INTRODUCTION:

Mesalamine is used to both alleviate the symptoms of ulcerative colitis, a disorder that results in swelling and ulcers in the lining of the colon (large intestine) and rectum. The group of drugs known as anti-inflammatory agents includes mesalamine.

Figure No. 1: Chemical Structure of Mesalamine

A non-steroidal anti-inflammatory medication (NSAIDS) like acetylsalicylic acid that has a structural connection to salicylates and is effective against inflammatory bowel disease. It is thought to be the primary portion of sulphasalazine. Although Mesalazine is unquestionably helpful in treating and sustaining remission from ulcerative colitis, it has actually encountered numerous issues relating to its lack of stability as a pharmacological agent. Important research organizations developed stable Mesalazine formulations in the middle of the 1970s and 1980s, such as the eudragit-S coating of Asacol brand Mesalazine and the encapsulation of Mesalazine in micro granules under the Pentasa brand. Mesalazine's ability to reduce inflammatory activity and, in turn, potentially lower the risk of colon cancer in disorders like ulcerative colitis, is still being researched today along with new ways to stabilize the drug.¹

Since their introduction in the early 1990s, solid lipid nanoparticles (SLNs) have been considered the most efficient lipid-based colloidal carriers. This is one of the most widely used methods for increasing the oral bioavailability of medicines that are less water-soluble. SLNs are physiologically indulged lipid components that are in a solid form at room temperature and are in the submicron size range of 50–1000 nm. Small size, broad surface area, high drug loading, phase interaction at the interface, and irresistible for their capacity to enhance the performance of pharmaceuticals are some of the special qualities that SLN provided.^2,3

An anti-inflammatory medication called 5-aminosalicylic acid (5-ASA), also known as mesalamine (USAN), Mesalazine (INN, BAN), or 5-aminosalicylic acid, is used to treat conditions such ulcerative colitis and mild-to-moderate Crohn's disease. It is a bowel-specific aminosalicylate medication that works locally in the gut, primarily by preventing the production of prostaglandins and leukotrienes, preventing bacterial peptide-induced neutrophil chemotaxis and adenosine-induced secretion, scavenging reactive oxygen metabolites, and possibly by inhibiting the activation of nuclear factor (kappa) B. This results in fewer systemic side effects like headache, malaise, abdominal pain, cramps, flatulence, and gas. An anti-inflammatory medication called 5-aminosalicylic acid (5-ASA), also known as mesalamine (USAN), Mesalazine (INN, BAN), or 5-aminosalicylic acid, is used to treat conditions such ulcerative colitis and mild-to-moderate Crohn's disease. It is a bowel-specific aminosalicylate medication that works locally in the gut, primarily by preventing the production of prostaglandins and leukotrienes, preventing bacterial peptide-induced neutrophil chemotaxis and adenosine-induced secretion, scavenging reactive oxygen metabolites, and possibly by inhibiting the activation of nuclear factor (kappa)B. This results in fewer systemic side effects like headache, malaise, abdominal pain, cramps, flatulence, and gas.⁴

The crucial steps in the development of new drugs include stability studies, which include stress tests and forced degradation tests. These tests evaluate the stability of drugs under various storage and shipping conditions, the shelf life of the dosage form, and the choice of primary packaging material. They develop a thorough understanding of a new molecule's chemical activity, its degradation mechanisms, and its intrinsic stability. Consequently, it is necessary to speed up the registration process for novel drug substances or products.⁵

SLNs were created in the current investigation utilizing the microemulsion technique. In order to characterize the prepared formulations and study their physicochemical properties in order to gauge the stability of SLN formulations, this work concentrated on developing novel SLN formulations that contain Mesalamine using various concentrations of solid lipids, surfactants, and other excipients.

Solid lipid nanoparticles are at the forefront of the rapidly developing field of nanotechnology with several potential applications in drug delivery, clinical medicine and research, as well as in other varied sciences and mesalamine is vital medication to treat many disease hence there is need to formulate mesalamine in loaded solid lipid nanoparticles.

