INTRODUCTION:

Owing to its natural origin, versatile biodegradability and biocompatibility, chitosan has been employed as a nanostructure material. The active moiety-loaded nanostructures have capacity to be employed for oral delivery of active molecules for management of numerous disorders. Delivery of acitve agents to the target site may permit for efficient tuberculosis management. Isoniazid is a synthetic antimycobacterial moiety used as the first-line drug in tuberculosis^[1]. Ever since, it was employed in the therapy in 1952, the drug stays at the front line in tuberculosis management mainly due to its high selectivity and potency against Mycobacterium tuberculosis^[2].

A German physician and microbiologist, Robert Heinrich Herman Koch got Nobel Prize in 1905 for discovery of the causative agent for tuberculosis (mycobacterium tuberculosis). After HIV/AIDS, tuberculosis is the most reason of death^[3]. Further research would be explored for the development of novel drug delivery systems for drug targeting.

Biodegradable nanostructures have gained considerable attention as significant drug delivery carriers. Chitosan (CS) is nontoxic, hydrophilic, favourable polymer which has been recently preferred as potential delivery carrier for management of tuberculosis^[4,5].

This investigation has explored the fabrication of chitosan nanostructures by simple and fast ionotropic gelation between CS (polymer) and Sodium tri-polyphosphate (STPP)^[6,7]. Furthermore, recently the fabrication of chitosan/carbon nanotubes microparticles as carriers for isoniazid to promote wound healing in bone tuberculosis has been reported^[8]. Moreover, oral administration of chitosan nanostructures provides shield against first pass metabolism and consequently, helping in the systemic circulation via absorptive transcytosis and reducing peripheral adverse reactions^{[9, 10]}. The aim of the current research was to fabricate and optimize formulations for oral delivery of isoniazid encapsulated in chitosan nanostructures, followed by evaluation of their sustained release characterstics.

MATERIAL AND METHOD:

Material:

Isoniazid was procured from High Purity Laboratory Chemicals (HPLC), India. Chitosan and Sodium tri-polyphosphate were procured from Himedia Laboratories Pvt. Ltd., Mumbai (India). Acetic acid was obtained from SD Fine-Chem Ltd, Mumbai (India).

Experimental design:

The optimization technique was applied to obtain an appropriate formulation design in order to reduce the number of experiment trials, and analyse the response surface to investigate the effect of independent variables on the response^[11]. The 2 factor 3 level (3²) factorial design was adopted for optimization of the formulation of nanostructures. Two factors A Chitosan and B Chitosan: STPP were taken at three different concentration levels i.e. low level, medium level, and high level (-1, 0, +1) as described in Table 1. The particle size (nm), entrapment efficiency (EE) and drug loading (DL) were taken as dependent variables. The response surface methodology (RSM) was applied for analysis using Design Expert software (Version-12.0.0.6). The software suggested 13 trial runs for the factorial design batches (F1-F13) are shown in table No.2

Table 1 Experimental Design of Chitosan nanostructures

Independent variables	Levels
Independent variables	Low (-1)	Medium (0)	High (1)
A Chitosan (mg/100ml)	40	50	60
B Chitosan:STPP	3:1	4:1	5:1

Method:

Fabrication of nanostructures:

The isoniazid nanostructures were fabricated by ionotropic gelation technique using Chitosan. Chitosan at various concentrations was dissolved in 2% acetic acid solution using magnetic stirrer and allowed to stand for 30 minutes. Then, the drug isoniazid was suspended in above chiotsan solutions with stirring. Various concentrations of Sodium tri-polyphosphate solution were added dropwise in the solution of drug isoniazid and polymer chitosan thus suspension of Isoniazid-Chitosan-Sodium tri-polyphosphate nanostructures was formed. STPP was added as a cross linking agent for the chitosan nanostructures to achieve sustained drug release. It was kept for sonication for 25 minutes. After sonication nanostructure suspension was centrifuged at 10, 000rpm (Remi, Mumbai) for 30 minutes and supernatant was discarded. The Pellet was redispersed in de-ionized water followed by sonication, centrifugation and lyophilisation [1]. Concentration of chitosan and STPP for the optimization were selected on the basis of literature [10] and optimization was carried out using 3² factorial design. 3 levels each of 2 factors, factor A (concentration of chitosan; %w/v) and B (ratio of chitosan: STPP (concentration); v/v), were used and the factor levels are shown in Table1. The 13 experimental runs carried out through various levels of the factors are shown in Table 2.

