Development of Rifampicin loaded Chitosan nanoparticles by 3² full Factorial design

 

Suchita Prabhakar Dhamane¹*, Dr. Swati Chagdeo Jagdale²

¹MAEER’s Maharashtra Institute of Pharmacy, Maharashtra, India

²Professor, Dept of Pharmaceutics, School of Pharmacy, Dr. Vishwanath Karad MIT World Peace University, MIT Campus, Kothrud, Pune, MH, India-411038

 

ABSTRACT:

 

 

INTRODUCTION:

Tuberculosis (TB) is a major cause of death worldwide as it is a highly communicable disease. Every year eight million peoples suffer from active tuberculosis and approximately two million deaths. Globally there were 1.3 million deaths due to TB among HIV negative peoples and additional 3.0 lakh deaths from TB among HIV positive peoples¹. The treatment for TB includes administration of multidrug combination of RIF, isoniazid (INH), pyrazinamide and ethambutol for 2 months followed by the administration of RIF and INH for 4 months.

 

Six months multidrug therapy effectively treats drug-sensitive TB². Before completion of treatment course patient discontinuing course is high and which leads to multidrug-resistant and extensively drug-resistant TB. The main reasons for non-adherence in anti-tuberculosis treatment are drug side effects, long duration of therapy. RIF is highly effective first line anti-TB agent and administered for at least 6 months. Continuous and long-term administration of RIF induces hepatotoxicity³. RIF is a BCS class II drug may be due to PS and crystalline form of drug which may obstruct accomplishment of therapeutic concentrations at the targeted site⁴. Delivery of RIF can be modified by development of nanoparticulate delivery systems which can improve efficiency and reduce toxicity. Nanoparticles are having PS in the range of 100nm to 1000nm and converts crystalline drug into amorphous form⁵. The aim of this study was to prepare RIF loaded chitosan nanoparticles using vanillin as a crosslinker. Nanoparticles prepared by using natural biodegradable polymer such as chitosan have been extensively studied. Crosslinking of chitosan by polyanionic polymer is due to presence of many functional groups such as amino groups and hydroxyl groups. Most commonly used methods for preparation of chitosan nanoparticles are covalent crosslinking, ionic crosslinking, sedimentation and self-assembly⁶. Crosslinking agent such as glutaraldehyde, aldehydes and glyoxal has a residual cytotoxicity. Glutaraldehyde is also neurotoxic in nature⁷. To overcome this problem, various researchers have used sodium tripolyphosphate or sodium sulphate as a crosslinker⁸. But, mechanical strength of chitosan nanoparticles using these polyanion is less which results in burst release of drug⁹. Hence, in the present work an attempt was made to formulate and evaluate chitosan nanoparticles of RIF using vanillin as crosslinker. Vanillin is a 4-Hydroxy-3-methoxybenzaldehyde which is most commonly used as flavouring agent in foods, drink and cosmetic. Few reports are available on crosslinking of chitosan by using vanillin^10,11,12,13. Objective of this work was to prepare chitosan nanoparticles using vanillin as a crosslinker and study effect of concentration of chitosan and vanillin on PS and EE by implementation of 3² full factorial design.

 

MATERIALS AND METHODS:

Materials:

Chitosan was purchased from Analab Fine Chemicals, Mumbai. RIF was obtained as gift sample from Lupin laboratories ltd. Pune. Vanillin was purchased from Loba Chem, Mumbai. All other chemicals agents used were of analytical grade.

 

Methods:

Preparation of chitosan nanoparticles:

Chitosan nanoparticles containing RIF were prepared by solvent evaporation method. Chitosan was dissolved in aqueous acetic acid solution. Vanillin and RIF was dissolved in ethanol. Vanillin and RIF solution was added dropwise in chitosan solution and the obtained solution was stirred for 5hrs to obtain chitosan nanoparticles. Obtained RIF nanoparticle suspension was stored for further characterization.

 

Factorial design of experiments:

To study effect of concentration of chitosan (X1) and vanillin (X2) a two factor, three level factorial design was implemented. The response variables selected were PS (Y1) and EE (Y2).

 

Characterization of RIF loaded chitosan nanoparticles suspension:

Prepared RIF loaded chitosan nanoparticle suspensions were evaluated for physical examination, pH of nanosupension, entrapment efficiency, PS, Zeta Potential and PDI.