The objective of the present work is to optimize drug delivery and reduce toxicity in enhancing the use of selected drug.

MATERIALS AND METHODS:

Materials:

Reagents and Chemicals:

A free sample of Mesalamine was received from Sun Pharma Ahmednagar in Maharashtra. Tween 80 was purchased from Mumbai's Merk Chemicals. The solvents GMS, Cremophor, and others were sourced from various places. Chemicals and reagents were all of analytical quality.

Preparation of Mesalamine solid lipid Nanoparticles (MES-SLNs):

To create a dispersion of solid lipid nanoparticles, hot homogenization was applied. This method involved melting the lipid in a water bath at a temperature 100 C above its melting point, adding the necessary amount of mesalamine, and stirring the mixture constantly until the mesalamine was entirely dissolved.⁶ Distilled water was used to dissolve Tween 80, which was then heated to the same temperature as the lipid mixture. Tween 80 made up 2.5% of the total weight of the SLN dispersion and was utilized as a stabilizer. The melted lipid-drug mixture was then mixed with hot surfactant solution, and the mixture was homogenized in a homogenizer at 2000 rpm for one hour. Following that, the mixture was taken out of the water bath and put through a vacuum filter assembly. The filtrate that included SLNs was delicately blended by constant shaking until it reached room temperature. Studies on the stability and characterization of solid lipid nanoparticles loaded with Mesalamine were carried out using this dispersion (MES-SLNs)⁷. Data on formulation are provided in Table No. 1.

Evaluation of Mesalamine solid lipid nanoparticles (MES-SLNs):

Measurement of particle size and Polydispersity index:⁸

Using a Zetasizer 3000 HSA, photon correlation spectroscopy was used to determine the size of MES-SLNs (Malvern, UK). From the formula, the polydispersity of solid lipid nanoparticles of non-uniform size was determined.

D90-D10

Polydispersity=--------------

D50

Where D90, D10, and D50 are, respectively, the diameters of particles that fall within the 90th, 50th, and 10th percentiles of undesirable particles. These values were used to classify the nanoparticles as monodisperse, homogeneous, and heterogeneous systems. A greater polydispersity index value denotes a high amount of non-uniformity.⁹ In Table No. 2, several trial outcomes are displayed.

Zeta potential:

One of the elements used to determine the physical stability of suspensions and emulsions is particle charge. Physical stability increases with increasing electrostatic repulsion between the particles. Typically, the so-called zeta potential, which is measured by a Zeta meter (Malvern Instruments Ltd., UK), is used to quantify particle charge. One milliliter of MES-SLNs was dispersed in one milliliter of distilled water using sonication, and the mixture was then run through a zeta potential analyzer.¹⁰

Determination of entrapment efficiency:

The formulated SLNs were dissolved in methanol and PBS (pH 7.4) in a water bath at 65⁰C for 30 minutes, then cooled to room temperature to selectively precipitate the lipid in order to test the efficacy of drug entrapment. Using a UV-VIS spectrophotometer, the amount of medication in the supernatant was calculated after centrifugation (1000rpm for 15min) (Shimadzu 1700). The following equation was used to determine the entrapment efficiency.¹¹

Analyzed weight of the drug in SLN

Entrapment efficiency = -------------------------- x 100

Theoretical weight of the drug loded in SLN

Particle shape and surface Morphology:¹²

SEM was used to examine the surface morphology, size, and shape of the formulations as well as the aggregation behavior of the mesalamine-loaded SLNs in the presence of carrier particles.

In- vitro release studies of MES-SLNs:¹³

A dialysis membrane (Hi-media, Mumbai, India) with a pore size of 2.4nm and a molecular weight cut off between 12,000 and 14,000 Dalton was used for the in vitro release tests. 1% HCl solution was used to activate the membrane for 12hours. The donor compartment received a volume of 2ml of MES-SLNs, and the receptor compartment received 100ml of phosphate buffer with a pH of 6.8 at 37±0.5°C, agitated at 800rpm. At predetermined intervals, an aliquot of 3ml of the sample was taken out of the receiver compartment and replaced with new medium. At a wavelength of 303nm, a UV-Visible spectrophotometer was used to evaluate the samples. Several kinetic equations were fitted to data from in vitro release tests to determine. The process by which MES is released from SLN. In this work, kinetic models such as zero order, first order, and Higuchi were used.

