FORMULATION AND EVALUATION OF CURCUMIN-LOADED CHITOSAN NANOPARTICLES AND ITS INTRANASAL EFFICACY FOR CNS DELIVERY

FORMULATION AND EVALUATION OF CURCUMIN-LOADED CHITOSAN NANOPARTICLES AND ITS INTRANASAL EFFICACY FOR CNS DELIVERY

Lucky Mangal *, Nitin Kaushik and Sanjar Alam

Department of Pharmaceutics, R. V. Northland Institute, Greater Noida, Uttar Pradesh, India.

ABSTRACT: Hydrophobic medications that are taken orally having poor bioavailability due to permeability issues. They experience a chemical and enzymatic breakdown in the digestive system and exhibit a significant amount of hepatic first-pass metabolism. Similar obstacles prevent the development of effective therapeutic molecule delivery systems and the discovery of new drugs. It has been revealing that curcumin, a key active lipophilic constituent of Curcuma longa (Zingiberaceae), has a variety of pharmacological properties, including cholagogues, anticancer, and anti-inflammatory properties. Based on its organoleptic characteristics, solubility, loss on drying, partition coefficient, infrared spectroscopy, and differential scanning calorimetry, Curcumin was physically and chemically characterized. The drug sample Curcumin was found to be genuine and pure based on the tests. For the purpose of determining in-vitro release and permeation study, an analytical method utilizing RP-HPLC was created and validated. Ionic gelation was used to create chitosan nanoparticles. The study includes the evaluation of various processing conditions, including surface morphology, particle size, particle size distribution, surface charge, percentage yield, entrapment efficiency, and drug loading, as well as ex-vivo permeation studies and in-vitro release models. For Curcumin-nanoparticles, the impact of drug concentration on particle size, polydispersibility index, entrapment efficiency, and loading capacity was optimized. It was found that as the drug: polymer ratio is raised; the average size of curcumin-loaded nanoparticles grows. As per ICH guidelines, the formulation's stability study was also carried out.

Keywords: Curcumin, Nanoparticles, Chitosan, Tripolyphosphate, Intranasal delivery

INTRODUCTION:

Curcumin: Turmeric is generated from curcumin (Curcuma longa- Haldi). Curcumin, like many herbal therapeutic compounds, was first ingested as a portion of food before being shown to have a significant pharmacological effect.

It has been used as a muscle relaxant and anti-inflammatory drug in Ayurveda (Indian school of medicine) for years to reduce inflammation and pain in the skin and muscles. It has also been shown to have melanoma effects.

Curcumin is revered in traditional Indian medicine as a "body purifier," and science is discovering a growing list of sick disorders that can be remedied by turmeric's bioactive constituents ¹. It may represent involvement in Alzheimer’s disease pathogenesis anti-inflammatory and anti-capabilities. In Monocytic THP-1 cells, Curcumin diminishes an induced Egr-1 production of proteins and Egr-1 DNA-binding activity. The significance of Amyloid peptide-induced cytochemokines gene expression in monocytes is mediated by Egr-1 was already studied. Curcumin lowers inflammation via inhibiting Egr-1 DNA-binding function. Curcumin inhibits monocyte chemotaxis, which can be initiated by chemokines secreted by stimulated microglia and astrocytes inside the brain. Curcumin appears to be a significant inhibitor of pro-inflammatory cytokine production in these trials, although this may vary depending on the nature of the target cells ².

FIG. 1: CHEMICAL STRUCTURE OF CURCUMIN

Chitosan: Chitosan is a natural polymer i.e., an N-deacetylated peptide that is a duplicate of chitin found in crustaceans, insects, fungus, and other organisms. Several fascinating properties of chitosan have been found, including the ability to form films, gelation capabilities, bio adhesion properties, and permeation-increasing effects, which are explained by opening epithelial cell tight intersections. Chitosan is also a polymer i.e., biodegradable and biocompatible that has attracted a lot of interest in the pharma industry, in addition to food science and cosmetic applications, due to its promising qualities. It has been converted into nanoparticles (NPs) and microparticles for the encapsulation of biological and pharmaceutical substances ³.

Due to its cationic nature as a polymer, CS has the ability to interact with molecules or polymers that are negatively charged. Recently, cationic chitosan and anionic counter ions like TPP have been used to create chitosan microparticles or nanoparticles through ionic interaction. Nanoparticles and microparticles are formed by flocculation of the Chitosan polymer in this known as the ionic gelation process. Ionic reaction should allow the cationic chitosan molecule in these particulate gel beads to bind anionic compounds. As a result, it is easy, harmless, and requires aqueous phases mixing at room temperature.