 

Entrapment efficiency (% EE):

EE (%) was determined by measuring the concentration of unentrapped drug after separation. To separate the nanoparticle, nanoparticle suspension was centrifugated at 12000rpm for 30 min. The supernatant was diluted with ethanol and analyzed by UV spectroscopy. Then the percent entrapment was determined using the following equation 1:

 

Weight of drug used in formulation-Weight of unbound drug in   supernatant

% EE = -------------------------------------------------------------------- ×100                  

                             Weight of drug used in formulation                       (1)

 

PS, Zeta potential and Polydispersity index (PDI)

 

Dynamic Light Scattering (MAL1098084, Zetasizer 7.12, Malvern Instruments, UK) was used to measure the PS and zeta potential (ZP) of all the RIF nanoparticles. All samples were diluted with distilled water to make up a suitable concentration. The Z-average PS, polydispersity index (PI), and zeta potential (ZP) were determined.

 

Statistical analysis:

The observed data of PS and EE of all formulations were subjected to statistical analysis by using design expert software and to obtain the optimized condition, the goal for each response (PS- 150nm to 170nm   and EE – 70% to 80%) was set in graphical optimization to create an overlay plot which highlights the operating window.

 

Model validation:

To validate the model, percent error was determined from observed and calculated values of PS and EE. Percent error was determined by using following equation 2

 

                         Calculated response-observed response

Percent Error= ––––––––––––––––––––––––––––––––– x100         (2)

                                           Observed response

 

In-vitro drug release studies:

For In Vitro drug release study, 2ml of optimized nanoparticle suspension was placed in dialysis bag with molecular weight cut off 1000. The dialysis bag was then immersed into 60ml of 7.4 phosphate buffer solution and shaken on rotary shaker at 60rpm at 37°C. A sample was withdrawn at different time intervals of 0, 2, 4, 6, 8, 12, 24hrs and analyzed simultaneously by UV spectrophotometry at 332.2 nm⁹.

 

Optimized nanoparticle suspension was freeze dried using freeze dryer (Alpha 1-2 LD plus), Christ) for further characterization Fourier Transform Infra-red Spectroscopy (FTIR), X-ray diffraction studies, Differential scanning calorimetry (DSC), Scanning electron microscopy (SEM).

RESULTS AND DISCUSSION:

RIF loaded chitosan nanoparticles were prepared by solvent evaporation method using 3² factorial design. Vanillin was selected as a crosslinker. Li et al (2014) reported that formation of chitosan nanoparticles is due to Schiff reaction between amino group of chitosan and aldehyde group of vanillin as well as formation of hydrogen bond between chitosan and vanillin. All formulations F1 to F9 were found to be red coloured. pH of all formulations were in the range of 4.5±0.41 to 4.9±0.28. EE of all the formulations was found to be in the range of 48.76± 0.95 to 83.21±3.25. PS was found to be in the range of 89.63±9.78 to 436.4±8.44nm.  Zeta potential was found to be in the range of 9.11±2.52 to 57.8±1.89. It was observed that as concentration of vanillin increases there is decrease in magnitude of zeta potential which may be due to negative charge on vanillin. Zeta potential of nanoparticles indicates stability. Zeta potential value greater than +20 or less than -20 indicates greater colloidal stability. As amino groups are present on the surface of chitosan nanoparticles, surface charge on chitosan nanoparticles is positive. Higher is the zeta potential value; higher is a stability of nanoparticles which is due to the electrostatic repulsion between the colloidal particles¹⁵. In 3² factorial design concentration of chitosan and vanillin was selected as independent variable whereas PS and EE was selected as response variable.

 

Observed data of PS, EE was fitted into different polynomial mathematical model and a best fit model was suggested by the design expert software. It was observed that the best fitted model for PS was 2FI (Factor Interaction) and quadratic for EE.

 

Parameters such as adjusted determination coefficient (adjusted R2), predicted determination coefficient (predicted R2) and PRESS (predicted residual sum of square) values are as given in table 1.

 

Table 1: Model summary statistics of response to select suitable model to fit data

PRESS: predicted residual sum of square

 

Value of predicted R² for PS (Y1) and EE (Y2) is in close agreement with adjusted R². Lesser is the PRESS, better is the model fitting. PRESS value of PS and EE is 33788.57 for 2FI and 372.13 for quadratic respectively. Though PRESS value of PS in quadratic model is less as compared to 2FI, model suggested is 2FI as p-value of 2FI is 0.0038 which is less as compared to quadratic model. F-value of 27.24 and p value of <0.0001 indicates 2FI model for PS is significant. Signal to noise ratio is measured by adequate Precision. Desired value of adequate precision should be 4 or greater than 4. Adequate precision value for PS is 17.25, which indicates model can be used to navigate the design space. Lack of fit value for 2FI is 0.2315 (p-value) which is non-significant. Non-significant lack of fit is good for model to fit.

