Design of Colon Specific Delivery of Sulfasalazine Loaded Nanoparticles for Inflammatory Bowel Syndrome: Application of Experimental Design

 

Sivakumar R*, Shafin P

Department of Pharmaceutics, Grace College of Pharmacy, Palakkad. Kerala - 678004, India.

*Corresponding Author E-mail: rrsk1879@gmail.com

 

ABSTRACT:

Sulfasalazine is the first line treatment for Inflammatory Bowel syndrome. Conventional formulation have poor targeting properties for colon site.Therefore designof colon specific nanoparticles (NP) is an ideal for better therapeutic outcome. The study aims to formulate and evaluate polymeric Nanoparticles loaded with Sulfasalazine to target the colon. The nanoparticles were prepared by Nano-precipitation method using pH dependent polymer and the prepared nanoparticles were evaluated for FTIR, morphology, pH, Viscosity, Entrapment efficiency, particle size and zeta potential etc. The nanoparticles were used to formulate core tablet and core tablets were coated with Eudragit S100. The tablets were evaluated for hardness, friability, thickness, weight variation and In vitro release study. Totally six formulations were developed for preliminary trial with varying concentration of polymers. From the preliminary trial, code F2batch was found to be best formulation. The F2batch was fitted to central composite design with two independent factors i.e. Concentration of Eudragit S100 (X1) and polyvinyl alcohol (X2) with four dependent variables i.e. Entrapment efficiency -Y1; Hardness- Y2; Friability-Y3 and In-vitro drug release- Y4. The formulation (code R6) was found to be the optimizedformulation. The optimized colon targeted tablets provide satisfactory release and might be suitable for colon targeting of sulfasalazine with better therapeutic effect.

 

KEYWORDS: Nanoparticles, Nano-precipitation, Sulfasalazine, Colon targeting, Experimental design.

 

 


INTRODUCTION:

Nanoparticles are novel dosage forms having size in Nano metric range. Nanoparticles can be utilized to deliver the drug to the target site without wasting the drug in a reduced dose1,2. Nanoparticles formulation, when converted into tablets which can be easily administrated to the patient. Hence, the nanoparticles-tablet combination would be a promising strategy in drug delivery systems3.

 

Inflammatory bowel disease includes two classes of diseases: Crohn’s disease and ulcerative colitis. The Crohn’s disease affects the entire gut whereas the ulcerative colitis affects the colonic part4.

 

Sulfasalazine is the first line drug used for the treatment of inflammatory bowel disease. It is a BCS - IV classified drug (Low solubility and low permeability). Hence, it is difficult to target the drug to the colonic region. Sulfasalazine, while loading to the polymeric nanoparticles which dissolve only in the colon pH, So that minimize the dose of the drug and target the drug to the colon with better patient compliance5.

 

A central composite design is an experimental design, helpful in response surface methodology, for building a second order (quadratic) model for the response variable without needing to use a complete three-level factorial experiment. After the designed experiment is performed, linear regression is used, sometimes iteratively, to obtain the accurate results6.

 

The aim of the research work is to develop and statistically optimize polymeric nanoparticles containing sulfasalazine tablets to target the colon.

 

 

MATERIALS AND METHODS:

Materials:

Sulfasalazine BP, Eudragit S100, Polyvinyl alcohol, Pluronic F127, Microcrystalline cellulose and Polyvinyl alcohol (Yarrow Chem products, Mumbai), Tween 20 and Acetone (Nice Chemicals, Cochin), Starch (Isochem, Cochin.All other chemicals were of analytical grade.

 

Drug excipient compatibility study:

Drug excipient compatibility was carried out using the blend of sulfasalazine and selected carriers using FT-IR.

 

Preparation of Drug Loaded Nanoparticles for Preliminary trial:

Different nanoparticle formulations containing sulfasalazine were prepared by Nano-precipitation method. Total amount of the drug, acetone and distilled water were constant. Totally six batch were developed for preliminary trial with varying concentration of excipients The drug and Eudragit S100 were dissolved in 20ml acetone. And the organic phase was quickly injected into an aqueous phase containing 40ml of 1% and 2% w/v of polyvinyl alcohol (PVA)/Tween 80/ Pluronic F127 with moderate magnetic stirring at room temperature.The organic phase to aqueous phase ratio was taken as 1:2 ratio. The nanoparticles were quickly formed and turned the solution which is slightly in turbid form. Then, acetone was completely removed by continuous stirring for 3-4h. The prepared nanoparticles were isolated by centrifuging the suspension at 40000rpm for 20 minutes at 50ºC. The isolated nanoparticles weresubjected for evaluation7.