This approach of producing drug-containing chitosan nanoparticles without the use of organic solvents has received a lot of attention. IR spectra, X- ray photoelectron spectroscopy, and velocimetry were utilized in separate articles to confirm the interaction or binding of opposite charged molecules of CS NP’s. The release profiles of CS NPs made using this technology revealed a high explosive effect within 1/2 hour and a rapid entire release of the drug. This behavior indicates that chitosan’s with an ionic reaction may have weak binding capabilities and that chitosan’s are not the best excipients in the formulation, as evidenced by the literature ⁴.

Nasal vaccine delivery using these nanoparticles were also investigated. The anti-tetanus IgG levels and anti-tetanus IgA titers elicited by tetanus toxoid-loaded nanoparticles were higher than those induced by solution vaccinations at 6 months following injection. The electrostatic attachment of drug-loaded molecules to the cationic CS polymer explains the carrier and stabilizer effect. Because of polymers have large mol. weight (70-150 kD) demonstrated an adverse immunogenic reaction following parenteral administration, commercially made chitosan can’t be used for parenteral preparations ⁵.

Nanoparticles: According to the history of development process, as novel challenges arise with greater size, their solutions must be determined at a level far deeper than previously recognized. From macroscale (>1 mm) to microscale (100–0.1 mm) to nanoscale (100–0.1 nm), now we have a better knowledge of science and technology (100-1 nm). With the introduction of biotech drugs, such as proteins, DNA, and RNA molecules, which can alter disease pathophysiology, the need for new drug delivery technology is pressing to overcome the size and stability issues associated with these new drugs, as well as to design methods to deliver these drugs in a controlled manner ⁶.

Drug nanoparticles are drug-containing particles with a diameter of less than 1m. These isolated and submicron-sized particles have been used for a different type of purposes, including targeted drug administration, controlled release, and increased bioavailability of water- insoluble medicines.

The goal of modern drug therapy is to improve the pharmacological properties of medications while reducing their negative side effects. Colloidal drug carriers that enable targeted drug delivery or site-specific delivery in a magic-bullet concept while ensuring optimum drug release profiles are a good choice. The most extensively studied colloidal carriers have been liposomes and nanoparticles. Polymeric nanoparticles have been proposed as an alternative drug carrier because liposomal formulations have some technological constraints, such as poor stability and limited drug entrapment effectiveness ⁷.

Polymeric nanoparticles (which act as drug carriers/ reservoirs) or nanosuspensions of the active therapeutic agent (which are made using various milling or homogenization methods to enhance the surface area and thus the solubility, dissolution, and bioavailability of insoluble drugs) are 2 types of nanoparticles used in drug delivery. For the manufacture of drug nanoparticles, a number of materials have been used, including biodegradable (e.g., PLGA, PLA, PGA, etc.) and non-biodegradable (e.g., sodium alginate, chitosan, etc.) pharmaceutically appropriate polymers. For the objectives of this study, representative polymers of both categories were utilized as model polymeric materials ⁸.

METHODS AND MATERIAL:

Materials: They also (Bangalore, India) provided chitosan and TPP with deacetylation degrees of 85% and average molecular weights of 40 kDa. Sigma-Aldrich supplied PVA (87-90% hydrolyzed, average molecular weight 30,000- 70,000 Da), dichloromethane, and sucrose Steinheim, Germany. The curcumin was provided by the Dabur Research Foundation, India, through Sigma Chemical Company. MERCK supplied absolute grade ethanol from Changshu Hongsheng Fine Chemical Co., Ltd., India). Fisher Scientifics provided HPLC quality acetone from RFCL Limited, India. IOL Chemical Ltd. (Mumbai, India) supplied GAA, while potassium dihydrogen phosphate and methanol were supplied by S.D. Fine Chemicals, Ltd. (Mumbai, India).

Solubility Determination: The Curcumin solubility was measured in distilled water and phosphate buffer with a pH of 7.4. To test the drug solubility, 25 ml of each solvent was separated from the excess curcumin and placed in separate conical flasks. In a mechanical shaker, the flasks were sealed and clamped. The shaker bath was set to a temperature of 25°C. Samples were taken in every 24 hours until equilibrium was attained (72 hrs.). Shimadzu UV-1601 was used to record absorbances after the samples were filtered and diluted (Shimadzu, Kyoto, Japan) ⁹.