Total drug content:¹⁴

A mixture of PBS (pH 7.4) and ethanol was used to dilute 1ml of the formulation to 10ml in order to estimate the assay. The mobile phase was used to create the final dilution, and the MES content was measured with a UV-Visible spectrophotometer at a wavelength of 303nm.

Differential Scanning Calorimeter:¹⁵

Shimadzu, Japan's DSC-60 differential scanning calorimeter was used to heat 4 mg of samples at a flow rate of 20ml/min in a nitrogen atmosphere. The samples were hermetically sealed in a flat-bottomed aluminium pan. The heating rate was 10°C/min with a temperature range of 20–200°C.

Stability studies:¹⁶

Physical stability studies:

For 90 days, it was determined whether the developed formulations were physically stable at ambient temperature, 40°C, - 2°C, and refrigerator temperature -20°C. Calculations were made of the average particle size, colour, and polydispersity at various time intervals of day 1, 2, and 3 months. Table No. 3 displays the results.

Chemical stability studies:

The chemical stability of MES-SLNs was investigated right away after formulation preparation. HPLC, Shimadzu LC solution, and the column was HYPERSIL-C18 (4.6 x 250mm) were used to analyze the MES-loaded SLNs that were standard for chemical stability. Acetonitrile and water were combined in the mobile phase at a flow rate of 1 ml/min at a 60:40 v/v ratio. The detection was carried out at 330nm in wavelength. Figure 7 shows the chromatogram that was produced from the fresh formulation.

RESULTS AND DISCUSSION:

Scanning electron microscopy of MES-SLNs:

The Scanning electron microscopy (SEM) photograph of the optimized formulation (F-7) is shown in Figure 2. In SLN dispersion, particlesize was found to be less than 100nm in size with a spherical shape and almost smooth surface.

Figure 2. SEM image of solid lipid nanoparticles

Particle size and Polydispersity index of SLNs:

It was discovered that the particle size of formulation (F-7) was 82.1±5.37nm. seen in Figure 3. The development of SLNs with smaller particle sizes was encouraged as a result of a reduction in the interfacial tension between the lipid matrix and the dispersion medium (aqueous phase) caused by an increase in surfactant content in SLN formulations. The stability of SLNs is also maintained by tween 80. To produce stable and smaller SLNs, tween 80 concentrations were changed from 1% to 3% (1gm to 3gm). It is clear from the data that using a concentration of 2.2% tween 80 was successful in achieving smaller particle sizes. The particle size was not reduced even when the concentration of tween 80 was increased further to 3%. These findings imply that a 2.2% tween 80 concentration is the ideal one.Enough to effectively coat nanoparticle surfaces and stop agglomeration during the homogenization process. Formulation information is provided in Table No. 1 and various experiment results are displayed in Table No. 2.

Figure 3: Particle size analysis graph of F-7

The improved SLNs were found to have a polydispersity index of 0.35. The polydispersity index (PI), which runs from 0 to 1, represents the width of the particle size distribution. Monodisperse populations theoretically show that PI = 0. However, a restricted size distribution is one where PI 0.2. Therefore, PI measurement was necessary to verify the particles' restricted size distribution.

Zeta potential:

The formulation's good physical stability is indicated by the SLNs' zeta potential (ZP), which was determined to be -13.9mV in Figure 4. Zeta potential has been demonstrated to influence how SLNs are distributed within cells in earlier research. Weakly basic pharmacological molecules interact electrostatically with the center of the nanoparticle matrix' negative charge. Therefore, the negative charge on nanoparticle surfaces may be a factor in the SLNs' negative zeta potential.

