Inhalable chitosan nanoparticles as antitubercular drug carriers for an effective treatment of tuberculosis

Abstract

The aim of this study was to prepare and characterize spray dried inhalable chitosan nanoparticles (CNPs) for sustained delivery of anti-tubercular drugs, isoniazid (INH) and rifampicin (RIF), to the lungs. CNPs were prepared by ionic gelation technique followed spray drying. Results showed that the CNPs obtained had a smooth spherical shape with an average size of 230 ± 4.5 nm, with a poly dispersity index of 0.180 ± 0.021. Both drugs, were detected in various organs (lungs, liver, spleen and kidney) until 24 h post nebulization. The chemotherapeutic efficacy of a single dose of drug-loaded CNPs suggested that they are more effective against the mycobacterium than free drugs.

Keywords::

• chitosan nanoparticle
• in vivo studies
• pulmonary drug delivery
• tuberculosis

Introduction

Tuberculosis (TB) is a contagious disease containing the tubercle bacilli, Mycobacterium tuberculosis (MTb) (Kaur et al. 2014a, Chaubey and Mishra 2014). It mainly spreads by inhalation of bacilli-containing airborne droplets (Kaur et al. 2014b, Patel et al. 2011). The failure of antitubercular drug (ATD) therapy is mainly due to patient non-compliance (Kaur et al. 2014c, Pourshahab et al. 2011), higher cost (Chaudhary et al. 2014, Saraogi et al. 2011), lengthy treatment duration (Gagandeep et al. 2014), and the side-effects caused (Garg 2014). In recent years, developments of nanoparticle-based formulations are being widely considered for direct delivery of ATDs to the lungs via the pulmonary route (Garg and Goyal 2014, Saraogi et al. 2010). Numerous biocompatible and biodegradable natural origin polymers (starch, gelatin, cellulose) are used as drug carriers, but chitosan was considered for this study due to its biocompatible and biodegradable nature as a non-toxic natural cationic polymer which has good mucoadhesive and membrane permeability-enhancing properties. It controls the drug release rate, prolonging the duration of therapeutic effects and delivering the drug to the specific sites in body. Chitosan also shows anti-microbial activity, which has also been reported for several years (Garg et al. 2012, Semete et al. 2010). The pulmonary route avoids the GI tract problems such as poor solubility, thereby enabling a reduction in the dose as well as in dosing frequency, and an improvement in patient compliance. The present study was planned to develop chitosan nanoparticles (CNPs) containing isoniazid (INH) and rifampicin (RIF), and characterized on the basis of morphology, entrapment efficiency and in vitro drug release. Chemotherapeutic efficacy and toxicity were also investigated against experimental murine TB.

Materials and methods

Materials

INH and RIF were received from BV Patel Centre (Ahmedabad, India) as gift samples. Chitosan with low molecular weight, Guar gum and Tripolyphosphate (TPP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mannitol and leucine were purchased from HiMedia Pvt. Ltd., Mumbai, India. All other reagents were of analytical grade, and were obtained from standard companies.

Animals

Female BALB/c mice (6–8 of weeks age, 20–25 g) obtained from the ISF College of Pharmacy, Moga, Punjab, India, were used in the study. The project was approved by the Institute Animal Ethics Committee. All efforts were made to reduce animal suffering.

Methods

Development and characterization of drug-loaded CNPs

CNPs were prepared by the ionotropic gelation technique (Kaur et al. 2014e). Briefly, TPP solution was added to chitosan solutions (0.5% w/v) containing different amounts of INH and RIF, under magnetic stirring. To prepare inhalable nanoparticles, the drug-loaded CNPs were spray-dried with mannitol (1% w/v) and leucine (0.25% w/v), using a spray dryer (LU 222 advanced, Labultima, Mumbai, India). The powders were then removed from the collector vessel and characterized (Kaur et al. 2014f).

CNPs were characterized for their size and polydispersity index (PDI) on a particle size analyzer (Beckman Coulter Pvt. Ltd., Delsa Nano C) (Kaur et al. 2014d). Scanning electron microscopy (SEM) was employed for visualization of the optimized nanoparticle shape. The amount of entrapped anti-tubercular drugs in the CNPs was calculated by estimating the amount of unentrapped drug recovered in the supernatant after centrifugation (Singh et al. 2014a, 2014b, Sharma et al. 2014c). Drug release from CNPs was determined using the dialysis tube diffusion method at 37 ± 1°C. In this technique, nanoparticulate dispersions (10 mL) were separately placed into the dialysis membrane (MWCO 12000–14000 Da, HiMedia, India), tied at both the ends, and suspended in a beaker containing 50 mL of PBS (pH 7.4). The formulations were stirred at 50 rpm using a shaking incubator (LSB-1005RE, Daihan Labtech. Co. Ltd., Korea). At predetermined time intervals, samples were withdrawn and analyzed spectrophotometrically (UV-1601 Shimadzu, Japan) for INH and RIF content, at 262 nm and 473 nm respectively. The studies were performed in triplicate (Sharma et al. 2014a, 2014b, Rohilla et al. 2014).

