INTRODUCTION:

Chitosan having certain limitation to use as nanoparticle drug delivery include hydrophilicity and high solubility in an acidic environment, which promotes the ready degradation of the chitosan in the harsh acidic environment of the stomach, proteolytic breakdown in the gastrointestinal tract, and poor permeability across the gastrointestinal mucosa. Hence need to overcome obstacles for delivery drug effective into the blood stream and for oral administration^1-3

Promoting modification such as acylation, alkylation, quatenization, thiolation, sulfation, phosphorylation, and graft copolymerization chitosan having presence of reactive amino group at C2, hydroxyl groups at C3 and C6 per glucosamine subunit⁴. Modification by hydrophobic alkyl groups onto the chitosan backbones can prompt the formation of chitosan more hydrophobic in the physiological aqueous environment. Sustain released was achieving by hydrophobically modified by cholesterol, 5bcholanic acid, tocopherol, galactosylated O-carboxymethyl grafting with stearic acid, N-octyl-O-sulfate-modified to deliver drugs, vitamins, steroids, and proteins.^5-6

In this study, chitosan was modified using Butyryl chloride and Lauroyl chloride and characterized using Fourier transform infrared spectroscopy (FTIR), 1H-nuclear magnetic resonance (NMR) spectroscopy, XRD and evaluated for solubility in solvents and degree of substitution. Zolmitriptan HCl loaded nanoparticles of modified N-Acyl chitosan were prepared by a ionotropic method using TPP and evaluated by particle size, zeta potential, PDI, TEM and SEM, drug loading, In-vitro-mucoadhesion and In-vitro drug release was also investigated.

MATERIAL AND METHODS:

Chitosan (55kDa; DDA 81.41%) was purchased from Merck while Butyryl chloride from sigma and Lauroyl Chloride from TCI. Zolmitriptan HCl was obtained from Yarrow-Chem Pvt.Ltd (Mumbai, India). All other reagents were of analytical grade and used without further purification.

Synthesis and Characterization of N Acyl Chitosan:

For synthesis, 2.0g chitosan was dissolved in 100ml mixing solution of 0.6% (w/v) acetic acid solution and 85ml of methanol. A molar equivalent (1.2) of Acyl Chloride include Butyryl chloride (C4H7OCl, Mol Wt. =106.55g) and Lauroyl Chloride (C12H23OCl, Mol Wt. =218.77g) were separately added slowly to the chitosan solution with magnetic stirring for 5 h, respectively. The mixtures were poured into the same volume of methanol and ammonia solution in volume ratio of 7 to 3. The precipitates were filtered and rinsed with distilled water, methanol, and ether. Then, they were dried in a vacuum at 500C overnight^7-8. Characterization of synthesize N-Acyl chitosan were carried out by FTIR, NMR, XRD, and evaluation of solubility in solvents and degree of Substitution using Ninhydrin Assay¹⁵. Preparation nanoparticles using synthesize N-Acylated chitosan100 mg of N-Butyryl chitosan and N-Lauroyl chitosan and chitosan were separately dissolved in 40ml of 1% acetic acid solution and 150mg of sodium tripolyphosphate was dissolved in 20ml distilled water with various concentrations at pH 5, based on the results of preliminary study. 250mg of Zolmitriptan HCl was dissolve in sodium tripolyphosphate solution and this solution was added drop-wise to N-acyl Chitosan solution under continuous stirring 500RPM (Magnetic Stirrer 1L, Remi Motors Ltd. India) at room temperature^9-10.

Standard Calibration Curve:

From solution having concentration 100μg/ml aliquots of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5ml were pipette out into 10ml volumetric flasks. The volume was made up to the mark with Phosphate buffer 6.8 to get the final concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/ml respectively. The absorbance of each concentration was measured at 230nm^11-15.

Evaluation of Nanoparticles:

Particle Size, Zeta Potential and PDI. The lyophilized nanoparticle were dispersed in deionised water and particle size, PDI, ZP was determined by dynamic light scattering (DLS) using Zetasizer (Malvern Instrument Ltd., UK, ZS 90) monitored at a 90° angle. All measurements were made in triplicate. % Drug loading (%DL) and % encapsulation efficiency (%EE) Weighted Zolmitriptan HCl loaded nanoparticles were dispersed in 10 ml of deionised water and vortexed for 5 min. The dispersion was centrifuged at 14000rpm for 30 min and separated supernatant filtered through 0.22μm filter (Millipore™) and analyzed at 250nm using UV-Visible spectrophotometer (UV 2401PC, Shimadzu Corporation, Japan). %DL and %EE were calculated using expressions previously described^16-18.