Characterization of nanostructures:

Particle size:

The nanostructures were dispersed in HPLC grade water. The average particle size of chitosan loaded isoniazid nanostructures was evaluated by Particle Size Analyzer (Malvern Instruments Ltd, Enigma Business Park, Grovewood Road, Malvern WR14 1XZ, United Kingdom). Observations of average particle diameter of fabricated nanostructures are depicted in table 2.

Determination of Encapsulation Efficiency (EE) of nanostructures:

10ml suspension of nanostructures was centrifuged at 10,000rpm (Remi, Mumbai) for 90 minutes at 10. After centrifugation, the clear supernatant procured was diluted 10 times with a solvent to quantify the amount of unbound isoniazid using UV-Visible spectrophotometer at λ_max=263 nm. Encapsulation Efficiency and drug loading of nanostructures were determined using following equations [12]: and data is given in table 2.

Fourier transform infrared spectroscopy (FTIR):

Infrared spectroscopy of the pure isoniazid, chitosan, physical mixture was carried out to determine drug loading and drug-excipient interaction^[13].

In vitro drug release study of Chitosan nanostructures:

The drug release study was carried out using dissolution medium (phosphate buffer, pH 7.4 (250ml)) at 50rpm at 37±0.5º C temperature. The nanostructures containing drug equivalent to 50mg were taken in dialysis bag and put into flask containing phosphate buffer (pH 7.4). 5ml aliquots of samples were withdrawn at specific interval i.e. 1, 2, 4, 6, 12, 14, 16, 18, 20, 22 and 24 hrs. The absorbance of samples was determined at λ_max 263 nm using UV-Visible Spectrophotometer after suitable dilutions and percent drug release at different time intervals was plotted against time.

RESULTS AND DISCUSSION:

Optimization using 3² factorial design:

Factorial design was applied for estimation of appropriate amount of chitosan and STPP on the basis of loading capacity, encapsulation efficiency and average particle diameter measurements.

Table 2 Experimental Design of Isoniazid Nanostructures and results for the different observed responses

Std	Run	Factor A Chitosan	Factor B Chitosan: STPP	Response 1 Drug loading (%w/w) ±SD	Response 2 Entrapment efficiency (%) ±SD	Response 3 Average particle size (nm)
7	1	0	-1	14.71±0.04	58.85±0.08	531.4
6	2	1	0	16.24±0.04	73.11±0.10	584.8
8	3	0	1	15.07±0.09	67.83±0.15	585.3
9	4	0	0	16.20±0.02	72.90±0.06	330.4
13	5	0	0	16.02±0.01	72.12±0.14	386.2
1	6	-1	-1	13.62±0.02	47.70±0.09	539.9
3	7	-1	1	16.62±0.05	74.80±0.02	254.8
4	8	1	1	12.70±0.05	69.88±0.07	632.1
2	9	1	-1	14.34±0.02	64.54±0.04	716.8
5	10	-1	0	17.31±0.05	69.25±0.04	359.2
10	11	0	0	15.75±0.13	70.88±0.04	234.2
11	12	0	0	16.41±0.50	73.88±0.04	280.8
12	13	0	0	17.77±0.07	79.97±0.03	380.8

Total13 formulations were prepared as per design and influence of 2 factors, i.e. Chitosan (A) and Chitosan:STPP (B) was examined on 3 responses viz. Drug loading, encapsulation efficiency and average particle diameter. The drug loading and encapsulation efficiency of all batches was found to be lying within range 12.70- 17.77 % and 47.7-79.97% respectively, whereas mean particle size was found to be between 234.2 and 716.8 nm. The obtained polynomial equations were employed for the calculation of the variance and responses were analysed for statistical parameters such as degree of freedom, F value, sum of squares and mean sum of squares through the software. Polynomial equations obtained through regression analysis for responses are as follows:

Drug Loading (DL) =+16.56-0.7114*A+0.2862*B-1.16*AB-0.0993*A²-1.98*B² (1)

Encapsulation efficiency (EE) = +73.45+2.63*A+6.90*B-5.44*AB-1.00*A²-8.85*B² (2)

Particle size (PS) = +346.20+129.97*A-52.65*B+50.10*AB+66.50*A²+152.85*B² (3)

Response surface methodology (RSM) plot depicting combined effect of both factors on DL, EE and PS of nanostructures is shown in Figure 1, 2 and 3, respectively. The importance was designated to mean PS than DL and EE. Variables as suggested by Design Expert software (Version-12.0.0.6) for optimized nanoformulation were chitosan 50 mg and Chitosan: STPP ratio 4:1 which led to formulation of nanostructures with average particle diameter of 380.8nm.

