INTRODUCTION:

Narcolepsy is a serious neurologic sleep disorder characterized by drowsiness and frequent day time sleepiness sometimes it may be associated with catalepsy. Its due to decreased level of hypocretin, which is known a small peptide neurotransmitter to control the sleep cycle. Modafinil is FDA approved first line drug for the treatment of narcolepsy with a minimal side effects and low potential for abuse, so it is preferred over other drugs. It is available as tablets only in the dose of 100mg and 200mg. But it has poor aqueous solubility; upon oral administration the drug is readily absorbed and peak plasma concentration (C_max) reaches in 2-3 hour.

The modafinil tablets have poor aqueous solubility which results in less bioavailability of drug. So, there was a need to develop a suitable formulation that can overcome the problems of insolubility and will provide better bioavailability and better targeting efficiency¹. Niosomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of phospholipids. Since the drug (modafinil) is highly lipophilic, it can be better entrapped in the niosomes. Niosomes were prepared by using cholesterol, soya lecithin, N Palmitoyl Glucosamine and different types of surfactants i.e. different grades of Span. It can entrap wide variety of drugs (e.g. hydrophilic and lipophilic), transport drug molecules to the site of action, overcome hepatic first pass metabolism, increases the retention time of drug in blood circulation, increases the permeability of drug across biological membranes, increases the bioavailability and increase the chemical and physical stability of drug. Niosomes have been used for diagnostic purpose, treatment and to modify the pharmacokinetic profile of drug. Due to their structure, it can deliver drugs to target site in sustain and control release manner². By changing surface charges or attachment of speciﬁc ligands (N Palmitoyl-L-Glucosamine), niosome can be made to cross the BBB and can deliver the drug at the particular site³. Chitosan coating confirm positive charges on niosomes and improving the interaction of the negative charged nasal mucosa with niosomes ultimately could modulate niosomal properties. Chitosan is well known natural polymer, biocompatible, mucoadhesive and responsible for enhancing drug permeation⁴. Nasal route offers direct delivery to brain via different mechanism like olfactory nerve transport, perineural drug transport and systemic circulation. This route is rapid, enhance safety, less systemic exposure and comfortable⁵. So, in present work attempt was made to entrap the modafinil in chitosan coated niosomes (mucoadhesive niosomes) and to deliver modafinil to brain by the nasal route to increases the bioavailability, to provide better targeting across the BBB and to overcome the problem of insolubility.

MATERIALS:

Modafinil drug was purchased from Yarrow Chem Products, Mumbai. Cholesterol, Soya lecithin, low molecular weight Chitosan, Span (40,60 and 80), Triton X- 100 NPG purchased from Sigma Aldrich (India) and other required chemicals were also purchased.

METHODS:

Appearance and Melting Point:

The powder drug was analyzed visually for its color and physical state; whether its amorphous or crystalline. Melting point of the drug was determined by digital melting point apparatus^6,7.

Preparation of calibration curve:

Ten mg of Modafinil was weighed and transferred in to 10 ml volumetric flask, then dissolved in methanol and made up to the volume with the water. After subsequent dilution the final concentration of 10µg/ml was scanned between 200 and 400nm using the same solvent as blank. The absorbance of 2,4,6,8,10,12,14,16,18, and 20 µg/ml were measured at 260nm. The calibration curve was plotted between concentration Vs absorbance^6,7.

Partition coefficient:

Partition coefficient of modafinil was determined by shake flask method. An excess of modafinil was added to equal quantities of n-octanol and water, it was shaken for 24 hrs till equilibrium was reached. The two phases had separated by separating funnel and analyzed for drug content using UV spectrophotometer^8,9.

Solubility Determination:

It was determined by Shake flask method. In this, excess of modafinil had taken and mixed with different solvents, which was then stirred for 24 hrs till equilibrium had reached. The solution was filtered and analyzed for drug content using UV spectrophotometer^8,9

Drug excipient compatibility studies by FTIR:

It was done by Spectrum Two Fourier Transform Infrared Spectrometer (Perkin Elmer, Inc, USA). In FTIR, the spectrum for pure modafinil, excipients and their physical mixtures were analyzed for identification of drug and any possible interactions of drug with excipients. Samples were scanned and spectrum was recorded from 4000 cm^-1 to 400cm^-1 to identify any modification in characters of the drug when it mixed with excipients^10,11.