Amount of Drug in Solution

Percent DL : –––––––––––––––––––––––––––– ×100

Amount of Nanoparticles

Amount of Drug in Solution

Percent EE: ––––––––––––––––––––––––––––––– ×100

Amount of Drug Incorporated in Nanoparticles

In-vitro Mucoadhesive Study:

1% (w/v) Mucin solution (1ml) was added to each 1% w/v nanoparticle preparation (19ml), with magnetic stirring (1L, Remi Motors Ltd. India) at 600rpm and mixtures were incubated at 37°C for 1h prior to analysis. The mucin-nanoparticle mixtures were then centrifuged (C24BL, Remi Motors Ltd. India) at 1000 RPM for 60 min and 1ml of supernatant diluted up to 10ml measured at 555nm using UV-Visible spectrophotometer (UV 2401PC, Shimadzu Corporation, Japan) and estimate free mucin concentration using the standard calibration curve.¹⁹ In addition, the mucoadhesiveness was expressed as the mucin binding efficiency of the nanoparticles and was calculated from the following equation:

Co-Cs ×100

Mucin Binding Efficiency (%): –––––––––––––

Where, Co is the initial concentration of mucin used for incubation, and Cs is the concentration of free mucin in the supernatant.

In-vitro Drug release study:

Zolmitriptan HCl loaded N-Acyl chitosan and chitosan nanoparticles were performed using a dialysis bag (12-14 kDa molecular weight cutoff; Himedia, India) containing 50ml of pH 1.2 pH HCl and 7.4 phosphate buffer separately. Nanoparticles of N-Acyl chitosan and chitosan comprising 1mg equivalent Zolmitriptan HCl were placed in the dialysis bag and both the ends were sealed. Then, the dialysis bag was kept in the receptor compartment containing dissolution medium (pH 1.2 HCl and 7.4 phosphate buffer) at 37±0.5°C, which was stirred at 100rpm using a magnetic stirrer (Remi Motors, India). At regular time intervals, 0.5ml samples were withdrawn and replaced with freshly prepared buffer up to 24h. Analyzed by spectrophotometrically at 250nm by using a UV-Visible spectrophotometer (UV 2401PC, Shimadzu Corporation, Japan) against the blank^20-24.

Kinetics Study:

To analysis, the mechanism for the release and release rate kinetics of the formulated dosage form, the data obtained from conducted studies were fitted into various models. In this by comparing the values obtained, the best-fit model was selected^25-26.

RESULT AND DISCUSSION:

Synthesis and Characterization of N-Acyl Chitosan:

The highly reactive acyl chlorides were used to react with glucosamine residue of chitosan, possess reactive amino group at C-2 position to form an amide bond. Product was precipitated with acetone and dried by lyophilization for further use.

IR spectroscopy N-Acyl Chitosan:

The FTIR spectrum of pure chitosan exhibited the characteristic hydroxyl group absorption (3440 cm^-1), along with the amide I band (1647 cm^-1), -NH2 bending (1591 cm^-1) and the C-O vibration (1026 cm^-1), while FTIR spectra of synthesized N-Acyl chitosan observed characteristic absorption peaks at 3000–3800 cm^-1for -OH and -NH2 stretching vibrations, and stretching vibration intensities of –(CH2)10– at 2800–2950 cm^-1and CO stretching vibration at 1068 cm^-1. Absorption of C-H stretching of N-Acyl chitosan at 2918 cm^-1and 2848 cm^-1increased with elongations of the alkyl side chain. Two major peaks at 1662 cm^-1and 1556 cm^-1were assigned to C=O stretching (amide I) and N-H bending vibration (amide II), respectively. Increasing amide II band in the IR spectra confirms the formation of an amide linkage between amino groups of chitosan and carboxyl groups. The reaction is highly selective toward N-acylation, as it can be confirmed by the absence of a band present at 1750 cm^-1.

Figure 1: FTIR Spectra A (Chitosan), B (N-Butyryl chitosan), C (N-Octanoyl Chitosan), D (N-Lauroyl Chitosan) and E (N-Palmitoyl Chitosan)

NMR of N-Acyl Chitosan:

Chitosan displayed two major peaks for three N-acetyl protons of N-acetyl glucosamine and the H-2 proton of glucosamine at 1.8ppm and 2.9ppm. The peaks at 3.1–3.9 ppm were given for (non-anomeric) ring protons of the chitosan (H-3, H-4, H-5, and H-6). The H-1 protons of the N-acetyl glucosamine and glucosamine residues were gives peaks at 4.6 and 4.8ppm respectively. The ¹H-NMR spectrum of N-Acyl chitosan showed new peaks at 0.75ppm for –CH₃ and 0.8–1.16 ppm for proton signals of –CH₃, 1.2-1.9 for -CH2- of acyl group, 3.0 for - CH3 of acetyl group of chitosan, 3.1–3.9 CH of carbon 2 of chitosan, CH of carbon 1 of chitosan (overlapping with the ring protons) and 4.5ppm for -CH2- (acyl protons)