Melting Point Determination: Curcumin was reduced in size to a very fine powder and dried at temperatures much below its melting point. It was then inserted into a glass capillary tube. A sufficient amount of dry medication powder was used to create a compact column with a height of 4-6 mm. This capillary tube, together with the thermometer, was then placed into the HICON melting point apparatus (Ningbo Hicon Industry Co. Ltd, Zhouxiang, China). The temperature at which the drug melted was recorded ¹⁰.

Analytical Methodology: For the preliminary and in-vitro research of curcumin, the reported HPLC and UV-spectrophotometric method was validated. As part of a typical research effort, a new HPLC method was refined and validated, and its application in plasma analysis was tested ¹¹.

UV Spectroscopy: To determine the λ_max, 100 mg Curcumin was diluted in 100 ml of distilled H₂O and scanned by the UV spectroscopy in the range of 200-800 nm.

Preparation of Calibration Curve in PBS with pH 7.4: To generate a 1 mg/ml stock solution, 50 mg of Curcumin dissolved in 50 ml of phosphate buffer with pH 7.4.Stock solution (1000 g/mL) was serially diluted at concentrations ranging from 2-20 g/ml, and absorbance was measured at 425 nm by using a Shimadzu UV-1700 instrument (Shimadzu, Kyoto, Japan). As a blank, a double beam spectrophotometer was used with phosphate buffer (pH 7.4). A graph was plotted between absorbance (y-axis) and concentration (x-axis) ¹².

Fourier Transform Infrared Spectroscopy: The FT-IR technique is used to get the infrared spectrum of absorption or emission of a gas, solid, or liquid. FT-IR spectrophotometer collects the data with a large spectral range with high spectral resolution.

This is a very important advantage over a dispersive spectrophotometer, which is limited in its ability to detect intensity over a limited range of wavelengths at once. The process of transform raw data into a spectrum using a (Perkin Elmer) Fourier- Transform is referred to as Fourier Transform Infrared Spectroscopy (mathematical strategy) ¹³.

DSC: Using a DSC Q2000 (TA Instruments, New Castle, USA) fitted with the TA Universal Analysis 2000 software, a thermogram of curcumin nanoparticles was obtained. Each sample was set inside a T- zero aluminum pan, covered with a T- zero cover, and studied at 10°C/min. scan rate between 30 and 350°C. A 50 mL/min. nitrogen stream was used to run the samples through an empty reference pan ¹⁴.

HPLC Method for Curcumin: The HPLC was validated for in-vitro analysis in PBS at pH 7.4.

HPLC Requirements: A reverse phase Phenomenex column C₁₈ (25 cm × 4.6, or 5 µm) with a mobile phase of water, methanol, and 2-propanol (50:45:5 v/v) and a flowing rate is 1 ml/min were used to analyzethe samples. The drug eluted after 10 minutes, and its amount was detected spectrophotometrically at 425 nm.

Curcumin Calibration Curves Prepared using the HPLC Method:

Instrumentation: Shimadzu HPLC with UV detector attached.

Column: Phenomenex C₁₈ reversed - Phase column (Particle size 5 µm, 25 cm x 4.6 mm).

Mobile Phase: Water: Methanol: 2-propanol.

Flow Rate: 1 ml/min.

Detection: 425 nm, UV detection.

Retention Time: 7.34 ± 0.1 min.

Injection: 20 µL.

Curcumin stock solution of 0.1 mg/mL was made by diluting 10 mg drug with 100 mL of methanol. From the stock solution (100 g/mL), serial dilutions for concentration ranging from 2-20 g/mL were prepared. A graph was drawn showing the relationship between the area under the curve (y-axis) and concentration (x-axis). A Calibration curve of Curcumin was created using the HPLC technique for drug content estimation and in-vitro study. Fig. 6 depicts a representative HPLC chromatogram of the drug ¹⁵.