Table 1: Formulation Data

Sr. No.	Ingredient	F-1	F-2	F-3	F-4	F-5	F-6	F-7	F-8	F-9
1.	Mesalamine (mg)	400	400	400	400	400	400	400	400	400
2.	GMS(mg)	1%	1%	1%	1%	1.2%	1.2%	1%	1.2%	1.4%
3.	Tween 80	1.5%	2%	2.5%	3%	1.5%	1.8%	2.2%	1%	1.5%
4.	Cremophor	1gm	1gm	1gm	1gm	1gm	1gm	1gm	1gm	1gm
5.	Water	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s

Table 2: Trial results

Sr. No.	Formulation code	Particle size	PDI(%)	%EE	Drug content
1.	F-1	1369	1.44	35.7±1.09	140.9±4.97
2.	F-2	702	1.43	43.5±2.93	171.73±3.23
3.	F-3	226	0.66	86.5±2.02	302.0±3.07
4.	F-4	92	0.95	76.5±1.74	341.5±5.78
5.	F-5	325	0.13	80.5±3.34	317.8±4.65
6.	F-6	280	0.66	79.6±2.09	314.26±3.8
7.	F-7	82.1	0.35	83.6±1.37	334.3±3.22
8.	F-8	88.20	0.97	73.3±1.98	289.38±5.03
9.	F-9	133.3	1.016	72.5±3.23	286.2±3.31

Figure 4: Zeta potential graph of nanoparticles

The entrapment efficiency of MES-SLNs (%EE):

The formulation F7 of the MES-SLNs was found to have the highest entrapment efficiency, at 83.6%± 1.37%, whereas formulation F1 had the lowest, at 35.7%±1.09%. The concentration of the lipid utilized in MES-SLN formulations has increased, which has led to an improvement in entrapment efficiency.

In-vitro release studies of Mesalamine SLN:

Figure 5 displays the in vitro drug release characteristics of MES-loaded SLNs of the F-3, F-5, and F-7 formulations. The release of MES from the lipid particles was studied for 10hours using phosphate buffer (pH 7.4) as a dissolution medium in order to assess the burst release potential of the tested formulations. The formulation F7 produced a maximum level of drug release, which was seen over an extended length of time. The medicine was released continuously from the seventh to the tenth hour. This might be caused by the formulation's greater lipid concentration. Most SLN formulations' drug release data were well-fitting into the zero-order release kinetics (r2 values found in rang 0.871). This indicates that the test product follows matrix diffusion-based release kinetics.

Figure 5. In vitro drug release profiles of MES-loaded SLNs

Total drug content:

From the prepared SLN formulation, 1ml of dispersion is dissolved in 10ml of phosphate buffer solution pH7.4 (PBS) and ethanol mixture. The amount of Mesalamine was determined using a UV spectrophotometer at 303 nm. The placebo formulation prepared similarly to drug-loaded SLN is used as blank. The total drug content was calculated and it was found to be 334.3±3.22mg in 100 ml.

Differential scanning calorimetric studies:

DSC thermograms of pure drug, lipids, and mixture are shown in Figure6. From the results obtained, there was no obvious change in the endothermic peak of the physical mixture at 254°C. DSC thermograms obtained suggested that there were no appearance of new peaks or disappearance of the existing peak. Hence there was no considerable effect on the thermal behavior of the drug with the lipid matrix under the experimental conditions.

Figure 6: DSC analysis of Mesalamine and GMS

Stability studies:

Physical stability:

For physical stability determination F-7 formulations were stored in amber-colored bottles at room temperature, 40⁰C, 4⁰C, and refrigerator temperature(-2⁰C). Different parameters analyzed during physical stability studiesare particle size, color, and PDI. Samples were subjected to evaluation at intervals of every 30 days up to 3months. It was observed that there was a change in the particle size, color, and PDI with SLNs stored at 4⁰C whileno significant changes were observed with SLNs stored at room temperature, 40⁰C, and -2⁰C. The results are shown in Table no. 3.