Figure 2: 1H-NMR Spectra a (Chitosan), b (N-Butyryl chitosan), c (N-Lauroyl Chitosan)

Figure 3: XRD of a (Chitosan), b (N-Butyryl chitosan), c (N-Lauroyl Chitosan)

Solubility:

Solubility was performing by placing 10 milligram of chitosan and N-Acyl chitosan sample into a test tube with each of 4 ml solvent. Mixing with a vortex mixer then ultrasonication, the mixture was stored at room temperature for 5 days, and visually observed. High crystallinity and strong inter or intra-molecular hydrogen bonding were responsible for poor solubility of chitosan. Therefore, hydrophobic substituents into chitosan backbone may likely disrupt the inter- or intra-molecular hydrogen bonding of chitosan and weaken its crystallinity.

Degree of Substitution:

N-acyl chitosan (0.3mg) were dissolved in an aqueous acetic acid (3% w/v, 1ml) and thoroughly stirred. Subsequently, 0.5ml of acetic acid/acetate buffer (4 M, pH 5.5) was added into 0.5ml of the prepared solution. Ninhydrin regent (1ml) was then added and solutions were placed in a boiling water bath for 20 min. Cooled and analyzed the absorbance at 570nm using acetic acid/acetate buffer as a blank and chitosan solution was used as a control. Ratio of absorbance by N-acyl chitosan to chitosan gives degree of substitution was found to be 12% to 14%.

Particle Size, Zeta Potential and PDI:

The particle size of nanoparticles by ionotropic gelation method was ranged from 54.1 to 724.3nm with a mean diameter of 324nm. Molecular size of N-acyl was higher than chitosan resulted increases the average particle size of N-acyl chitosan moreover as the length of acyl group increase with respect to average particle size also increases. Chitosan was cationic in nature because nanoparticle having positive zeta potential.

Figure 5: TEM of N Acyl Chitosan

N-acyl substituted decrease the positive charge hence having less zeta potential compared with chitosan. Along of concentration of TPP in preparation of nanoparticle also decrease of zeta potential due to the charge neutralization reaction between amine groups of chitosan and free chains and negative charges of TPP. PDI for both chitosan and N- acyl chitosan nanoparticles were ranged 0.39 to 0.45 which signified a fairly monodispersed pattern of size distribution. The slightly increased size of N acyl chitosan could be due to its longer acyl chain group. The nanosize and spherical nature with good structural integrity of the nanoparticles was confirmed by TEM and SEM analysis.

% Drug loading (%DL) and % Encapsulation Efficiency (%EE):

Loading drugs into nanoparticles by ionotropic method was carried out by either incorporation or incubation. Drug loading capacities and EE of ionotropic chitosan nanoparticle depend on polyphosphate crosslinker contents, chitosan-to-drug loading ratios contribute to effects EE and DL²³. Drug was entrapment in nanoparticle matrix by hydrophobic interactions, hydrogen bonding and other physiochemical forces. As the hydrophobic modification to chitosan by N-Acyl developed hydrogen bonding also responsible for cross linking indirectly drug loading capacity. As increases the length of acyl group entrapment and loading get enhanced. Zolmitriptan HCl entrapment efficiency was increase up to 81.26±3.4 while drug loading was 28.89±1.2 as compared with chitosan.

Table 1: % DL and % EE of Nanoparticles

Product	% DL	% EE
Chitosan	24.80±1.1	54.96±3.8
N-Butyryl Chitosan	25.37±1.8	62.31±4.1
N-Lauroyl Chitosan	28.89±1.2	81.26±3.4

In vitro Mucoadhesive Study:

Sialic acid present in mucin is distributed throughout human tissues, is present in several fluids, including, cerebrospinal fluid, serum, urine, amniotic fluid saliva, and breast milk. Depending on the physiological conditions and physiochemical properties such as pH, the carboxylate group of sialic acid residues on mucin can interact with the positive charge on the chitosan particles, due to the protonated amino group (NH₃⁺) to form electrostatic and hydrogen bonds by hydrophobic and hydrophilic interactions^26,27. Presence of Charge on N-acyl chitosan and chitosan nanoparticle should be estimated by zeta potential. The value of zeta potential was decreases as the attachment of acyl group. However mucin binding efficiency of N-acyl chitosan was increases with increasing the length of acyl group due to hydrogen bonds by hydrophobic interactions.