 

Experimental Design:

A32 factorial design was employed to study the effect of concentration of Eudragit S100 and poly vinyl alcohol on the quality of nanoparticles, ‘K’ value was taken as response, the lower value as coded as -1, middle level as 0, and higher level as +1(Table1).

 

Data obtained using design expert software were subjected to fit quadratic model as shown in following equation.

 

Y= b0 + b1X1 + b2X2 + b12X11 + b22X22 + b12X1X2

 

Where, b is co-efficient in polynomial equation which shows the magnitude of effect on independent variables.

 

Development ofcoated tablets with optimized nanoparticles:

The optimized nanoparticles (R6) weight equivalent to 40mg of the drug was mixed withmicrocrystalline cellulose 200mg, magnesium stearate 5mg and talc 5mg. The tablets were prepared by the direct compress method using 8mm convex punch8. The core tablets were coated by enteric polymer. The coating solution containing Eudragit S100 (8%) was dissolved in acetone and isopropyl alcohol mixed solvents (1:1) and then polyethylene glycol (2%), titanium dioxide (1%) and Tartrazine yellow (0.5%) was added, stirred and filtered. The coating solution was applied on the tablets by dipping and drying method9.

 

Evaluation of nanoparticle for preliminary trial batches:

Particle size and zeta potential:

Particle size analysis and zeta potential was performed using a zeta sizer (Nano ZS Malvern instrument UK)

The zeta potential of nano suspension is measured using Zeta sizer Nano ZS at 25±0.5°C. A potential of ±150 mV is set in the instrument in 0.75ml capacity disposable cuvettes10.

 

Entrapment efficiency (%)

The entrapment efficiency of the prepared nanoparticles was calculated by the following formula10:

 

                             Total drug – Drug in supernatant

Entrapment (%) = -------------------------------------- x 100

Efficiency                             Total drug

 

Evaluation of coatedtablets:

Hardness Test:

Tablets were selected in a random and individual hardness of each tablet was determined by Pfizer hardness tester. The average hardness (Kg/cm2) was calculated and recorded (n = 3).

 

Thickness test:

Tablets were taken in a random and individual and thickness of each tablet was determined by screw gauge. The average thickness was calculated and recorded (n = 3).

 

Weight Variation test:

Twenty tablets were collected randomly and weighed individually. The individually weighed tablets compared with an average weight for the determination of weight variation. The percentage of deviation was calculated11.

 

Friability test:

Six tablets were initially weighed and transferred into Roche Friabilator and was operated at 25 rpm for 4 min. Then the tablets were weighed after rotation. The formula given below

 

Friability (%) = initial weight – final weight / initial weight X 100

 

Drug content:

Twenty tablets were taken, weighed and finely powdered. An accurately weighed quantity of this powder was taken and suitably dissolved under sonication at pH 7.4 phosphate buffer and filtered. The samples were analyzed by UV visible spectrophotometer after making appropriate dilutions12.

 

In vitrorelease study:

In vitro release study was carried out in using USP dissolution test (Paddle type) apparatus. The set condition was 50rpm, 900ml of pH 6.8 phosphate buffer, 37.0±0.5⁰C. Samples were withdrawn at predetermined intervals and analysed by UV spectrophotometer at 359 nm13.

 

Release kinetic study:

Drug release data were fitted in kinetic model equations for determining the mechanism of drug release from optimized nanoparticle containing colon specific tablet. Zero order, First order, Higuchi and Korsmeyer-Peppas equations used to understand the rate of drug release mechanism of the prepared formulations14.

 

Table 1: Layout and results of Sulfasalazine NP loaded tablets -Dependent variables

Runs

X1

X2

Y1

Y2

Y3

Y4

R1

-1(450)

-1 (450)

67.3 ± 4

3.9 ± 1

0.92 ± 3

39.5 ± 3

R2

-1 (450)

0 (500)

64.3 ± 3

5.3 ± 1

0.74 ± 4

45.5 ± 3

R3

-1 (450)

+1 (550)

65.4± 5

7.3 ± 2

0.41 ± 6

48.3 ± 2

R4

0(500)

-1 (450)

74.1 ± 4

3.6 ± 4

0.93 ± 2

42.3 ± 4

R5

0(500)

+1 (550)

74.8 ± 2

6.9 ± 2

0.34 ± 3

48.5 ± 2

R6

0(500)

