitosan, physical mixture, placebo nanoparticles, and RNPs using a FTIR spectrophotometer.The samples were prepared and scanned in the range of 4000 cm −1 to 400 cm −1 .The IR spectrums were compared to investigate the interaction between NP and rotigotine.

Determination of Entrapment Efficiency (%EE)
The entrapment efficiency was determined using ultracentrifugation.Ten mL RNPs was ultracentrifuged (Beckman Coulter, Optima LE 80K, Brea, CA, USA) at 4 • C at 25,000 rpm for 50 min and a clear supernatant was obtained.The supernatant was diluted and analyzed by UV spectrophotometry (Lambda 25 UV/VIS spectrometer) to determine the presence of free drugs in the solution.The exact concentrations of free drugs were obtained from the standard curve of rotigotine.The %EE was calculated using equation as below:

%EE =
Total drug − Free drug Total drug × 100

Mucoadhesive Strength of Nanoparticles
The binding efficiency of nanoparticles to mucin, i.e., the mucoadhesive strength, was determined by employing a method modified from Yin et al. [17].Briefly, 5 mL of porcine mucin suspension (0.5 mg/mL in PBS pH 7.4) was added to the same volume of nanoparticles.The mixture was mixed well first by vortex and then left to shake at 37 • C for 60 min.Following this, the mixture was centrifuged (60,000× g for 1 h) and the concentration of free mucin in the supernatant was determined by reading the absorbance at 255 nm using a UV spectrophotometer.
The mucin binding efficiency of nanoparticles was calculated using the following equation Mucin binding efficiency (%) = Total Mucin − Free Mucin Total Mucin × 100

In Vitro Release of Rotigotine from Polymeric Nanoparticles
The in vitro release kinetics of rotigotine from polymeric nanoparticles was assessed using a dialysis technique, whereby samples of drug-loaded nanoparticles were contained within dialysis tubing and suspended in a release medium simulating nasal fluid (PBS 7.4).Drug release was recorded at hourly intervals under sink conditions over a 24 h period.The concentration of rotigotine in the outer release medium was analyzed using high-performance liquid chromatography (HPLC).All experiments were carried out in triplicate.Data obtained from the in vitro release studies of rotigotine-loaded polymeric nanoparticles were fitted to various kinetic models to assist in explaining the mechanism and kinetics of drug release.

Ex Vivo Drug Permeation Study Using Goat Nasal Mucosa
Nasal mucosa was carefully removed from the nasal cavity of goat.Tissue samples were mounted in a Franz diffusion cell and immersed in PBS medium.To ensure oxygenation and agitation, a mixture of 95% O 2 and 5% CO 2 was bubbled through the donor and receptor compartments.The temperature was maintained at 37 • C ± 0.5 • C.After a pre-incubation time of 20 min, a suspension (2 mL) of rotigotine-loaded nanoparticles in PBS (pH 7.4) containing a known weight of nanoparticles was placed in the donor chamber.At predetermined time points of 1 h over 24 h, 1 mL samples were withdrawn from the receptor chamber and replaced with an equivalent amount of PBS medium [18].The collected samples were analyzed for permeated drugs using HPLC.

Histopathological Examination of Nasal Mucosa
The short-term effect of rotigotine-loaded polymeric nanoparticles on the structural integrity of nasal mucosa was investigated by histopathological examination of untreated goat mucosa and goat mucosa subjected to 24 h of the drug permeation experiment.Normal mucosa (negative control), mucosa treated with isopropyl alcohol (positive control), mucosa treated with rotigotine solution, and mucosa treated with rotigotine-loaded polymeric nanoparticles were fixed immediately in 10% formalin and embedded in paraffin.Sections (5 µm) were cut and de-paraffinized using xylene and ethanol, followed by staining with hematoxylin and eosin.The sections were inspected for tissue damage using optical microcopy at 10× magnification [19].