Method of preparation:

In present work niosomal formulations were prepared by using Span, cholesterol with soya lecithin, N palmitoyl-L-Glucosamine and drug. All the lipid components were dissolved in 10mL chloroform and introduced in round bottom flask. The organic solvent was evaporated using a rotary flash evaporator and reduced pressure at a temperature of about 65°C till a lipid film was formed inside the flask. After hydration of the thin layer with 10ml PBS (pH 7.4), 4 cycles of heating 3 min at 65°C and vortex mixing for 3 min were done. The temperature of 65°C was selected since it was above the phase transition temperature of span 60 which was the component of lipid mixture with highest value of transition temperature (53°C). The niosomal formulation were centrifuged at 4000rpm for 15 min than sonicated for 5 min with the instrument set at 60% of its maximum power. All the samples were stored at 4°C and protected from the light. Then a solution of chitosan at different concentrations (0.5, 1.0, 2.0 and 3.0mg/ml) in 1% v/v acetic acid (adjusted with sodium hydroxide 1M at pH 4.5) in water, was filtered (pore size 0.45µm) and drained on niosomal suspension, it leads to the formation of chitosan coated niosomes^3,5,12,13.

Visual Size measurement by optical microscopy:

Optical microscopy method was used to determine average vesicle size of the formulated niosomes (N10 and C10) using optical microscope (Olympus SZ51 stereo microscope body with 45°C) and the size distribution also studied for the both ucoated and coated optimized niosomes by measuring randomly selected100 niosomes vesicles from each formulation¹².

Scanning electron microcopy:

Optimized formulation was also studied by scanning electron microscope to check their shape and surface morphology (ZEISS GeminiSEM). Niosomes were mounted onto the stub using doubled sided sticking tape and coated with the gold film of thickness 200nm under reduced pressure of 0.001mmHg after that at different magnification photographs were taken^10,11.

Study of zeta potential:

In vivo performance of the vesicles majorly affected by surface charges. Stability of vesicular formulation is significantly depending on value of zeta potential. SurPASS was an electrokinetic analyzer which predict electrostatic attraction or repulsion between solutes and solid surfaces (between particles). Zeta potential can be determined at 25ºC of suitable diluted niosomal formulation by using SurPASS from Anton Paar Gmbh, Austria. This equipment generally worked on the principle of the electrophoretic light scattering (ELS), which determines electrophoretic movement of charged particles under an applied electric field from doppler shift of scattered light for determining zeta potential^12,13,14,15.

Percentage Entrapment efficiency:

It was determined by centrifugation method. The 10ml of uncoated and coated optimized formulation (N10 and C10) was centrifuged at 14,000rpm for 15min at 25 and the supernatant will be separated and the cake left behind will be ruptured using triton-X solution (10%w/v) and both supernatant and ruptured cake were analysed for drug content at λmax 260nm spectrophotometrically. Percent entrapment efficiency will be calculated by the formula given¹⁶:

Total drug added - free drug

% Entrapment efficiency = ----------------------------------- ×100

Total drug

In vitro drug release study of niosomes:

The uncoated (N10) and coated niosomal (C10) formulations with best entrapment efficiency were selected for in vitro drug release study and compared with drug suspension. In vitro drug release study was assessed by using the dialysis bag method. Ten mL of niosomal formulation (20mg/mL) was placed in cellulose acetate dialysis bag hydrated, then immersed in simulated nasal fluid at 37°C under magnetic stirrer. Aliquots of samples were withdrawn periodically at predetermined interval up to 8hrs and immediately replenished with same volume of fresh simulated nasal fluid. Concentration of modafinil was determined at 260 nm spectrophotometrically. Results were mean of three experiments for each formulation. Separate study was done for each formulation¹².

Stability study:

Stability studies of coated optimized formulation of niosome were carried out by storing the sample at 4°C and 25°C after sealing and protected by light. Particle size, zeta potential, PDI and encapsulation efficiency were determined immediately after the preparation and after every month upto 12 weeks. For assessing leakage coated optimized formulation (C10) also stored at 4°C, 25°C and also on 37°C for a period of 12 weeks. After every month % entrapment efficiency were calculated taking initial entrapment efficiency as 100%. Vesicles were visually and microscopically observed for disruption, aggregation and sedimentation³.

RESULTS AND DISCUSSION:

Appearance and Melting point:

The powder drug was analyzed visually for its color and physical state and it was white amorphous crystalline solid. Melting point of the drug was determined by digital melting point apparatus and it was found to be 165°C.

Preparation of calibration curve and Partition coefficient:

UV spectrum of modafinil was obtained, the λ_maxwas found to be 260nm and was selected as analytical wavelength. Modafinil was linear within the concentration range of 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20µg/ml at λ_max260nm. It was found that determined log P value of modafinil in n-octanol-water system is 5.3165 which showed good solubility in lipophilic solvents.

