INTRODUCTION:

Nimesulide is a preferential COX-2 inhibitor that has been effectively used for the treatment of a variety of inflammatory and painful conditions, including osteoarthritis. Nimesulide is almost completely absorbed orally, 99% plasma protein bound and have a half-life of 2-5 hours.^[1] Nimesulide upon oral administration cause liver failure. It has a volume of distribution (Vd) of 12-27 L. Thus it is not highly distributed and remains on the systemic circulation mainly, which may be a reason for liver failure.^[2] The drug has low extraction ratio of 0.1^[2], which indicates the drug is presenting more into the eliminating organ like liver – it is again harmful. This made the situation to bypass the route of administration from oral to topical, in diseases accompanying with symptoms like pain and inflammation. The drug loaded magnetite nanoparticles can be localized at a specific targeted area by application of external magnetic field where the drug molecules are gradually released.

Thus the therapeutic efficacy of the drug is improved by lowering the toxic side effects on healthy tissues^[3]. The aim of the present study was to formulate and evaluate nimesulide loaded magnetite nanoparticles.

MATERIALS AND METHODS:

Nimesulide was purchased from Research lab fine chem industries, Mumbai. Ferrous sulphate and ferric chloride was purchased from Nice chemicals (P) LTD, Kochi. All the other chemicals used were of analytical grade.

Preparation of magnetite nanoparticles ^[4]

Magnetite nanoparticles were prepared by co-precipitation of Fe²⁺ and Fe³⁺ ions in the presence of a base. 30g of ferrous sulphate and 40g of ferric chloride were dissolved in 100ml deionized water. The solution is stirred using mechanical stirrer and added 3M NaOH solution quickly at 30⁰ C until the mixture reached a pH around 11. Kept it aside without stirring for 30min. After 30min, the mixture was heated to 80⁰C for 30min.

Preparation of drug loaded magnetite nanoparticles

Solvent evaporation method ^{[5, 6]}:

In solvent evaporation method the drug- polymer solution was initially prepared by dissolving the required quantity of polymer in acetone followed by the drug nimesulide to the above solution and dissolved. The required quantity of magnetite was added to the drug-polymer solution and stirred well to form a homogeneous mixture. This organic phase was poured drop wise to 1:1 mixture of light and heavy liquid paraffin with vigorous stirring over a mechanical stirrer for 8 hrs. To the stirred contents, hexane was added and filtered. This is again washed with hexane and finally with water. Then the product is dried. The detailed formula is given in table No 1.

Table 1: Formula for drug loading by Solvent evaporation method

Sl. No.	Ingredients	Batch
Sl. No.	Ingredients	F1
1	Nimesulide	100mg
2	Magnetite	50mg
3	HPMC K15M	100mg
4	Heavy liquid paraffin	12.5 ml
5	Light liquid paraffin	12.5 ml
6	Acetone	10ml
7	hexane	20ml

Powder coating method ^[7]

In powder coating method, the polymer solution is prepared by adding the polymer in small quantities under stirring, to get a clear solution. The Mixture of drug and magnetite was loaded into the Pelletizer and heated for few minutes to dry the particles and to ensure immediate drying during the coating process. The polymer solution is sprayed over the drug magnetite mixture at a temperature of 60^oC. The quantified volume of polymer solution is sprayed over core material to coat the core at different levels of polymer concentration such as 10, 20, 30, 40 and 50 mg using HPMC E5 and PVP K30. Directly 50mg was of HPMC K15M was coated for another batch to compare the process with Solvent evaporation method. The unit formula for Powder coating method is given in table No 2.

Table 2: Formula for drug loading by Powder coating method

Sl. No.	Ingredients	Batches
Sl. No.	Ingredients	F2	F3	F4
Core Layer
1	Nimesulide	1g	1g	1g
2	Magnetite	1g	1g	1g
Coating Layer
3	HPMC K15M	200mg	-	-
4	PVP K30	-	200mg	-
5	HPMC E5	-	-	200mg
6	Distilled water	20 ml	20 ml	20 ml

Evaluation Test:

Preformulation Study:

Pre-formulation studies such as miscibility study, melting point determination and drug excipient compatibility studies of drug was carried out.

Evaluation of Magnetite nanoparticles.

Percentage yield, particle size analysis ^[9,
10] and SEM ^{[6, 8]} of magnetite particles were carried out

Evaluation of DLMNPs:

Drug content and dissolution study of the drug loaded magnetite particles ^{[11, 12]} were carried out as per pharmacopoeial procedure.

RESULTS AND DISCUSSION:

Miscibility study

Miscibility study of the solvents used for solvent evaporation method is carried out and the results are given in the table 3.