In vivo drug disposition studies

For in vivo studies, female Balb/c mice (20–30 gm) were used for organ distribution studies of optimized formulations. A compressor nebulizer system (Pan Century, USA) was used in this study. For single dose drug disposition studies, female mice were grouped as follows, with six animals in each group: Group 1, free drug, aerosol; and Group 2, nanoparticle-loaded ATDs, aerosol. Animals were sacrificed at different time points, and drug levels were determined in lungs, liver, kidney and spleen tissue homogenates (Pabreja et al. 2014, Morie et al. 2014, Modgill et al. 2014).

Experimental infection and chemotherapy

Experimental infection and chemotherapy studies were done in the National Jalma Institute for Leprosy and other Mycobacterial Diseases (JALMA), Agra, UP, India. The animals were infected with 4.2 × 10⁷ bacilli/ml of M. tuberculosis H₃₇Rv in 0.1 ml of 0.9% NaCl, through the aerosol route (Glas-Col inhalation exposure system, USA). The animals were then divided into the following groups: Group 1, control group; Group 2, naïve group; Group 3, free drug, aerosol; and Group 4, nanoparticle-loaded ATDs, aerosol. The animals were sacrificed after 2 and 4 weeks of chemotherapy; lungs and spleen were removed, homogenized in sterile normal saline solution and plated on Middlebrook 7H10 Agar plates for colony-forming unit (CFU) estimation on the 14th and 28th days post inoculation (Marwah et al. 2014, Malik et al. 2014, Kaur et al. 2014h).

Statistical analysis

Statistical tests were performed with GraphPad Instat Software. A difference with a value of p ≤ 0.05 was considered statistically significant.

Results and discussions

Physicochemical characterization of chitosan nanoparticles

CNPs had a smooth spherical shape with an average size of 230 ± 4.5 nm, with a PDI of 0.180 ± 0.021 (Figure 1). Smooth-surface nanoparticles are easily captured by the alveolar macrophage of lungs, and the low PDI of nanoparticles suggested a homogenous dispersion (Kaur et al. 2014g, Kataria et al. 2014).

Figure 1. SEM images (A) and particle size distribution (B) of optimized CNPs.

Drug encapsulation efficiency was observed to be 70.8 ± 6.62 for RIF and 68.8 ± 7.02 for INH. The higher drug encapsulation observed for CNPs in this study can be attributed to the higher drug/polymer ratio, as well as an excellent ionic gelation between TPP and chitosan (Joshi et al. 2014, Johal et al. 2014, Hussain et al. 2014). The release behavior of RIF and INH from the nanoparticulate formulation followed a biphasic pattern, that is, an initial burst (40–50% within 4 h) followed by a slower sustained release (90–95% for up to 72 h) (Figure 2). Kinetic analysis of drug release from optimized formulations indicates that the drug is released from the nanoparticles by a diffusion mechanism. An initial burst release of RIF and INH from nanoparticles could be due to rapid dissolution of drug crystals located on the surface or present beneath the nanoparticle surface (Goyal et al. 2013b, 2014a, 2014b).

Figure 2. In vitro drug release profile of optimized nanoparticulate formulations.

In vivo drug disposition studies

When the CNPs were administered through nebulization, the superiority of the ATD-loaded CNPs was seen in comparison to the plain drug in increasing the accumulation of ATDs within the organs rich in macrophages, that is, lungs. Both drugs were detected in tissues, that is, lungs, spleen, liver, and kidney, for up to 24 h (Figure 3), thus prompting us to design the nebulized drug-loaded CNPs for chemotherapy. The higher lung uptake of CNPs may be attributed to preferential phagocytosis by macrophages and localization in alveolar macrophages (Goyal et al. 2013a, Garg et al. 2013, 2014b).

Figure 3. In vivo organ distribution of ATDs administered as plain drug solution and ATD-loaded CNPs.

Chemotherapeutic efficacy

Administration of nebulized CNPs for 28 days to MTb- infected mice resulted in undetectable mycobacterial CFUs in lung and spleen homogenates (Table I). These experimental observations suggested that CNPs are more effective against the mycobacterium than free drugs (Figure 4). The efficacy of chitosan nanoparticle formulations shows an effective targeting to macrophage-rich organs in treating the disease (Garg et al. 2014a, Garg and Goyal 2014).

Figure 4. Chemotherapeutic efficacy of various formulations.

Table I. Chemotherapeutic efficacy of various formulations (mean ± S.D., n = 6).

	Log10 CFU
Groups	Lungs	Spleen
Untreated control	6.51 ± 1.15	4.22 ± 0.29
Free RIF-INH	5.99 ± 1.34	3.78 ± 0.19
ATD-loaded chitosan nanoparticles (CNPs)	1.23 ± 0.19	0.75 ± 0.08

Conclusions

In this study, drug-loaded CNPs were successfully synthesized and characterized as an effective delivery carrier for alveolar macrophage targeting. The optimized formulations showed lower cytotoxicity and significant reduction in the number of bacilli in the lungs, as compared to free drug. Finally, it may be concluded that CNPs may be exploited as a prospective tool for drug delivery to direct drugs to the lung tissues for the treatment of TB.

Acknowledgements

The author Mr. Tarun Garg is thankful to the Punjab Technical University (PTU), Kapurthala, Punjab, for providing an opportunity to carry out the research work. The author Dr. Amit K. Goyal (under IYBA scheme; BT/01/IYBA/2009 dated 24/05/2010) is thankful to the Department of Biotechnology (DBT), New Delhi, India, for providing financial assistance to carry out research work.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.