Figure 6: %Mucin Binding Efficiency of Nanoparticles

In Vitro Drug Release Study:

N-acyl chitosan and chitosan nanoparticles showed an initial burst release of Zolmitriptan HCl 33.13±1.84%, 30.16±2.23%, 24.22±2.34% in pH 1.2 and 31.25±2.48%, 27.19±2.96%, 23.13±3.54%, in pH 7.4 by chitosan, N-Butyryl-, N-Lauroyl chitosan nanoparticle respectively. This initial “burst effect” release is due to the fact that some amounts of Zolmitriptan HCl were localized on the surface of nanoparticles by adsorption which could be released easily by diffusion. N-acyl chitosan was retard released due to increased hydrogen bonding with drug. Burst effect diffuse the drug which are adsorbed on surface and loosely interact with polymer and resulted 72.66±1.59%, 66.41±1.58%, 60.16±1.84% at pH 1.2 in 9 h while 68.44±2.98%, 64.06±3.65%, 60.16±3.24% at pH 7.4 in 12 h released by chitosan, N-Butyryl-, N-Lauroyl chitosan nanoparticle respectively.

Later on sustain released of the drug which are present at core diffuse slowly. Aqueous media penetrated into nanoparticle diffuse the drug. As the hydrophobic attached group to chitosan retard the penetration of media into core and enhanced the sustain release. Hydrophobicity of N- Acyl chitosan nanoparticle increase with length of acyl group with respect to its sustain release property also enhanced as 92.19±1.45% and 84.38±2.87% at ph 1.2 while 84.69±1.89% and 78.13±3.24% at pH 7.4 Zolmitriptan HCl released at 24 h by N-Butyryl and N-Lauroyl Chitosan respectively. While chitosan was release 96.57±3.01% at pH 1.2 after 18 h and 90.31±3.64% pH 7.4 after 24 h because of high solubility in acidic media due to protonation.

Kinetic Data:

Release data of N-Acyl chitosan fitting to the Korsmeyer’s-Peppas model demonstrated a higher value of correlation coefficient as given in table 4. While n value 0.5 > for Korsmeyer’s-Peppas model was suggesting an fickian transport mechanism for Zolmitriptan HCl release by N- Butynoyl-, N-Lauroyl Chitosan and Chitosan. This suggested that the release of Zolmitriptan HCl from N-Acyl Chitosan nanoparticles was not only governed by diffusion, but also included polymer swelling.

Figure 7: a) % Release Profile at pH 1.2 of N Acyl Chitosan b) % Release Profile at pH 7.4 of N Acyl Chitosan

Table 2: Kinetics Study of the Prepared Nanoparticles Loaded with Zolmitriptan HCl

Kinetic		Chitosan		N-Butynoyl chitosan		N-Lauroyl chitosan
Kinetic		pH 1.2	pH 7.4	pH 1.2	pH 7.4	pH 1.2	pH 7.4
Zero order		0.5975	0.5674	0.6087	0.6095	0.6312	0.6566
First order		0.5979	0.5679	0.6091	0.6099	0.6316	0.6570
Higuchi		0.9890	0.9862	0.9900	0.9892	0.9919	0.9925
Peppas		0.9970	0.9963	0.9976	0.9962	0.9980	0.9970
Hixon-Crowell		0.5978	0.5678	0.6089	0.6097	0.6315	0.6568
Korsmeyer- Peppas	n	0.3960	0.4066	0.4179	0.4179	0.4204	0.4398
Korsmeyer- Peppas	K	0.0263	0.0247	0.0267	0.0267	0.0230	0.0241

CONCLUSION:

Hydrophobically modification of chitosan was successfully done with Butyryl and Lauroyl and was characterized by FTIR, NMR, XRD evaluating successful attachment of acyl group at amino position, also improved solubility in various solvent. Zolmitriptan HCl was loaded efficiently in N-acyl chitosan nanoparticles by ionotropic method using TPP. Particle size of N-Acyl chitosan nanoparticle increases with length of acyl group while decreasing zeta potential was observed. PDI exhibited uniformed monodispersed particle size also proved by TEM while SEM revealed spherical particles with smooth surfaces. The loading efficiency ranged improved up to 28.89% which directly correlated to increasing the length of N-acyl side chain. N-acyl chitosan demonstrated sustain drug release at pH 1.2 and pH 7.4 as compared to chitosan. In Vitro exhibited the biphasic released pattern that followed Korsmeyer’s-Peppas model with 0.5 > n value suggesting a fickian transport mechanism.

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Received on 28.07.2022 Modified on 10.12.2022

Res. J. Pharma. Dosage Forms and Tech.2023; 15(2):85-90.

DOI: 10.52711/0975-4377.2023.00015