Formulation Development:

Ionic Gelation Method: Chitosan was used as a mucoadhesive polymer. The Chitosan nano-particles were created by the ionic gelation method with tripolyphosphate anions. For this experiment, CS was dissolved in glacial acetic acid (2% v/v) at different concentration (0.05, 0.1, 0.15, 0.175. 0.2, 0.25 and 0.3% w/v). TPP was dissolved in distilled H₂O at concentrations of 0.1, 0.15, 0.2, and 0.3% w/v. Finally, 4 ml of TPP solution was dropped into 10 mL of CS solution and stirred at room temperature for 30 min. Visual examination of the samples revealed three distinct systems: aggregates, opalescent suspension (nanoparticles), and clear solution. The particles in an opalescent suspension are very tiny, in the nano range. Opalescent suspensions (nanoparticles) were chosen from the preparations with various concentrations of chitosan and TPP, and their mean particle sizes were determined ¹⁶.

FIG. 2: FORMULATION DEVELOPMENT OF CURCUMIN- LOADED CHITOSAN NANOPARTICLES

Characterization of Placebo NPs: The mean size, polydispersibility index (PDI), and zeta potential of the nanoparticles was determined by the zeta sizer (Nano ZS 90, Malvern, UK). Curcumin-chitosan nanoparticle characterization was examined using a TEM (JEM-3011, JEOL, Tokyo, Japan) and an SEM (JSE- 5200, JEOL, Tokyo, Japan) ¹⁷.

Preparation of Drug-loaded Chitosan Nanoparticles: Curcumin was incorporated into the nanoparticles by combining the drug with chitosan solution and storing the solution for 24 hrs. To obtain nanoparticles, the TPP was added drop wise to the drug-containing chitosan solution while stirring after 24 hrs.

Determination of Nanoparticle Encapsulation Efficiency, Loading Capacity, and Percentage Yield Determination: The encapsulation efficiency and loading capacity of nanoparticles were revealed by ultracentrifugation at 14000 rpm for 45 minutes at 4°C from an aqueous medium containing non- associated Curcumin. The amount of free Curcumin in the supernatant was determined using a UV spectrophotometer set to 425 nm. The encapsulation efficiency (EE) and loading capacity (LC) of Curcumin NP’s were calculated using the formulae provided below and all measurements were performed in triplicate ¹⁸.

EE = (Total drug-Free drug) / (Total drug) × 100

LC = (Total drug-Free drug) / (Nanoparticles weight) × 100

The percentage yield was determined as the total dry weight of initial materials (W2) and the weight of dried NP’s recovered (W₁) as:

Percentage yield (%) = W₁ / W₂ ×100

Evaluation of Developed Formulation and their Optimization:

Dynamic Light Scattering is used to Determine Particle size: DLS measurements were taken with a Nanosizer 90ZS to measure the size distribution and average size of the polymeric micelles (Malvern Instruments, Southborough, MA). The intensity of dispersed light was measured in relation to an incident beam at 90°C. After filtering the micellar aqueous solution with a membrane extruder with a pore size of 0.2 mm, the freeze-dried powder was dispersed in an aqueous buffer, and measurements were taken (Millipore, St Charles, MO). All of the data analysis was done automatically. The size that was measured was displayed as the mean of 20 runs with three measurements taken during each run ¹⁹.

Zeta Potential: At 25°C, the zeta potential of chitosan nanoparticles was calculated using a Zeta Plus Zeta Potential Analyzer (Brookhaven, USA). The samples were adjusted to pH 4.5 with 0.1 mM NaCl solution and automatically monitored to maintain a consistent ionic strength. Each sample was measured 3 times, and the values shown are the average of 2 replicate samples ²⁰.

Surface Morphology of Drug-loaded Chitosan Nano-particles by TEM: By using TEM, the surface morphology and size of the generated nanoparticles were determined. The nanoparticle was given one minute to adhere to the carbon substrate after being dropped a drop of nanosuspension onto a paraffin sheet. The remaining drug suspension was eliminated by adsorbing the drop on the corner of some filter paper. Following that, the grid was left on the phosphotungstate drop for 10 sec. The sample was air dried after filter paper was used to absorb any remaining liquid. The sample was studied by TEM (Morgagni 268D TEM, Massachusetts, USA) ²¹.

In-vitro Release Study: Using the dialysis method, the in-vitro release of curcumin from the curcumin solution was calculated. 40 mL of PBS containing 0.1% Tween-80 were used to dissolve 4 mL of each formulation in a dialysis bag (MWCO 3500 Da). A 100-rpm swirl was applied to the dialysis bag at 37°C. 1 mL of the sample was taken out and the same volume of fresh media was added at the predetermined intervals (0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, and 72 hrs.). The HPLC technique was used to determine the concentration of curcumin ²².