Preparation and Optimization of Placebo Chitosan Nanoparticles
Placebo chitosan nanoparticles (CS NPs) were successfully developed using an ionic gelation method [20].For this purpose, CS was dissolved in acetic acid (2% v/v, pH 3.5) at various concentration ranges (0.05, 0.1, 0.15, 0.175, 0.2% w/v).Tripolyphosphate (TPP) was dissolved in purified water at the same concentration range as that of chitosan solution (Figure 1).All the preparations were analyzed visually, and three different systems were identified: clear solution, opalescent suspension, and precipitates.The zones of clear and opalescent suspensions were difficult to differentiate as compared to the precipitate suspension.Particle size (PS), PDI, and zeta potential (ZP) were determined for each formulation.The particle size, PDI, and zeta potential for the different CS/TPP ratio, obtained by PCS are shown in Figure 1.The criteria for initial selection of a good formulation for further optimization and characterization study were a particle size less than 200 nm, PDI value less than 0.5, and zeta potential more than 15 mV.From Figure 1, formulations in the I and J groups were completely rejected due to very large particles size and PDI, which make them unsuitable brain targeting through the intranasal route.The large particle size may be due to the higher CS concentration.Others have shown that the particle size increased with increasing the concentration of either CS or TPP [21].Formulations in groups F, G, and H meet our initial criteria.Therefore, formulations F5-F7, G5-G6, and H5 were selected; these have CS/TPP ratios 2-1.43:1 and show better particle size, PDI, and zeta potential.We also observed that, in general, decreases in the CS/TPP ratio were associated with decreases in the particle size, PDI and ZP; there were few exceptions to this, for example, formulation H4, where the zeta potential increased to 60 mV.However, above a certain CS/TPP ratio the particle size increased.As we know from previous publications, formation of the NPs was only possible for some specific concentrations of CS and TPP.Ionically, cross-linked NPs were produced due to the formation of inter-and intra-molecular linkages between the phosphate groups of TPP and the amino groups of CS [20].This method results in the spontaneous formation of NPs of smaller size without using any organic solvents or surfactants [22,23].The ratio between CS and TPP is important, as it controls the particle size and size distribution of the NPs.Therefore, from Figure 1, we inferred that the ratio of CS and TPP should be not more than 2 and not less than 1.43 to achieve the desired particle size, PDI, and ZP.Above and below a ratio of CS/TPP of 2-1.43:1, the formation of NPs is not possible; moreover, any NPs formed would have an unsatisfactory size.To exclude the effect of the TPP concentration, Figure 2 shows the effect of increasing the TPP concentration and reducing the volume of TPP on PS, PDI, and ZP at a fixed concentration of CS.Here, we observed a similar pattern, i.e., with decreases in the CS/TPP ratio the particle size decreased, but below a certain ratio, the particle size increased.Figure 2 shows that the CS/TPP ratios in the range 2.5-1.67:1yielded better particle sizes, PDIs, and zeta potentials.Therefore, CS/TPP ratios in the range 2-1.1.43: 1 were chosen for further optimization and were selected for the preparation of rotigotine-loaded chitosan nanoparticles.Represent optimization of placebo chitosan (CS) nanoparticles (NPs) using different concentration of CS and TPP (% w/v), different CS:TPP ratio.There were a total 32 formulations run for optimization purposes.Formulation code F represents 0.05% CS and TPP (% W/V), G represents 0.1% CS and TPP (% W/V), H represents 0.15% CS and TPP (% W/V), I represents 0.175% CS and TPP (% W/V), and J represents 0.2% CS and TPP (% W/V).Sub-codings F1, G1, H1, I1, and J1 (1 mL TPP, CS: TPP ratio 10:1), Sub-codings F2, G2, H2, I2, and J2 (2 mL TPP, CS: TPP ratio 5:1), Sub-codings F3, G3, H3, I3, and J3 (3 mL TPP, CS: TPP ratio 3:1), Sub-codings F4, G4, H4, I4, and J4 (4 mL TPP, CS: TPP ratio 2.5:1), Sub-codings F5, G5, H5, I5, and J5 (5 mL TPP, CS: TPP ratio 2:1), Sub-codings F6, G6, H6, I6, and J6 (6 mL TPP, CS: TPP ratio 1.67:1), Sub-codings F7 and G7 (7 mL TPP, CS: TPP ratio 1.43:1).

Development and Optimization of Rotigotine-Loaded Chitosan Nanoparticles (CS NPs)
The rotigotine-loaded CS NPs were optimized based on particle size, PDI, zeta potential, and entrapment efficiency.The effect of CS and TPP concentrations on particle size, PDI, and ZP after addition of a fixed concentration of drugs are shown in Table 1.For the selection of drug-loaded NPs, we set additional stringent criteria, these being a particle size less than 100 nm, a PDI value less than 0.3 for more homogeneity, and a ZP greater than 25 mV for better formulation stability.Formulation F5D with a CS/TPP ratio of 2:1 showed smaller particle size, a lower PDI, and a higher ZP compared with other formulations (Table 1), and this formulation was thus selected.