Solubility Determination:

Preliminary solubility study of modafinil was performed in various solvents like distilled water, 0.1N HCl, ethanol, methanol and Phosphate buffer (pH 7.4) which shows appreciable solubility of modafinil in 0.1 HCl and PBS 7.4. The results are shown in Table 1.

Drug excipient compatibility studies by FTIR:

Modafinil pure drug is having characteristic peaks at 1684.12 cm^-1 due to amide functional group (N-H bending vibration). This amide group is confirmed by the presence of peak at 3399 cm^-1 (N-H stretching vibration). Peak at 2879.72 cm^-1 indicates presence of (C-H stretching vibration) of alkane. Peaks at 1493.05 cm^-1 represents alkene (C=C stretching vibration). Peak at 1165.19 cm^-1 and1078.70 cm^-1 represents sulfinyl group (S=O stretching vibration). Peaks in the range of 900-600 cm^-1 indicates presence of aromatics rings. All these peaks matched with reference spectra thus confirming the purity of the drug. As shown in Figure 1 (a) and (b) the peaks of FTIR spectra of modafinil and physical mixture showed no significant changes when compared with peaks of pure drug spectra which means there is no interaction between any of the excipients with modafinil drug. Figure 1 (c) FTIR spectra had peaks different from standard spectra of modafinil drug which means complete entrapment of the drug in noisome.

Table 2: Optimization of various formulations of niosomes

S. No.	Surfactant	Ratio (C+S:* NPG:S)	% Drug Entrapment	Zeta Potential (mV)	Mean Diameter of Vesicles Size (nm)
N1	Span 40	1.0:0.1:2.5	48.34 ± 0.34	-13 ± 0.44	522 ± 0.67
N2		1.0:0.1:2.0	53.09 ± 1.01	-12 ± 0.25	440 ± 0.23
N3		1.0:0.1:1.5	64.22 ± 1.23	-12 ± 0.82	374 ± 0.95
N4		1.0:0.1:1.0	82.99 ± 1.51	-11 ± 0.52	334 ± 0.65
N5		1.5:0.1:1.0	84.81 ± 1.61	-11± 0.44	423 ± 0.74
N6		2.0:0.1:1.0	84.31 ± 1.32	-10 ± 0.25	514 ± 0.63
N7	Span 60	1.0:0.1:2.5	52.26 ± 1.42	-9.0 ±0.26	396± 0.81
N8		1.0:0.1:2.0	54.38 ± 1.27	-7.0 ± 0.44	260 ± 0.99
N9		1.0:0.1:1.5	68.41 ± 1.41	-4.0 ± 0.39	257 ± 0.42
N10		1.0:0.1:1.0*	90.76 ± 1.36	-3.0 ± 0.21	221 ± 0.65
N11		1.5:0.1:1.0	90.66 ±1.02	-3.0 ± 0.38	324 ± 0.78
N12		2.0:0.1:1.0	90.23 ± 1.11	-2.0 ± 0.52	337 ± 0.74
N13	Span 80	1.0:0.1:2.5	50.64 ± 1.29	-5.0 ±0.67	362 ± 0.58
N14		1.0:0.1:2.0	55.31 ± 1.34	-4.0 ± 0.69	260 ± 0.67
N15		1.0:0.1:1.5	64.22 ± 1.81	-3.0 ± 0.92	257 ± 0.92
N16		1.0:0.1:1.0	71.99 ± 1.24	-2.0 ±0.25	255 ± 0.85
N17		1.5:0.1:1.0	72.41 ±1.61	-2.0 ± 0.30	318 ± 0.88
N18		2.0:0.1:1.0	72.03 ± 1.22	-2.0 ± 0.52	329 ± 0.49

*1.0:0.1:1.0 – Cholesterol + Soya Lecithin (90mg + 10mg): NPG (10mg): Surfactant (100mg)

Each formulation contains 200mg of modafinil and coated with 2% Chitosan solution in Acetic acid 1% v/v (adjusted with 1 N NaOH at pH 4.5).

Table 3: Effect of change in concentration of chitosan coating on selected optimized formulation (N10).