Table 3: Results for Miscibility study

Experiment	Observation
Miscibility of heavy liquid paraffin and acetone	Not miscible
Miscibility of light liquid paraffin and acetone	Not miscible
Miscibility of heavy liquid paraffin and n-hexane	Miscible
Miscibility of light liquid paraffin and n-hexane	Miscible
Miscibility of n-hexane and acetone	Miscible

Heavy liquid paraffin and light liquid paraffin were found to be immiscible with acetone. So, in solvent evaporation method, mixture of heavy liquid paraffin and light liquid paraffin can be used as the continuous phase and acetone can be used as the organic solvent containing drug, polymer and magnetite. The immiscible nature of acetone with heavy liquid paraffin and light liquid paraffin will helps to load drug over the magnetite particles. The miscibility of heavy liquid paraffin, light liquid paraffin and acetone with n-hexane will helps to wash out these solvents from the drug loaded magnetite particles.

Melting Point Determination:

The melting point of pure drug nimesulide was found to be 147^oC which was close to the reported melting point (147-151⁰C) of nimesulide. ^[9] Since the melting point of nimesulide is high, nimesulide will not melt during temperature conditions of powder coating method.

Drug excipient compatibility study

The compatibility study of pure drug (nimesulide) and magnetite with HPMC and PVP were carried out. The physical changes of initial sample and the final samples were verified and the results are given in the table 4. No physical changes were observed for both initial and final samples.

FTIR spectroscopy analysis:

The FTIR spectra of mixture of nimesulide with polymers such as HPMC and PVP before and after the stability study are given in figure 1-5. Major peaks of nimesulide are present in the FTIR spectrum of initial samples of nimesulide and polymer mixture. The FTIR spectrum of final samples of nimesulide with HPMC and PVP has no significant variation of peaks in the graph. So, no chemical changes are occurred between nimesulide and polymers such as HPMC and PVP during the stability condition. Therefore HPMC and PVP are compatible with nimesulide. So these polymers are suitable for drug loading.

The FTIR spectra of mixture of magnetite with polymers such as HPMC and PVP before and after the stability study are given in figure 6-10. Major peaks in the spectrum of magnetite are present in the FTIR spectrum of initial samples of magnetite and polymers. The FTIR spectrums of final samples of magnetite with HPMC and PVP have no significant variation of peaks. So no chemical changes are occurred between magnetite and polymers such as HPMC and PVP. So these polymers are compatible with magnetite and they are suitable for drug loading.

Figure 1: FTIR spectrum of Nimesulide

Figure 2: FTIR spectrum of initial sample of Nimesulide and HPMC

Figure 3: FTIR spectrum of stability sample of Nimesulide and HPMC

Figure 4: FTIR spectrum of initial sample of Nimesulide and PVP

Figure 5: FTIR spectrum of stability sample of Nimesulide and PVP

Figure 6: FTIR spectrum of magnetite

Figure 7: FTIR spectrum of initial sample of magnetite and HPMC

Figure 8: FTIR spectrum of stability sample of magnetite and HPMC

Figure 9: FTIR spectrum of initial sample of magnetite and PVP

Figure 10: FTIR spectrum of stability sample of magnetite and PVP

Results for evaluations of magnetite particles:

The percentage yield of the prepared magnetite nanoparticles was found to be 95%. This ensures the optimal process. The particle size analysis of the prepared magnetite particles were carried out by optical microscopy and was found to be less than 1µm (Figure 11). The SEM images of magnetite particles and drug loaded magnetite particles prescribed in figure 12 and figure 13 shows both the particles were mostly rounded in shape.

Drug content:

By solvent evaporation method, the drug content was found to be 53%. Powder coating method using the polymers PVP K30 and HPMC E5 was found to be more efficient in drug loading compared with solvent evaporation method. Among these polymers, HPMC E5 has more drug loading efficiency. Figure 14 shows the effect of PVP K30 and HPMC E5 polymer concentration in drug content. With increase in the concentration of PVP, there is only slight change in the drug content. But, with increase in the concentration of HPMC E5, the percentage drug content also increased. So, the concentration of PVP K30 have less effect in drug content and have less drug loading efficiency than HPMC E5. HPMC E5 also have more percentage drug content than HPMC K15M. So, polymer HPMC E5 have more drug loading efficiency than HPMC K15M and PVP.

Figure 11: Particle size analysis of magnetite

Figure 12: SEM image of magnetite particles

Figure 13: SEM image of drug loaded magnetite particles

Figure 14: Effect of PVP K30 and HPMC E5 concentration in drug content

Drug release profile:

Figure 16 shows the comparative graph of dissolution profile of the drug loaded magnetite particles prepared by solvent evaporation method and powder coating method using HPMC 15 M. The study was conducted for a duration of 48 hours. Both the method produces a similar drug release near to 95% at the 48 hr, but the initial release pattern of both the method are different. The one prepared by powder coating method produces higher initial drug release then the other prepare using solvent evaporation method. An initial burst effect of drug is always required to produce the minimum effective concentration and produces the onset of action at this time. The batch with solvent evaporation technique has slower dissolution profile and it has increased only after 20 hours. The powder coating producers a constant drug release pattern from the 5th hour to the 48 Hour. This constant release pattern makes the powder coating method better as compared with that of the solvent evaporation method.