Effect of Increase in CS Concentration at Fixed Drug Concentration
Table 2 shows the effect of an increase in the chitosan concentration (0.05 to 0.15% w/v) on particle size, polydispersity index, zeta potential, and entrapment efficiency at a fixed drug concentration in rotigotine-loaded nanoparticles in formulations F, G, and H; an increase in particle size was observed as the CS concentration increased.This is because a high concentration of CS results in more CS chains per volume, thus forming larger particles when added with the cross-linking agent, TPP.It will also cause the cross-linking density between CS and TPP to decrease, resulting in particle aggregation and the formation of larger particles [24][25][26].The PDI scores of less than 0.3 (except H5D) showed that the RNPs developed from these three formulations are well-distributed and that particle sizes are similar among the batches.On the other hand, the zeta potential and %EE of rotigotine decreased with the increase in the CS concentration.F5D shows the optimum PS (64.93 ± 0.87 nm), PDI (0.266 ± 0.20), ZP (25.40 ± 0.20 mV), and %EE (93.29 ± 0.03%).Thus, the F5D formulation was chosen to determine the effect of rotigotine concentration.Different concentrations of rotigotine (0.25, 0.50, 1.00, and 1.5 mg/mL) were incorporated into formulation F5 (Table 3).Increases in drug concentration from 0.25 to 1.5 mg/mL resulted in an increase in the particle size from 73.37 ± 0.17 to 146.4 ± 3.3; this was probably due to a decrease in the interaction of CS/TPP [20,27].On the other hand, an increase in drug concentration slightly decreased the PDI, while the decreased in zeta potential and %EE was similar, as reported previously by other researchers [20,27].The probable explanation for the decrease in %EE is that as concentration of rotigotine increased, more rotigotine molecules became adsorbed onto the surface of the chitosan which had been separated from the nanoparticles due to centrifugation.This causes rotigotine molecules to be separated from each other, resulting in a reduction in %EE [28].However, increasing the rotigotine concentration did not produce any significant changes to these characteristics among formulations F5D1, F5D2, and F5D3.Hence, the initial drug concentration of 0.5 mg/mL was optimized based on particle size and %EE.Lastly, amongst all formulation, F5D2 was chosen as the best, considering all the parameters analyzed; optimized formulation parameters are shown in Table 4.

Characterization of Optimized Nanoparticles
The particle size, particle size distribution, and zeta potential of optimized placebo CS NPs and rotigotine-loaded CS NPs were characterized by PCS and TEM (Figures 3-5).The particle size of placebo CS NPs and rotigotine-loaded CS NPs were 63.96 and 65.22 nm based on PCS and 40-60 nm based on TEM, respectively.This difference between PCS and TEM results could be due to surface charges and their interaction with water when using PCS, whereas in TEM imaging, the dehydration of CS NPs during sample preparation leads to measurement of the actual particle which is smaller than hydrodynamic particles [29].The PDI value of placebo and drug-loaded nanoparticles were less than 0.5, indicating a narrow particle size distribution and a homogenous formulation [30].The zeta potential represents an index for cell membrane interaction and particle stability [31].The mean zeta potential of placebo CS NPs and rotigotine-loaded CS NPs, at around +25 mV (Figure 4) were close to 30 mV, which is ideal for stability, indicating that the prepared NPs were stable.The zeta potential of rotigotine-loaded CS NPs was less than that of placebo NPs; this small difference, which did not reach statistical significance (p ≥ 0.05) may be due to substitution of the amino group of chitosan [32].Prepared CS NPs were all positively charged due to the presence of the residual amino groups which are not neutralized by their interaction with the negative charge of TPP molecules [33,34].The positively-charged surface of the formulation is preferred to a negative charged surface, as the negative charge on cell membranes will attract the positively-charged formulation, facilitating mucoadhesion, prolonging residence time and reducing mucocilliary clearance of NPs from the nasal cavity [32].The FESEM images of placebo and rotigotine-loaded CS NPs (Figure 6) show that CS NPs were mostly uniform, and nearly spherical in shape, with smooth surfaces and showed little aggregation; the minimal aggregation of the NPs may be due to the use of freeze drying [35].The greater particle size of the rotigotine-loaded CS NPs compared with placebo CS NPs as revealed by this method is consistent with the data obtained using TEM.The size and morphological appearance of the placebo and rotigotine-loaded CS NPs were examined by AFM (Figure 7).Surface morphological analysis of the particles using AFM also confirmed the size range of approximately 14.36 nm for placebo and 16.04 nm for rotigotine-loaded NPs.The 3-dimensional AFM images of the formulations (Figure 8) indicate that the placebo and rotigotine-loaded CSNPs on glass surfaces maintain their spherical morphology and homogeneous particle distribution [36].The mucoadhesive strength for optimized placebo and RNPs was determined by calculating their binding efficiency to mucin.The placebo CS NPs showed 92.60 ± 0.90% and rotigotine-loaded CS NPs showed 80.98 ± 1.78% mucoadhesion; this decrease in mucoadhesive strength of the drug-loaded NPs relative to the placebo may be explained by a reduction in the availability of CS for interaction with mucin, which would decrease the penetration of NPs into the mucosal layer [20,37].3D AFM images of (A) placebo nanoparticles and (B) rotigotine-loaded nanoparticles.In both formulations, particle distribution is homogeneous and the particle shape is seen to be round shaped.Topography image 1 micrometer × 1 micrometer of the same region.