0 (500)

73.5 ± 2

4.4 ± 3

0.69 ± 3

45.4 ± 1

R7

+1 (550)

-1 (450)

85.6 ± 1

4.1 ± 2

0.88 ± 4

41.8 ± 3

R8

+1 (550)

0( 500)

83.9 ± 4

4.0 ± 2

0.95 ± 2

44.9 ± 2

R9

+1 (550)

+1 (550)

80.4 ± 2

7.5 ± 4

0.46 ± 2

53.4 ± 2

Note: X1 = Concentration of Eudragit S 100(mg) X2= ConcentrationofPVA (mg) Y1= Entrapment Efficiency of NP (%) Y2=Hardness (Kg/cm2), Y3= Friability, (%) Y4=in- vitro drug release (%)

 

RESULTS AND DISCUSSION:

All the results are presented in Table 1-3 and Figure 1and 2. FTIR study indicated that the drug was compatible with all the excipients. The nanoparticles were prepared by Nano-precipitation method. Totally six formulations are prepared for preliminary trailsbased on the evaluation F2 formulation showed highest entrapment efficiency and agreeable particle size and zeta potential, hence that the formulation selected for optimization. In the present study process variable kept constant and formulation variable have been optimized using statistical design.

 

The colon targeted tablets containing sulfasalazine loaded nanoparticles were prepared by direct compression method and prepared tablets were subjected to all the quality control test.

 

All the important peaks are present in the FTIR spectra indicate FT-IR spectrum of Drug and Excipients did not differ with major peaks of Sulfasalazine, i.e.; all the major peaks of the drug appeared on the blend indicate that there is no interaction between drug and excipients.

 

The prepared nanoparticles were subjected to preliminary evaluation study. The pH the NP product were 4.0 - 5.5. The highest pH was found in the F6 formulation. Hence, the formulation wassuitable for colon targeting. The highest viscosity was found in F2 formulation due to the increased amount of Eudragit S100 (500mg) and poly vinyl alcohol (2%). The entrapment efficiency of the formulations was in the range of 65.3 - 78.8. When the concentration of polymer is increased, the binding ofthe drug to the core is increasing, and hence greater entrapment efficiency was seen in the F2, F4 and F6 formulations. The particle size was in the range of 150 -480nm.The least particle size was found in F3 formulation due to the less amount of Eudragit S100, because the polymers unable to swell when comes into contact with the aqueous phase. The zeta potential values were in the range of -26.6 to 4.28 mV.


Table 2: Results of Evaluation of Sulfasalazine loaded Nanoparticles – Preliminary Trial

S. No

Parameters

Formulation Code

F1

F2

F3

F4

F5

F6

1.

Entrapment efficiency (%)

75.6±3

78.8± 5

65.3± 4

69.6± 7

71.2± 6

74.2±4

2.

Particle size (nm)

152±3

206.4±2

480.7± 5

266.9±4

272± 6

165.2± 7

3.

Zeta Potential (mV)

-19.8

-26.6

-1.11

-1.19

4.28

1.78

 


The prepared nanoparticles were isolated by centrifugation,dried and blended with the excipients for tablet preparation. The hardness of the formulations was found to be higher in F2 formulation. Hardness of F2, F4 and F6 was foundhigher than F1, F3 and F5. This indicates that nanoparticles containing the highest amount of polymer (EudragitS100) showed better hardness than the other batches. The thickness of the formulation was in the range of 1.56 – 1.84mm. All the formulated tablets were passed weight variation test and the weight variation was within the IP limits of ±7.5% of the weight. The friability of the formulations were in the range of 0.48 – 0.76 %. The least friability was found in F2 formulation. This is due to the increased amount of polymer in the nanoparticles. The drug content was in the range from 94-99%. The drug content were increased in F2, F4 and F6 formulations. The results showed that increased in polymer concentration results in increased drug content of the nanoparticles.


 

Table 3: Results of evaluation of colon targeted tablets containing sulfasalazine loaded nanoparticles

S. No

Parameters

Formulation Code

F1

F2

F3

F4

F5

F6

1.

Colour

Yellow

Yellow

Yellow

Yellow

Yellow

Yellow

2.

Shape

Round Flat

RoundFlat

Round Flat

RoundFlat

Round Flat

Round Flat

3.

Hardness (kg/cm2)

5.3 ± 0.1

7.5±0.06

6.5±0.23

6.9±0.33

5.8±0.13

7.1±0.23

4.