 

The best fit 2FI model equation generated for PS (Y1) is as given in equation 3.

 

Y1= 175.23+78.20X1+76.38X2+58.82X1X2              (3)

 

In above eq. 1, Y1 is response variable (PS), 175.23 is average coefficient 78.20 (X1) and 76.38 (X2) is the average coefficient indicating main effect due to changing one factor at a time from low level to high level. The interaction effect of X1 and X2 is 58.82 which indicate change in response when two factors are changed simultaneously.  Polymer, crosslinker and interaction of both are having significant effect on PS. PS of chitosan nanoparticles depends on concentration of chitosan and crosslinker concentration¹⁵. It was observed that PS increases with increase in concentration of chitosan and vanillin. In case of EE, F-value of 19.13 and p-value of 0.0001 indicates quadratic model is significant. Adequate precision value for EE is 13.3382 indicating quadratic model can be used to navigate the design space. Lack of fit value for quadratic model is 0.6113 which is non-significant. The best fit quadratic model equation generated for EE (Y2) is as given in equation 4.

 

Y2=73.37+12.09X1+5.36X2+2.03X1X2-8.75X12       (4)

 

Coefficient for chitosan and vanillin was found to be 12.09 and 5.36 respectively. Coefficient data for individual factor indicates chitosan is having more effect on PS, EE as compared to vanillin. The average percent error for PS and EE was found to be 2.10% and 0.24% respectively. Optimized formulation was selected from graphical optimization process. In optimization process PS was set to 150 to 180nm and EE 70 to 80 %.

 

Overlay plot of (fig. 1) optimization process suggested 0 (1%) and 0 (0.8%) levels of X1(Polymer) and X2(Crosslinker) respectively. Optimized formulation was prepared according to the suggested levels of independent variables. Observed response values were close to the predicted values in the optimized formulation. Prediction error of EE and PS of optimized formulation was 2.42% and 0.45% respectively which further demonstrates suitability of optimization procedure in development of RIF loaded chitosan nanoparticles.

 

Fig 1: Overlay plot to select optimized conditions

 

 

Fig 2: FTIR spectrum of A) Rifampicin B) Chitosan C) Vanillin D) Rifampicin loaded chitosan nanoparticle.

 

 

Fig.3: XRPD of rifampicin and rifampicin loaded chitosan nanoparticles nanoparticles

 

In FTIR spectrum of pure RIF (Fig.2A)  absorption band at 3447 cm^-1 was due to presence of –OH and –NH group, peak at 1726 cm^-1 corresponds to C=O acetyl stretching, peak at 1620cm^-1 indicates presence of C=N stretching, 1620cm^-1 indicates C-O-C stretching and peak at 811cm^-1 is due to C-H stretching. In FTIR spectrum of pure chitosan (Fig.2B), stretching vibrations at 3300-3500cm-1 indicates presence of –OH and –NH group. Stretching vibrations at 1589 cm-1 is due to presence of amino group. Peak at 1028cm-1 indicates stretch vibrations of C-O. Peak at 1317cm-1 indicates stretching vibrations of C-N bond. In FTIR spectrum of pure vanillin (Fig.2C) peak at 3150cm-1 is due to stretching vibrations of –OH, peak at 1675 cm-1 corresponds to stretching vibrations of C=O of aldehyde group, peak at 1590, 1514 and 812 cm-1 is due to presence of benzene ring. In FTIR spectrum of RIF loaded chitosan nanoparticles (Fig.2D) peak at 1697 cm-1 corresponds to characteristics vibrations of C=N which indicates formation nanoparticles due to interaction between the aldehyde group of vanillin and amino group of chitosan.

 

X-ray diffractogram (Fig.3) of RIF indicates crystalline nature of drug and RIF loaded chitosan nanoparticles indicates absence of crystalline peaks of drug. Absence of crystalline peaks of drugs indicates drug is converted into amorphous form.

 

DSC thermogram (Fig.4) reveals a sharp endothermic peak of vanillin at 81.3°C, endothermic peak of RIF at 196.23 °C, broad endothermic peak of chitosan at 84.0°C. In the drug-loaded nanoparticles, melting endotherm was observed at 282.3° with broad peak. This change in DSC of RIF loaded nanoparticles indicated reduction in crystallanity of RIF in the formulation of nanoparticles.

 

 

Fig.4: DSC thermogram of A: Vanillin B:Rifampicin C: Chitosan D: Rifampicin loaded chitosan nanoparticles.

 

Fig.5 Scanning electron microscopy of RIF loaded chitosan nanoparticles.