XRD and FTIR Studies
The comparative XRD patterns of rotigotine, CS, physical mixture, placebo CS NPs, and RNPs are shown in Figure 9. Chitosan exhibits characteristic peaks at 2θ of 9 • and 20.43 • (Figure 9), indicating the high degree of crystallinity [20].The pure rotigotine shows a strong characteristic peak at 2θ at 10.29 • and 19 • .The physical mixture of chitosan and rotigotine showed peaks indicating that there was no interaction between drug and polymer.A reduction in intensities and broadening of peaks as compared to individual diffraction pattern indicates a reduction in crystallinity.The diffraction pattern of placebo CS NPs showed a single reduced peak of CS at 9 • , indicating the poorly crystalline nature of the polymer.In RNPs, the sharp peaks at 10.29 • and 19 • were reduced and there was broadening of the peaks, showing a reduction in the crystallinity for rotigotine.This reduced crystallinity was due to cross linking between CS and TPP, into which rotigotine was entrapped, thereby showing lower or negligible intensities for rotigotine in the diffraction pattern of RNPs [19,20,35].The comparative FTIR patterns of rotigotine, CS, physical mixture, placebo CS NPs and RNPs are shown in Figure 10.The FTIR spectra showed characteristic peaks for rotigotine at 3540, 1588, 1277, 774, 375, and 356 cm −1 , respectively.The FTIR spectrum of CS showed intense peaks at 1650 and 1575 cm −1 , confirming the presence of amide I and amide II.The CS peak at 3472 cm −1 indicates that the O-H stretching overlapped with and N-H stretching vibration.The physical mixture showed no shift of the bands, indicating no interaction between drug and chitosan.In the case of placebo CS NPs, there was a shift of the band for O-H and N-H stretching vibration and the appearance of a new peak P = O as compared to CS individual spectra, as well as an increase in the intensity of the amino group; this is indicative of NP formation through the ionic gelation method.The characteristic peaks for rotigotine were absent in the case of RNPs when compared to the spectra of pure drug, confirming that the rotigotine was encapsulated into the nanoparticles [20,35].

In Vitro Release Studies
The percentage drug release profile showed a sustained release of the drug from the formulation (Figure 11).The drug release rate for RNPs was faster in the first 3 h (19.39 ± 0.81); this was followed by slow drug release for 24 h (49.45 ± 2.09), whereas rotigotine solution show complete release after 24 h (95.96 ± 1.79%).The initial slight rapid release of the rotigotine could be due to rotigotine adsorbed on the surface of the NPs [38].The drug release rate was slower from the nanoparticles compared with drug solution because higher encapsulation of drug favors increased interaction with the drug and chitosan polymer, which retards the drug release from the nanoparticle [20,35,38].The release kinetics data appeared to fit best in Higuchi kinetics as indicated by higher r 2 values of 0.96.When the release data were analyzed using the Korsemeyer-Peppas model, the value of the diffusional exponent n was found to be 0.72, demonstrating that the drug release by CSNPs exhibit a non-Fickian diffusion mechanism [35,38,39].