Ex-vivo Nasal Permeation Study: Skin attaches to the cell body (receptor) and cell cap (donor). The shot of isotonic saline solution was given through a specially designed passageway. The lower part of the water jacket surrounding the isotonic saline solution chamber receives temperature-controlled water, which circulates out through the upper part to ensure a constant temperature of 32°C. A Teflon-coated magnetic bar provides agitation and promotes uniform distribution of temperature throughout the saline bathing solution.

A syringe is used to withdraw the sample, and the drug content is determined using a UV spectrophotometer. The receptor cell was filled with medium, and 1 ml of nanoparticle was applied to the skin surface of donor compartment. The receptor media was kept at 35.5 ± 0.5°C and agitated magnetically at 600 rpm. After the test formulation was applied to the donor side, 0.5 ml of an aliquot was taken from the receiver cell at specified intervals (i.e., 0, 1, 2, 3, 4, 5, 8, 10, 12 and 24 hrs.) for a period of 24 hrs and immediately replaced with the same amount of fresh media maintained at 35 ± 0.5°C. The samples were filtered through a 0.45 m membrane filter after the necessary dilutions, and the amt. of drug in the receptor medium was determined using a UV spectrophotometer at 425 nm ²³.

Data Analysis: Measurement and plotting the rate of curcumin permeation through the skin (Q, g/cm²) as a function of time (hrs.) was carried out. The drug flux at steady state was calculated using the slope of the linear component of the curve (Js, g/cm2/ hr) (Misra et al., 2016)

Cumulative amount of drug permeated = (Concentration (μg) × Volume of diffusion cell) / (Area (cm^²))

Volume of diffusion cell = 20 ml

Area of diffusion cell = 3 cm²

Flux = Slope of steady-state portion of the cumulative amount of drug permeation v / s time (µg/cm2 / hrs.)

Permeability coefficient (Pb) = Flux / (Drug concentration in donor compartment (μg/ml))

Drug concentration in donor compartment (Cd) = 20 µg/ml.

RESULTS:

Solubility Record:

TABLE 1: CURCUMIN’S SOLUBILITY DATA WERE AS FOLLOWS

Curcumin is freely soluble in methanol, insoluble in water, readily soluble in ethyl acetate and acetonitrile and sparingly soluble in hexane.

Melting Point Determination:

TABLE 2: MELTING POINT OF CURCUMIN FOUND AS FOLLOWS

Curcumin was discovered to have a melting point of 189°C. This value approximates the 183°C value mentioned in the literature.

Analytical Data:

Calibration Curve of Curcumin:

TABLE 3: WAVELENGTH OF CURCUMIN

FIG. 3: CALIBRATION CURVE OF CURCUMIN

FTIR Spectra of Curcumin showing Characteristics Band and Stretching: IR spectra of curcumin Fig. 4 display stretching vibrations at around 1628 cm^-1, primarily attribute to the overlapping stretching vibrations of alkenes (C=C) and carbonyl (C=O).

FTIR Spectrum:

FIG. 4: FTIR SPECTRA OF CURCUMIN

The ligands IR spectrum exhibits stretching vibrations caused by O- groups at 3200- 3500 cm^-1, C=C aromatic stretching vibration at 1427 cm^-1, and a high intensity band at 1512 cm^-1 that includes mixed vibrations of stretching carbonyl bond vibrations (C=O) and in- plane bending vibrations around aliphatic CC-C, CC=O.

DSC:

FIG. 5: DSC ANALYSIS OF CURCUMIN

Fig. 5 displays Curcumin DSC thermograms. The drug showed an endothermic peak at 178.152°C and the melting point of Curcumin is 183ºC according to literature citation.

HPLC:

FIG. 6: REPRESENTATIVE HPLC CHROMATOGRAPH OF CURCUMIN

TABLE 4: CALIBRATION CURVE OF CURCUMIN IN PBS AT pH 7.4

FIG. 7: CALIBRATION OF CURCUMIN BY RP- HPLC METHOD

A calibration curve was prepared using 2 - 10 µg/ml stock solution to determine the linearity of the proposed method. The retention time of curcumin was found by 7.34 ± 0.1 min. The chromatogram showed good peak symmetry with no tailing as already shown in Fig. 7. Peak area v/s concentrations were plotted to create a calibration curve, and the regression equation was computed using the average of 3 triplicate observations with a coefficient of correlation i.e., R²= 0.9998, the regression equation, y = 29362x, where y denotes the peak area and x denotes the concentration in g/ml, provided the mean calibration curve.