 

SEM analysis of freeze dried nanoparticles (Fig.5) of optimized nanoparticle suspension showed agglomerated irregular shaped nanoparticles with spongy surface.

 

Fig. 6 Release profile of RIF through optimized nanoparticles.

 

In vitro drug release shows sustained release of RIF (Fig.6) over a period of 24hrs. Drug release profile indicates burst release of RIF (22.58%) during first 2 hr. At the end of 24hrs drug release was found to be 65.70%.

 

CONCLUSION:

RIF loaded chitosan nanoparticles were successfully prepared by crosslinking with vanillin. Full factorial design of response surface methodology can be used to determine optimum conditions for preparation of RIF loaded chitosan nanoparticles using vanillin as crosslinker. Study concludes that as concentration of both variable increases PS and EE increases. FTIR, DSC, XRPD study reveals crystalline nature of rifampicin is converted to amorphous nature.  Optimized formulation has 170.23nm and 78.23% PS and EE respectively which can be further developed various other solid dosage forms.

 

ACKNOWLEDGEMENT:

Authors are thankful to MAEER’s Maharashtra Institute of Pharmacy,Pune for providing technical assistance during research work.

 

CONFLICTS OF THE INTEREST AND STATE OF HUMAN AND ANIMAL RIGHTS:

S. P. Dhamane and S. C. Jagdale declare that they have no conflict of interest. This article does not contain any studies with human and animal subjects performed by any of the authors.

 

REFERENCES:

1.      WHO (2018) Global Tuberculosis Control. Geneva, Switzerland.

2.      WHO (2010) Treatment of tuberculosis guidelines. 4^th ed. Switzerland.

3.      Mekonnen HS, Mekonnen AWA. Non-adherence to anti-tuberculosis treatment, reasons and associated factors among TB patients attending at Gondar town health centers, Northwest Ethiopia. BMC Research Notes. 2018; 11:691.

4.      Henwood SQ, Liebenberg W, Tiedt LR, Lotter AP, Villiers MM. Characterization of the Solubility and Dissolution Properties of Several New RIF Polymorphs, Solvates, and Hydrates. Drug Development and Industrial Pharmacy. 2001; 27(10): 1017–1030.

5.      Burgess DJ, Jog R. Pharmaceutical Amorphous Nanoparticles. Journal of Pharmaceutical Sciences 2017;106 (2017): 39-65.

6.      Ahmed TA, Aljaeid BM. Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery. Drug Design, Development and Therapy. 2016;10: 483–507.

7.      Gurnyb R, Peppas N, Felt O, Mayera J, Reista M, Bergera J. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics. 2004; 57: 19–34.

8.      Lopez-Leon T, Carvalho E, Seijo B, Ortega-Vinuesa J, and Bastos-González D Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behaviour. Journal of Colloid and Interface Science. 2005; 283(2): 344–351.

9.      Li PW, Wang G, Yang ZM, Duan W, Peng Z, Kong LS, Wang QH  Development of drug-loaded chitosan–vanillin nanoparticles and its cytotoxicity against HT-29 cells. Drug Delivery. 2014;1–6.

10.   Xiong H, Peng H, Li J, Xie M, Liu Y, Bai C, Chen L. Vanillin cross-linked chitosan microspheres for controlled release of resveratrol. Food chem. 2010;121(1):23-28.

11.   Shang Z, Zhang Y, Shi X, Yu Y, Zhao S, Song H, Chen A. Preparation and Characterization of Vanillin Cross-Linked Chitosan Microspheres of Pterostilbene, International Journal of Polymer Analysis and Characterization. 2014; 19(1): 83-93.

12.   Li Y, Zou Q, Li J. Preparation and characterization of vanillin-crosslinked chitosan therapeutic bioactive microcarriers. International Journal of Biological Macromolecules. 2015; 79:736-747.

13.   Li QX, Song BZ, Yang ZQ, Fan HL. Preparation and characterization of chitosan microcapsules crosslinked by vanillin. Chinese Journal of Process Engineering. 2006;6(4): 608–613.

14.   Valizadeh H, Ghanbarzadeh S, Milani PZ. Application of response surface methodology in development of Sirolimus liposomes prepared by thin film hydration technique. Bioimpacts. 2013; 3(2): 75-81.

15.   Gan Q, Wang T, Cochrane C, McCarron P. Modulation of surface charge, particle size and morphological properties of chitosan– TPP nanoparticles intended for gene delivery. Colloids and Surfaces B: Biointerfaces. 2005; 44(2): 65-73.

 

 

Accepted on 22.11.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6):2545-2550.