Ex Vivo Nasal Permeation Study
Figure 12 shows comparative nasal permeation as the cumulative rotigotine permeated versus time (h); the percent rotigotine permeated from NPs was greater at all time points compared to rotigotine solution.The cumulative quantity of rotigotine permeated through the nasal mucosa from RNPs was 92.15 ± 4.74%, whereas only 58.22 ± 1.75% was found to permeate from rotigotine solution (Figure 10).The steady-state flux and permeability coefficient of drug solution through the nasal mucosa was 0.018 µg cm −2 h −1 and 7.31 × 10 −5 cm −2 h −1 , respectively, whereas steady-state flux and permeability coefficient of drug-loaded CS NPs was 0.080 µg cm −2 h −1 and 1.95 × 10 −4 cm −2 h −1 , respectively.The overall improvement ratio for flux and permeability coefficient were 4.88 and 2.67.The RNPs showed high values of all parameters related to permeation when compared with rotigotine solution.This could be due to the presence of positively-charged chitosan which interacts with the negative surface of sialic acid residues in the cell membrane, this helping in the modulation of tight junction components on the mucosa, which will consequently enhance the paracellular transport and permeation across the nasal surface [18,19].

Nasal Histopathology Study
Nasal histopathology is useful to determine the effects of the formulation on the integrity of nasal mucosa.Figure 13 shows (A) normal mucosa (negative control), (B) mucosa treated with isopropyl alcohol (positive control treated), (C) mucosa treated with rotigotine solution, and (D) mucosa treated with rotigotine nanoparticles.Normal mucosa showed intact, well-preserved epithelial lining and numerous mucous glands.The mucosa treated with isopropyl alcohol showed extensive damage with alteration in the epithelial lining, absence of mucous glands and necrosed tissue.Mucosa treated either with rotigotine solution or rotigotine nanoparticles showed preservation of the overall structure, without evidence of tissue necrosis or ulceration [18,19].The viability of mucosa specimens was ascertained and the histopathological study showed that mucosal barrier function was unaffected by the nanoparticular formulations.

Conclusions
In the present study, RNPs were successfully developed, optimized, and evaluated.The desired particle size, PDI, and entrapment efficiency for nose-to-brain delivery were achieved.The smaller particle size, positive zeta potential, and ability for mucoadhesion for RNPs indicated their potential for increased nasal residence time and reduced mucocilliary clearance from the nasal cavity.The FTIR and XRD analysis of NPs demonstrated the crosslinking between chitosan and TPP into which rotigotine was entrapped successfully.Data obtained from in vitro release kinetics demonstrated the sustained release of the drug from the formulation.No toxicity or structural damage was seen upon histopathological examination of nasal mucosa.However, studies using an in vitro cell line, as well as pre-clinical and clinical studies are required to establish the potential of rotigotine-loaded chitosan nanoparticles to be an efficient carrier for nose-to-brain delivery as an alternative to conventional routes.

Figure 2 .
Figure 2. Represents the effect of increasing the TPP concentration and reducing the volume of TPP on (A) PS, (B) PDI, and (C) ZP at a fixed concentration of CS to exclude the effect of the TPP concentration.

Figure 7 .
Figure 7. Atomic force microscopy images of (A) placebo NPs and (B) rotigotine-loaded NPs.Surface morphological analysis of the particles using AFM also confirmed the size range approximately 14.36 nm for placebo and 16.04 nm for rotigotine-loaded NPs nm.Topography image 1 micrometer × 1 micrometer of the same region.

Figure 8 .
Figure 8. 3D AFM images of (A) placebo nanoparticles and (B) rotigotine-loaded nanoparticles.In both formulations, particle distribution is homogeneous and the particle shape is seen to be round shaped.Topography image 1 micrometer × 1 micrometer of the same region.

Table 1 .
Effect of CS and TPP concentration on particle size, polydispersity index, and zeta potential after addition of fixed concentration of drug (n = 3).

Table 2 .
Effect of increase in chitosan concentration on particle size, polydispersity index, zeta potential, and entrapment efficiency of rotigotine-loaded nanoparticles (n = 3).

Table 3 .
Effect of increase in rotigotine concentration on particle size, polydispersity index, zeta potential, and entrapment efficiency (n = 3).

Table 4 .
The optimized formula for placebo and rotigotine-loaded chitosan nanoparticles.