S. No.	CS (mg/ml)	Size	PDI	Zeta potential
1	0.0	146.70 ± 2.6	0.245 ± 0.001	-11.0 ± 1.8
2	0.5	236.75 ± 3.1	0.258 ± 0.004	+22.9 ± 2.0
3	1.0	238.03 ± 2.0	0.271 ± 0.010	+27.4 ± 1.4
C10	2.0	241.60 ± 2.9	0.290 ± 0.002	+29.5 ± 2.34
5	3.0	310.14 ± 2.1	0.339 ± 0.007	+31.6 ± 2.02

Visual Size measurement by optical microscopy and Zeta Analyzer:

All the formulations were spherical in shape and their sizes were ranging from 522nm to 221nm. Size distribution for all the formulations were shown in Table 2, 3 and Figure 2(a), (b) and (c). HLB value affects the size of vesicles, as the HLB value move towards the hydrophilicity, the vesicles were found to be increasing. Niosome vesicles formulated with span 40(HLB 6.7) got higher range vesicles then the vesicles prepared with span 80(HLB-4.7). Further, the size of the vesicles of span 60(HLB-4.3) and span 80(HLB-4.7) was almost same this may be due to adjacent HLB values of these surfactants in HLB scale. The HLB value effects surfactant in two ways first could be explained the surface energy increases with increasing the hydrophilicity and second water uptake also increases with moving towards the hydrophilic region and due to these both reason the larger size particle size of vesicles was found with span 40. Further, after the study of various batches it has also been recorded that changing cholesterol ratio results in increase in size of the vesicles. Cholesterol increases membrane stability, alters the membrane permeability of membrane and decreases the fluidity of membrane of the niosomes by eliminating the phase transition temperature peak of the vesicles. Soya lecithin has the wetting property which helps in dissolution of immiscible substance which provide suitable release from niosomes. As shown in Table 3, increasing chitosan concentration in coating solution, increases the particle size and polydispersity index of niosomes. The optimal concentration was 2mg/ml when tested on N10 optimized formulation.

(a) (b) (c)

Figure 2: Photographs (a) Span 40 (b) Span 60 (c) Span 80

Scanning electron microscopy:

The SEM photomicrograph of optimized niosomal C10 formulation at different magnification revealed that shape of niosome was nano size, spherical and with no aggregation as shown Figure 3. Some unevenness may be there due to drying under normal condition taking photographs under scanning electron microscopy.

Figure 3: Scanning electron photomicrograph of Niosomes at 50X, 100X, 200X, 500X and 1.0KX magnification

Zeta potential study:

Particles with zeta potential more positive than +30mV or more negative than -30mV are generally considered as stable colloidal dispersion system. However, if particles with different density than dispersant (dispersion medium) may have tendency to get settle down and may form packed sediment (hard cake). In present study zeta potential of all the uncoated formulations results ranging from -13mV to -3mV. Optimized formulation N 10 and C10 (after coating) had shown -3mV and +30.0mV zeta potential as represented in Figure 4(a) and (b). The positive charges are contributed by the chitosan present in coating solution.

(a)

(b)

Figure 4: Zeta potential of Optimized formulation (a) before coating (b) after coating with Chitosan

Polydispersity index of the niosomes:

It’s used to describe the degree of non-uniformity in size distribution of vesicles and polydispersity is dimensionless and scaled as follows – 1. PDI value < 0.05 its mainly seen with highly monodisperse standard 2. PDI value >0.7 it indicates sample has very broad particle size distribution 3. PDI = 0.0 it indicates perfectly uniform sample 4. PDI = 1.0 it indicates polydisperse sample with multiple particle size 5. PDI ≤ 0.3 it indicates (niosomal or formulation) homogeneous lipid vesicles. The C10 formulation showed polydispersity index 0.290±0.002 which finally indicates the homogeneity of the formulation (Table 3).

Entrapment Efficiency of Niosomes:

All the batches of modafinil niosomes formulations were characterized for percentage entrapment efficiency were shown in Table 2 which clearly shows that the formulations containing span 60 showed highest entrapment efficiency of 90.76% followed by span 40 containing formulation i.e., 82.99%. while span 80 containing formulations showed the least entrapment efficiency 71.99%. As per literature its due to the structure, orientation and packing behavior of the surfactant span 80. Span 40 and 60 have the same head group but different alkyl chain as well as in case of span 80 unsaturated alkyl chain was present. In span 80 due to presence of double bond in chain, the chain bends which means that the adjacent molecule cannot come together tightly while making the membrane. So, membrane becomes more permeable and shows less entrapment efficiency. But in case of span 60 it has long saturated alkyl chain due to which it showed highest entrapment efficiency. Highest entrapment efficiency was also due to solid nature, hydrophobicity and high phase transition temperature of the span 60[Tc = 53] amongst all span [Span 40, Tc = 42 and span 80, Tc = -12]. As acyl chain increases gel transition temperature also increases. Moreover, HLB also affects the entrapment efficiency lower the HLB of surfactant higher the entrapment efficiency and stability. Span 60 (HLB 4.7) formulations showed higher entrapment efficiency than span 40 (HLB 6.7). N10 had showed maximum entrapment efficiency as shown in Table 2.