Visual Observation:

TABLE 5: BASED ON VISUAL OBSERVATION, THE EFFECT OF VARIOUS CHITOSAN CONCENTRATIONS IS DETERMINED

Loading Capacity, Encapsulation Efficiency, and Percentage Yield Determination of Nanoparticles:

TABLE 6: DRUG CONCENTRATION ON ENTRAPMENT EFFICIENCY

TABLE 7: EFFECT OF DRUG CONCENTRATION ON LOADING CAPACITY

Characterization of Nanoparticles:

TABLE 8: CHARACTERISTICS OF THE DEVELOPED NANOPARTICLES

 Particle size measurement by Dynamic Light Scattering (DLS):

TABLE 9: PARTICLE SIZE DISTRIBUTION AND PARTICLE SIZE OF PLACEBO FORMULATIONS

TABLE 10: PARTICLE SIZE DISTRIBUTION AND PARTICLE SIZE OF THE DRUG-LOADED FORMULATION

Particle size: The particle size of the optimized NPs (LF-21D, LF-22D, and LF-23D) was estimated using a DLS instrument and has been given in Fig. 8, 9, and 10 respectively.

FIG. 8: PARTICLE SIZE ANALYSIS OF DRUG-LOADED FORMULATIONS                               

FIG. 9: ANALYSIS OF PARTICLE SIZE OF DRUG-LOADED FORMULATION

FIG. 10: ANALYSIS OF PARTICLE SIZE OF DRUG-LOADED FORMULATION

Zeta Potential: Zeta potential research is a crucial component of nanoparticle characterization because surface charge plays a crucial role in enabling particle systems to adhere to biological surfaces and ensuring the stability of suspensions. A prominent property of chitosan/TPP particles is that chitosan nanoparticles are all positively charged. The process by which particles form can be used to explain this; specifically, how positively charged -NH₂ groups interrelate with the negatively charged tripolyphosphate molecules to neutralize their charges. The positive potential would be brought on by the lingering amino groups. Chitosan nanoparticles must be more stable because they have a greater zeta potential in an optimum range. Long amino groups appear to impede anion adsorption and maintain a high electrical double layer thickness, avoiding the formation of aggregates. The zeta potential of CS-Nanoparticles is to be -35.8 mv.

FIG. 11: ZETA POTENTIAL OF THE DRUG-LOADED FORMULATION

Surface Morphology of Drug-loaded Chitosan Nanoparticles by TEM:

FIG. 12: TEM IMAGE OF THE OPTIMIZED CURCUMIN NANOPARTICLES

Fig. 12, which depicts TEM of the polymeric NPs, shows that the particles have sphere-shaped morphology, a smooth surface, and a size range of about <100 nm, which is equivalent to the size determined by Dynamic Light Scattering.

Ex-vivo Nasal Permeation Study:

TABLE 11: COMPARATIVE EX-VIVO NASAL PERMEATION STUDY

FIG. 13: IN-VITRO PERMEATION STUDY OF SELECTED FORMULATION                      

FIG. 14: PERMEATION OF CONTROL

In-vitro Release Profile:

TABLE 12: CURCUMIN DRUG RELEASE IN-VITRO AT pH 7.4 IN PHOSPHATE BUFFER

FIG. 15: CURCUMIN-LOADED CHITOSAN NANOPARTICLES RELEASED IN-VITRO

Fig. 15 depicts the curcumin release profile from chitosan nanoparticles. The obtained kinetic profile accounts for the existence of two distinct nanoparticle release mechanisms. Curcumin released in vitro demonstrated a quick initial release followed by a gradual drug release. The first quick release of medication might be attributed to the release of curcumin from the nanoparticle surface, however later on, as a result of chitosan hydration and swelling, curcumin was regularly released from the core of nanoparticles.