Table 3: Results of Physical Stability Studies of F-7

Sr. No.	Time storage	Room temp.			40^oc			4^oc			-2^oc
Sr. No.	Time storage	color	Size	PDI	color	size	PDI	color	size	PDI	color	size	PDI
1.	0 day	L.B.	82.1	0.35	L.B.	82.1	0.35	L.B.	82.1	0.35	L.B.	82.1	0.35
2.	30 days	D.B.	89.10	0.87	L.B.	88.20	0.37	L.B.	4102.9	1.02	L.B	83.7	0.53
3.	60 days	D.B.	91.40	0.89	L.B.	93.3	0.42	L.B.	4205.7	1.32	L.B.	89.7	0.59
4.	90 days	D.B.	96.57	0.93	L.B.	98.7	0.43	L.B.	4275.9	1.24	L.B.	97.4	0.64

L.B. - Light Brown color, D.B. - Dark Brown color

Particle size analysis:

Particle analysis of F-7 at different time interval result is shown in Table no. 4

Chemical stability:

Chromatograms of the stored samples obtained were compared with the chromatogram of pure drugs and peaks other than the solvent peaks and the main drug peaks were considered as degradation products. No degradation was found with samples stored at 40⁰C, and room temperature after 3 months of storage.Slight degradation was found in samples stored at -2⁰C and 4⁰C. Results are shown in Figure 7.

Inthepresentstudy, anattempthasbeenmadetodesign the characters and develop the parameters for the stability study of Mesalamine drug in the loaded solid lipid nanoparticles. The study has been carried out as per the ICH guidelines.

Figure 7: Chemical stability results F-7 (3months)

CONCLUSION:

Thepotential dispersions as carriers for delivery of Mesalamine drug was formulated and exploited for characterization and stability studies. Solid Lipid Nanoparticles were prepared by hot homogenization method using bio-acceptablelipids such as GMS and Tween80as an emulsifier. Drug-loaded SLNs showed average diameters in the colloidal size range, a good loading capacity with drugburst release property. Formulated solid lipid nanoparticles showed good physical stability at Room temp, 40⁰C, and -2⁰C but poor stability at 4⁰C. In chemical stability studies no degradation was found with samples stored at 40⁰C, and room temperature after 3 months of storage. Slight degradation was found in samples stored at -2⁰C and 4⁰C. The results strongly support the potential application of SLNs in Mesalamine therapy as a drug delivery system.

The usage of SLNs has a number of benefits, including low toxicity, high bioavailability of pharmaceuticals, diversity in the inclusion of hydrophilic and lipophilic medications, and the potential for mass manufacture of the carrier systems. These nanoparticles' properties can be modified to deliver drugs in precise dosages to targeted tissues while reducing leakage and binding to non-target tissues.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this research.

ACKNOWLEDGMENTS:

The authors are very thankful to KLE College of Pharmacy Belagavi, ICMR Belagavi, and Basic Science Research Centre Belagavi for providing all the facilities for completing the research work.

REFERENCES:

1. Komal S. Lavhate, Rahul S. Solunke, Kore K. J. Rajkumar V. Madhuri S. Deshmukh T. Development and Characterization of Mesalamine Microsphere for Colon Specific Drug Delivery. Research J. Pharm. and Tech. 2020; 13(4): 1747-1751.doi: 0.5958/0974-360X.2020.00315.7.

2. Venugopal D. Karadi A. Arshad M. Raju A. Spectrophotometric Determination of Mesalamine by Diazotization Coupling Reaction in Solid Dosage Forms. Asian J. Research Chem. 2011; 4(6): 968-70. doi: 10.5958/0974-4150.

3. Motwani S. Khar R. Ahman F. Chpra S. Kohli K. Application of a validated stability-indicating densitometric thin-layer chromatographic method to stress degradation studies on moxifloxacin. Analytical Chemist. 2006; 582(1):75-82. doi: 10.1016/j.aca.2006.08.053.

