Development and Characterization of Nanoembedded Microparticles

Article pubs.acs.org/molecularpharmaceutics

Development and Characterization of Nanoembedded Microparticles for Pulmonary Delivery of Antitubercular Drugs against Experimental Tuberculosis Amit Kumar Goyal,*,† Tarun Garg,†,‡ Goutam Rath,†,‡ Umesh Datta Gupta,§ and Pushpa Gupta§ †

Nanomedicine Research Centre, Department of Pharmaceutics, ISF College of Pharmacy, Moga, Punjab, India Punjab Technical University, Kapurthala, Punjab, India § National Jalma Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India ‡

ABSTRACT: The foremost objective of the present research study was to develop and evaluate the potential of rifampicin (RIF) and isoniazid (INH) loaded spray dried nanoembedded microparticles against experimental tuberculosis (TB). In this study, RIF-INH loaded various formulations (chitosan, guar gum, mannan, and guar gum coated chitosan) were prepared by spray drying and characterized on the basis of in vitro as well as in vivo studies. Results showed that guar gum spray dried particles showed uniform size distribution with smooth surface as compare to mannan formulations. Guar gum batches exhibited excellent flow ability attributed to their optimum moisture content and uniform size distribution. The drug release showed the biphasic pattern of release, i.e., initial burst followed by a sustained release pattern. The preferential uptake of guar gum coated formulations suggested the presence and selective uptake capability of mannose moiety to the specific cell surface of macrophages. In vivo lung distribution study showed that guar gum coated chitosan (GCNP) batches demonstrated prolonged residence at the target site and thereby improve the therapeutic utility of drug with a significant reduction in systemic toxicity. Optimized drug loaded GCNP formulation has resulted in almost 5-fold reduction of the number of bacilli as compared to control group. Histopathology study also demonstrated that none of the treated groups show any evidence of lung tissue abnormality. Hence, GCNPs could be a promising carrier for selective delivery of antitubercular drugs to alveolar macrophages with the interception of minimal side effects, for efficient management of TB. KEYWORDS: tuberculosis, nanoembedded microparticles, guar gum coated chitosan, alveolar macrophages, pulmonary drug delivery, challenge studies

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INTRODUCTION Contagious or transmissible infections prompted by intracellular microorganisms may be a sign of a trial for existing antimicrobial usages owing to the need that antibiotics spread therapeutic attentiveness at the infection site.1 Currently, mycobacterial infection frequency has enlarged quickly. Onethird of the world’s population is infected with the second most common leading infectious disease, tuberculosis (TB), which causes the death of youth and adults worldwide.2 TB predominantly spreads by inhalation of the tubercle bacilli, Mycobacterium tuberculosis (MTb). In alveolar macrophages, MTb lives, persists, and divides every 16 to 20 h.3 Conventional TB treatment protocol is a long-lasting and complex process involving combination antibiotic therapy given over long periods of 6−9 months.4 The poor cell penetration of antibiotic makes it inactive against intracellular bacteria and further develops multidrug resistant (MDR) strains.5 Directly observed therapy short courses (DOTS) are commonly effective but have numerous operational complications, principally in developing countries.6 © XXXX American Chemical Society