Thickness (mm)

1.86 ± 0.03

1.76 ± 0.02

1.56 ± 0.03

1.62 ± 0.02

1.84 ± 0.03

1.79 ± 0.02

5.

Weight Variation (%)

2.3 ± 2

3.4 ± 3.2

2.8 ± 3

4.1 ± 2.5

3.5 ± 1

4.4 ± 0.5

6.

Friability (%)

0.74 ± 1

0.69 ± 1.5

0.71 ± 1.5

0.70 ± 2.3

0.68 ± 1.7

0.75 ± 1.5

7.

Drug Content (%)

95 ± 0.21

99 ± 0.25

96.3 ± 0.03

96.8 ± 0.17

94.9 ± 1.2

98.3 ± 0.32

 


The in vitro drug release is affected by both the composition of the nanoparticles as well as the composition of the tablets. As the concentration of polymer increases, the release of the drug will be slow, as there will be more amount of the polymer to bind to the drug particles. But the F2 formulation showedhighest release of the drug at the 10th hour (51.73±2.3%).

 

 

Figure 1: In vitro drug release profile for all prepared controlled release tablets of sulfasalazine loaded nanoparticles

 

From the evaluation studies, F2 was found to be the best formulation. F2 was fitted to central composite design with two independent variables, X1( Concentration of Eudragit S 100) and X2 (Concentration of Microcrystalline cellulose) Entrapment efficiency (Y1), Hardness (Y2), Friability (Y3) and In vitro Dissolution (Y4) were response variables. From the 9 runs, the constant regression values for Entrapment efficiency was 73%, hardness 4.7 kg/cm2, Friability 0.6%, in vitro drug release 46%. From the data it was observed that R6 formulation was the best formulation with 73.5% entrapment efficiency, 4.4kg/cm2 hardness, 0.69 friability and 45.4% in vitro drug release.

 

 

A

 

B

 

C

 

D

Figure2: Surface plot of (A) Y1 – Entrapment efficiency (B) Y2- Hardness (C)Y3-Friability (D)Y4- In-vivo drug release

 

Effect of independent variables on Entrapment efficiency of nanoparticles:

High entrapment efficiency is an essential factor in nanoparticle system. Thedecreases the dose of treatment related side effects, type amount and molar ratio of polymeris vital factor that directly imparts on entrapment efficiency. The results of the study showed that increase the X1 and X2 concentration leads to the increase in entrapment efficiency,

 

Y1= 73.881 + 8.82 X1 -1.07 X2 + 0.16 X1*X1 + 0.51 X2*X2 - 0.82 X1*X2

 

Effect of independent variables on Hardness

The hardness of the tablets significantlyincrease with increase the concentration of PVA, but influence of Eudragit S100 did not showed significant influence on hardness of the tablet

 

Y2=4.787 -0.150 X1 + 1.683 X2 + 0.123 X1*X1 + 0.723 X2*X2 + 0.000 X1*X2

 

Effect of independent variable on Friability

The friability of the tablet decreased with increase in concentration of Eudragit and PVA

 

Y3= 0.6747 - 0.0233 X1 - 0.2517 X2 + 0.0020 X1*X1 - 0.0230 X2*X= + O.O225 X1*X2

 

Effect of independent variable on invitro drug release

When the concentration of Eudragit S100 increases, decreases the drug release pattern. Also PVA concentration showed the decrease the in vitro release of drug from the formulation

 

Y4= 46.986 + 1.133 X1 + 4.433 X2 - 0.83 X1*X1 - 0.63 X2*X2 + 0.70 X1*X2

 

Based on the statistical optimization by 32 factorial design the R6 formulation was found to be optimized formulation. Theformulation was subjected to kinetic study. The result indicated that the formulation followed controlled release with Case II transport mechanism.

 

CONCLUSION:

Nanoparticle containing sulfasalazine successfully develop and incorporated in to a coated tablet for delivery into colon. In conclusion that the prepared nanoparticle based colon specific tablet is a promising approach and carrier for the delivery of sulfasalazine with controlled and targeted effect.

 

CONFLICT OF INTEREST:

The authors declare there is no conflicts of interest to disclose.

 

ACKNOWLEDGEMENT:

The author would like to acknowledge Grace College of Pharmacy, Palakkad, Kerala for providing necessary laboratory facilities for this study.

 

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Received on 17.11.2022           Modified on 06.01.2023

Accepted on 22.03.2023         © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(3):1272-1276.

DOI: 10.52711/0974-360X.2024.00199