Determination of Release Rate Order:

Investigation of the Drug Release Profile of Drug-loaded Chitosan Nanoparticles in Phosphate Buffer at pH 7.4:

TABLE 13: KINETICS ANALYSIS OF CURCUMIN LOADED CHITOSAN NANOPARTICLES

In-vitro Release Kinetics Study:

FIG. 16: THE RELEASE PROFILE OF CURCUMIN BY ZERO ORDER RELEASE MODEL OF OPTIMIZED NANOPARTICLES

FIG. 17: THE RELEASE PROFILE OF CURCUMIN BY FIRST ORDER RELEASE MODEL OF OPTIMIZED NANOPARTICLES

FIG. 18: THE RELEASE PROFILE OF CURCUMIN BY HIGUCHI MODEL OF OPTIMIZED NANOPARTICLES

FIG. 19: THE RELEASE PROFILE OF CURCUMIN BY THE PEPPAS MODEL OF OPTIMIZED NANOPARTICLES

TABLE 14: CHITOSAN NANOPARTICLES WITH AN OPTIMIZED COEFFICIENT OF CORRELATION

Fig. 19 depicts the curcumin release profile from chitosan nanoparticles. The % drug release profile demonstrated the drug's sustained release from the formulation. Curcumin released from the in-vitro formulation revealed an instantaneous release for 1 hr. (greater than 30%) followed by slow drug release for 24 hours. The first instant release of medication may be attributable to the release of Curcumin from the nanoparticle surface, whereas later on, as a result of chitosan hydration and swelling, curcumin may be constantly release from the core of nanoparticles. To determine release order, a kinetic analysis of the in-vitro profile of the optimized chitosan nanoparticles was performed. Because the correlation coefficient (R²) for the Peppas model of release was closer to unity, that is, (0.9863) for curcumin-loaded chitosan nanoparticles, the Peppas model was the best fit model for chitosan nanoparticles.

The Peppas equation was used to assess the release data, revealing a range of 0.43 to 0.85 for the release exponent 'n'. This suggests that drug release was governed by a combination of swelling and diffusion, also known as anomalous transport.

CONCLUSION: The drug curcumin was freely soluble in methanol. The melting point was reported to be 183°C which was in accordance with the literature cited value i.e., 183°C. The calibration curve of curcumin in PBS with pH 7.4 shows an R² value of 0.9895 at 425 nm. HPLC of curcumin was carried out using water: methanol: 2-propanol with the rate of flowing 1ml/min and retention time reported to be 7.34 ± 0.1 min.

The DSC thermogram of curcumin indicated an endothermic peak at 178.152°C and a melting point of 183°C. The selected optimum formulation which depends on visual observation is further evaluated for particle size, percentage yield, entrapment efficiency (EE), drug loading (LC), surface morphology, in-vitro release models, and ex-vivo permeation studies. The average size of Curcumin-loaded CS NPs was found to be 183.7 ± 8.4nm (LF-21D), 255.4 ± 5.6nm (LF-22D), and 341.3 ± 4.7nm (LF-23D). The EE and LC were found to be 81.6 ± 4.2 and 70.69 ± 1.8 respectively, for the drug: polymer ratio (2:1). In-vitro kinetic investigation of the optimized chitosan NPs was done to determine release order. The Peppas model was the best appropriate model for CS-NP’s because its correlation coefficient (R²) was closer to unity, that is (0.9863) for curcumin-loaded CS NPs. The Peppas equation was used to analyze the release data, and the results indicated that both swelling and diffusion- i.e., anomalous transport- were responsible for controlling the drug release. The release exponent, or “n”, ranged from 0.43-0.85. The maximum flux in the nasal permeation of curcumin from nanoparticle NP₂ (L22) is to be 332.9 µg/cm²/hr. as compared to the flux of control i.e., 31.495 µg/cm²/hr. The enhancement ratio of the curcumin nanoparticle formulation (L21, L22, and L23) was found to be 9.76, 10.57, and 9.69 respectively. The Arrhenius method and ICH guidelines were followed in the accelerated stability study of Cur-NP, and a shelf life of 1.58 years was obtained. We can therefore reach the conclusion that the optimized Curcumin nanoparticles were resistant to physical stress.

As a result, sustained release formulations for intranasal delivery systems were shown to be potentially effective.

ACKNOWLEDGMENTS: We would like to express our gratitude to our supervisor, participants, institution, and families and friends for their support throughout this research project. Our supervisor's guidance and feedback were invaluable, and the participants' contributions were essential to the success of this study. The resources and facilities provided by our institution were crucial to the smooth execution of our research.

CONFLICTS OF INTEREST: There is no any conflict of interest among the authors.

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    How to cite this article:

    Mangal L, Kaushik N and Alam S: Formulation and evaluation of curcumin-loaded chitosan nanoparticles and its intranasal efficacy for CNS delivery. Int J Pharm Sci & Res 2023; 14(12): 5916-29. doi: 10.13040/IJPSR.0975-8232.14(12).5916-29.

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