In vitro Drug Release Study:

The in vitro modafinil release study was performed by the dialysis bag method using simulated nasal fluid (pH 6.4) at 37°C for 4 hrs. the percentage drug release was calculated and plotted the graph and whole experiment was performed three times. The in vitro drug release for drug suspension, N10 and C10 are shown in Figure 4 provides prediction for release pattern of drug from drug suspension, N10 and C10. It reveals that the drug release from the modafinil suspension was very low (38.63 ± 1.23%) as compared to N10 (54.95±0.79) and C10 (70.01±1.29) within 4 hr of time as shown Figure 5. C10 with chitosan coating exhibited somewhat steady state release pattern upto 24hr.

Figure 5: In vitro Percentage Drug Release Vs Time in Min

Stability studies:

It was conducted on C10 at 4°C and 25°C in PBS 7.4 and analyzed in terms of size polydispersity index and zeta potential. After visual inspection it was reported that there is no mold growth and sediment. Zeta potential study revealed that there were not significant changes observed with respect to freshly prepared sample. It was obtained that the formulations were stable after 12 weeks. The size was also acceptable, there was very slight change in dimension but without a statistically significant difference. Throughout the stability study polydispersity index did not show statistically significant difference. Percentage entrapment efficiency also checked and 89.96% was observed after 12 weeks. All the results were shown in Table 4.

Table 4: Effect of storage on chitosan coated optimized formulation at 4°C and 25°C after 12 weeks.

S. N.	Size (nm)		PDI		Z Potential		EE%
	0 days	90 days	0 days	90 days	0 days	90 days	0 days	90 days
4°C	221±0.65	223±1.32	0.290±0.002	0.295±0.002	+31±2.1	+30.1±2.1	90.76 ± 1.36	89.96± 2.31
25°C	221±0.65	225±1.04	0.290±0.002	0.298±0.008	+31±2.1	+29.8±1.4	90.76 ± 1.36	88.01±1.41

Table 5: Drug leakage study of chitosan coated optimized formulation at various temperature conditions.

S. No.	% Drug remaining Entrapped		% Drug remaining Entrapped		% Drug remaining Entrapped
	4°C	Lost	25°C	Lost	37°C	Lost
Initial	100	-	100	-	100	-
4 weeks	99.390	0.61	99.150	0.850	98.867	1.133
8 weeks	98.810	1.19	98.642	1.385	95.902	4.098
12 weeks	98.501	1.499	97.980	2.02	93.149	6.851

Results of drug leakage study shown in Table 5 revealed that a minimum loss at refrigerated condition i. e. 4°C. The drug loss at this temperature 98.501 at the end of 12 weeks as compared to storage at 25°C and 37°C which led to 97.980 and 93.149% drug loss respectively. Increase in temperature shows higher drug loss due to degradation of the lipid bilayer and defects in membrane packing and ultimately loss of overall rigidity that makes the niosomes leaky.

CONCLUSION:

Modafinil Loaded mucoadhesive niosomes were prepared successfully. The preformulation studies of the drug confirm its purity and compatibility with other ingredients. Niosomes have particle size less than 300nm and had PDI less than 0.3 former is a critical parameter for nasal transport of drug and latter is required for uniformity in particle size. Niosomal formulations should be stored at lower temperature to prevent drug loss after this study. It is concluded that good capability of the developed mucoadhesive modafinil niosomes for brain targeting through nasal route, even if further in vivo-ex vivo studies are required to support this hypothesis.

ACKNOWLEDGEMENT:

The authors are thankful to the Head of Institution, Dept of Pharmacy, Banasthali Vidyapith, Rajsthan, India, for providing necessary facilities to carry out this work.

CONFLICT OF INTEREST:

Declared none.

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Received on 24.01.2024 Modified on 29.03.2024

Research J. Pharm. and Tech 2024; 17(5):2365-2371.

DOI: 10.52711/0974-360X.2024.00370

Solvent	Distilled Water	PBS pH 7.4	0.1N HCl	Ethanol	Methanol
Solubilities	0.632mg/ml	0.810mg/ml	0.92mg/ml	0.25mg/ml	1.00mg/ml