Figure 16: Comparative dissolution profile of drug loaded by solvent evaporation technique and Powder coating method

Figure 17 shows the dissolution data of drug loaded magnetite particles coated with different polymer concentrations of PVP K30. The batches behave differently in terms of percentage relative release. Percentage cumulative release found to be increased with increasing polymer concentration this is absolutely a different observation. At the initial time points the percentage CR highest for 50 mg coated particles than 10 mg coated. At the 48 Hour the 50 mg batch gives the maximum drug release near to 95% whereas 10mg batch producers only 70% of drug release even it has produced at 24^th hour. This result shows the difference in drug content at lower concentration of polymer. Since it is powder coating method using a 10mg polymer the drug might not be completely coated over the magnetite particles whereas at 50 mg polymer concentration the drug might be fully coated this is the reason why there is a higher release at the initial time with the batch having a higher polymer concentration. The same scenario is observed in another experiment conducted using HPMC E5 as the polymer figure 18, there also the higher concentration produces the better results it is 50 mg of HPMC E5 produces a burst effect at the initial time. Maximum release of 98% at the 48 hour. Based on this comparative study it is concluded that irrespective of the nature of the polymer a minimum 50 mg must be coated to get the maximum drug concentration over the magnetic particles. While comparing the 50 mg concentration of PVP with HPMC both 50Mg produces an initial burst release as well as an extended release upto 48 hours with a release of 95%. There is no great difference between both the groups at 50mg level. But a 5% higher release is observed with HPMC, it cannot be neglected as it releases the maximum drug. Based on this observation the batch with HPMC E5 is considered as the best one

Figure 17: Dissolution data of DLMNPs coated with different concentrations of PVP

Figure 18: Dissolution data of DLMNPs coated with different concentrations of HPMC E 5

CONCLUSION:

In this work, an attempt was made to formulate and evaluate nimesulide loaded magnetite nanoparticles. Nimesulide was subjected to pre-formulation studies and identified the compatibility of drug and magnetite with excipients by FTIR spectroscopy. Magnetite particles were prepared by co-precipitation method. The prepared magnetite particles were evaluated for particle size, shape and flow property. Drug loading over the magnetite was done by powder coating method using HPMC E5 polymer and found to have more drug loading efficiency and dissolution profile.

ACKNOWLEDGEMENT:

This study was supported by Department of Pharmaceutics, Nirmala College of Pharmacy, Kerala, India.

REFERENCES:

1. Tripathi KD. Essentials of medical pharmacology. 7th ed: 203-04.

2. Bernareggi A. Clinical pharmacokinetics of nimesulide. Clinical Pharmacokinetics. 1998; 35(4): 247-274.

3. Silambarasi T, Latha S, Thombiduarai M, Selvamani P. Formulation and evaluation of curcumin loaded magnetic nanoparticles for cancer therapy. International Journal of Pharmaceutical Sciences and Research. 2012; 3(5):1393-1399.

4. Vinod KR, Sridhar D, Sandhya S et al. International Journal of Pharmaceutical Sciences and Nanotechnology. 2012; 4(4): 1519-1520.

5. Kakar S, Batra D, Singh R. Preparation and evaluation of magnetic microspheres of mesalamine (5-aminosalicylic acid) for colon drug delivery. Journal of Acute Disease. 2013; 2(3):226-231.

6. Kakar S, Batra D, Singh R et al. Magnetic microspheres as magical novel drug delivery system: a review. Journal of Acute Disease. 2013; 1(2): 1-12.

7. Du Z, Wen S, Wang J et al. The review of powder coatings. Journal of Material Science and Chemical Engineering. 2016; 4: 54-59.

8. Chandna A, Kakar S, Batra D et al. A review on target drug delivery: magnetic microspheres. Journal of Acute Disease. 2013: 189-195.

9. Scherer F, Anton M, Schillinger U. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Therapy. 2002; 9: 102-109.

10. Zhang X, Chen F. A novel method to prepare magnetite chitosan microspheres conjugated with methotrexate for the controlled release of methotrexate as a magnetic targeting drug delivery system. Drug Delivery. 2009; 16(5): 280-288.

11. Dhanaraju MD, Thirumurugan G. Dissolution profiling of nimesulide solid dispersion with polyethylene glycol, talc and their combinations as dispersion carriers. International Journal of Pharmaceutical Technology and Research. 2010; 2(1): 480-484.

12. Asmatulu R, Fakhari A, Wamocha HL et al. drug-carrying magnetic nanocomposite particles for potential drug delivery systems. Journal of Nanotechnology. 2009: 1-6.

Received on 21.02.2019 Modified on 05.04.2019

Research J. Pharm. and Tech. 2019; 12(10):4651-4656.

DOI: 10.5958/0974-360X.2019.00801.1

Sample No	Sample	Initial		Stability Sample 40°C ± 2°C/75% RH ± 5% RH		Observation
Sample No	Sample	Colour	Texture	Colour	Texture	Observation
1	Nimesulide and HPMC	Pale yellow	Powder	Pale yellow	Powder	No change
2	Nimesulide and PVP	Pale yellow	Powder	Pale yellow	Powder	No change
3	Magnetite and HPMC	Pale yellow	Powder	Pale yellow	Powder	No change
4	Magnetite and PVP	Pale yellow	Powder	Pale yellow	Powder	No change