4. Sagar D. Kadam, Shashikant Dhole, Sohan Chitlange. Formulation and Evaluation of Sustained Release Colon Targeted Mesalamine Tablet. Research J. Pharm. and Tech 2020; 13(5):2241-5. doi: 10.5958/0974-360X.2020.00403.5.

5. ICH Stability Testing of New Drug Substances and Products. Q1A (R2). ICH. 2006.

6. Maruthi R, Chandan R.S, Barath M, G Naveen Datta, Merryl D’silva, Kajal Kumari M, Farhan Ahmad, Geetha R. Analytical Method development and Validation of Teneligliptin by RP-UFLC. Research J. Pharm. and Tech. 2020; 13(9):4035-4040. doi: 10.5958/0974-360X.2020.00713.1.

7. Khaledi S. Jafari S. Hamidi S. Molavi O. Devaran S. Preparation and characterization of PLGA-PEG-PLGA polymeric nanoparticles for co-delivery of 5-Fluorouracil and Chrysin. J Biomater Sci Polym. 2020; 31(9): 1107-26. doi: 10.1080/09205063.2020.1743946.

8. Patle L. Formulation and Evaluation of Ondansetron Floating Tablet. Res. J. Pharm. Dosage Form. & Tech. 2017; 9(4): 173-179. doi:10.5958/0975-4377.2017.00028.3.

9. Ravi Kumar P. Padmavathi Y. Niveditha P. Babu N. Development and Validation of Difference Spectrophotometric Method for the Quantitative Estimation of Mesalamine in Bulk Drug and Dosage forms. Asian J. Pharm. Ana. 2017; 7(4): 225-8. doi: 10.5958/2231-5675.2017.00036.9

10. Mitri K, Shegokar R, Gohla S, Anselmi C, Müller RH. Lipid nanocarriers for dermal delivery of lutein: preparation, characterization, stability and performance. Int J Pharm. 2011; 414(1-2): 267-75. doi: 10.1016/j.ijpharm.2011.05.008.

11. Lim SJ, Kim CK. Formulation parameters determining the physicochemical characteristics of solid lipid nanoparticles loaded with all Trans’ retinoic acid. Int. J. Pharm. 2002; 243: 135-46. doi: 10.1016/s0378-5173(02)00269-7.

12. Huang G, Zhang N, Bi X, Dou M. Solid lipid nanoparticles of Temozolomide: Potential reduction ofcardiacl and nephric toxicity. Int J Pharm. 2008; 355: 314–20. https://doi.org/10.1016/j.ijpharm.2007.12.013.

13. Bhalekar MR, Pokharkar V, Madgulkar A, Patil N, Patil N. Preparation and evaluation of miconazole nitrate-loadedsolid lipid nanoparticles for topical delivery. AAPS Pharm Sci Tech.2009; 10: 289–96. doi: 10.1208/s12249-009-9199-0.

14. Mohammad Mojeeb Gulzar Khan, Atul Arun Shirkhedkar. New, Sensitive, Simple and Validated Stability Indicating TLC/ Densitometric Method for the Estimation of Anti-Ulcer Drug Mesalamine in Bulk and Tablet Formulation. Asian J. Research Chem. 2018; 11(6): 871-875. doi: 10.5958/0974-4150.2018.00152.9

15. Gupta M, Bhargava HN. Development and validation of a high-performance liquid chromatographic method for the analysis of budesonide. J Pharm Biomed Anal. 2006 Feb 13; 40(2):423-8. doi: 10.1016/j.jpba.2005.06.038.

16. Barath M, Chandan R. S, Maruthi R, N Paramakrishnan. Analytical Method Development and Validation of Etravirine by RP-UFLC. Research Journal of Pharmacy and Technology. 2021; 14(7): 3537-2. doi:10.52711/0974-360X.2021.00613.

Received on 12.09.2022 Modified on 28.01.2023

Research J. Pharm. and Tech 2023; 16(10):4767-4773.

DOI: 10.52711/0974-360X.2023.00773

Sr. no	Temp	1month	2months	3 months
1.	-2⁰C
2.	4⁰C
3.	Room temp.
4.	40⁰C