Particle size:

Particle size of the prepared nanoformulations is shown in Table 2. The observed particle size varied from 234.2 to 716.8 nm for chitosan nanostrcutres and particle size of optimized formulation (F-13) was shown in Figure 4.

Figure 1: Response surface plot depicting combined effect of Chitosan and Chitosan: STPP upon loading capacity (%) of isoniazid nanostructures.

Figure 2: Response surface plot depicting combined effect of Chitosan and Chitosan: STPP upon encapsulation efficiency (%) of isoniazid nanostructures.

Figure 3: Response surface plot depicting combined effect of Chitosan and Chitosan: STPP upon particle size of isoniazid nanostructures.

Figure 4. Particle size distribution of the Isoniazid loaded chitosan nanostrucutres (F-13 optimized).

For chitosan nanostrucutres, the quantity of STPP has a significant role in the design of nanostructures.

Determination of Encapsulation Efficiency (EE) of nanostructures:

The drug loading and encapsulation Efficiency of the fabricated nanostructures are shown in Table 2. As the concentration of chitosan was enhanced the encapsulation Efficiency and drug loading was also increased owing to increase in viscosity of the aqueous phase with enhancement in quantity of chitosan and leading to minimum drug loss. The encapsulation Efficiency and drug loading was also increased with increase in concentration of STPP.

Fourier transform infrared spectroscopy:

Drug compatibility studies using FTIR were conducted for the polymer, pure drug, sodium tripolyphosphate, physical mixture and the spectral data are given in Figure 5-8. The results indicated no chemical incompatibilities between pure drug and polymer used in nanostructure.

Figure 5. FTIR spectrum of the polymer.

Figure 6. FTIR spectrum of pure drug.

Figure 7. FTIR spectrum of Sodium tripolyphosphate.

Figure 8. FTIR spectrum of physical mixture.

In vitro drug release studies:

The release pattern from isoniazid nanostructures displayed cumulative drug release was found to be in the range of 72.77 %– 94.23% as shown in Figure 9.

Figure 9. In vitro drug release profiles of Isoniazid nanostructures (F1- F13).

The nanostructures showed a biphasic active moiety release profile with outburst release initially, which was followed by a sustained release of isoniazid. The outburst release may be due to the association of drug with the surface of nanostructures. Initial release of drug may be linked to the drug dispersing from close to the nanostructure surface. The active moiety release may depend on chitosan amount. The optimized formulation (F13) showed drug release of 72.77±0.02% within 24 hours providing a sustained release profile. In first hour, nanoformulation provided a burst release of the drug and subsequently a sustained release was obtained. Literature reports suggest that macrophages take 2 h to achieve their maximum engulfment capacity. Therefore, it can be deduced that the majority of drug would be released inside the cell following endocytosis of the carrier system [14, 15].

CONCLUSION:

The nanocarrier based systems can enhance drug delivery to patients as described by in vitro analysis. A number of nanodelivery systems have been explored for bioavailability enhancement of antitubercular activity of isoniazid along with minimising their adverse effects. The above-mentioned objectives could be attained by fabricating isoniazid loaded chitosan nanostructures by ionotropic gelation technique for tuberculosis management by improving the bioavailability. The optimization was carried out by investigating the effect of concentration of chitosan and Chitosan: STPP on loading capacity, encapsulation efficiency and mean particle size. The obtained framework for optimized formulation (F-13) was found to be significant in comparison to software values. Thus, the developed nanoformulation has displayed the sustained release of isoniazid for optiomal tuberculosis management with the potential for minimising dose, and therefore the adverse effects.

ACKNOWLEDGEMENT:

The authors are grateful to Chairperson, Department of Pharmaceutical Sciences, for providing all necessary facilities and the University Grants Commission (UGC) for financial support.

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Received on 09.12.2019 Modified on 19.01.2020

Research J. Pharm. and Tech 2020; 13(9):4219-4223.

DOI: 10.5958/0974-360X.2020.00745.3