TB management may be improved by local pulmonary delivery of antitubercular drug (ATD) loaded controlled release formulations, which reduced the dose frequency and shortened treatment period as compared to standard oral or systemic treatment protocols.7 At present, numerous polymeric and lipid based carrier systems of encapsulated ATDs are successfully used via oral and parenteral routes against TB.6,8 The promising assistance of these carriers includes controlled and sustained release, drug targeting, drug protections, biocompatibility, and biodegradability without toxic end products.9 Natural polymers are frequently used as drug carriers to control drug release rate and to increase the therapeutic effect duration and site specific delivery. In this study, we used chitosan and guar gum polymer to encapsulate ATDs for an effective treatment of TB. Chitosan is a natural cationic Received: January 7, 2015 Revised: September 10, 2015 Accepted: September 25, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00016 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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MATERIALS AND METHODS Chemicals and Drugs. INH and RIF were received from BV Patel Centre (Ahmadabad, India) as a gift sample. Chitosan, guar gum, mannan, Karl Fisher reagent, dimethyl sulfoxide, dry methanol, acetonitrile, eosin, hematoxylin, Lowenstein−Jensen medium, Middlebrook 7H10 agar, FITC dye, glacial acetic acid, and tripolyphosphate (TPP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mycobacteria 7H11 agar medium and Middlebrook OADC enrichment medium were purchased from Biohouse Solutions Pvt. Ltd., Chandigarh, India. Mannitol, leucine, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and potassium chloride were purchased from Himedia Pvt. Ltd., Mumbai, India. All other materials and reagents used in this study were of analytical grade and were used without any further modification. Methods. Preparation of ATD Loaded Spray Dried Nanoembedded Microparticles. In this study, various ATD loaded spray dried nanoembedded microparticle formulations were prepared by using different techniques. Chitosan based nanoembedded microparticles (CNPs) were prepared by an ionotropic gelation technique.5 Briefly, TPP solution was added dropwise through a buret to the chitosan solution (0.5% w/v, pH = 2.8) under magnetic stirring (Spinot RQ-122, Tarsons Products Pvt. Ltd., India) (TPP to chitosan weight ratios of 1:3). Guar gum based nanoembedded microparticles (GNPs) were prepared by a precipitation technique using ethanol as antisolvent.19 In this technique, guar gum solution (0.5% w/v) was prepared by dissolving guar gum in cold distilled water with proper stirring for 15 min. Surfactant (Tween 80, 0.4%) solution was added to the above solution. Both suspensions (CNPs and GNPs) were stirred for 60 min at room temperature before being exposed to additional analysis and applications. The milky suspension was filtered through membrane filter (0.45 μm) to remove insoluble residue aggregates. Mannan based nanoembedded microparticles (MNPs) were prepared by spray drying technique. Briefly, mannan (0.5% w/v) obtained from Saccharomyces cerevisiae was dissolved in purified water. Guar gum coated chitosan nanoembedded microparticles (GCNPs) were prepared by dissolving guar gum (0.5% w/v) in deionized water and added dropwise through a buret to the prepared above chitosan formulation under magnetic stirring (guar gum to chitosan weight ratios of 1:3).3 For the preparation of drug-loaded microparticles, optimized concentrations of INH (2 mg) and RIF (2 mg) were separately added to the same solution. To prepare inhalable nanoembedded microparticles, all formulations were spray dried individually by using mannitol (1% w/v) and leucine (0.25% w/v) by using a lab scale spray dryer (LU 222 advanced, Labultima, Mumbai, India). In the existing experiments, the inlet air temperature was 120 °C, the outlet temperature was 80 °C, the feed rate was 1 mL/min, and the aspirator was set at 60%.12 The process yield of spray-dried powder can be calculated as follows:

polymer which helps in intracellular delivery of the drugs to explore the maximum therapeutic outcomes.10 Further, chitosan was selected for its native antibacterial activity.11 Guar gum is a natural nontoxic, biodegradable polysaccharide composed of sugars such as galactose and mannose. The presence of mannose moiety in guar gum imparts it a great affinity toward mannan receptors (MMR).12 Mannose is commonly used as a ligand for active targeting of macrophage.13 Earlier reports suggested that mannosylated receptors present on macrophage actively participate in the receptor endocytosis process.14 Based on the earlier reports, the current hypothesis was undertaken to explore the ligand specification of guar gum, polymer of 1,4-β-galacto mannan for the mannosylated receptor located on alveolar macrophage. Generally, nonionic behavior of guar gum (alone) does not allow controlling the drug release under different physiological pH. Considered above, better intracellular targeting of the drug is made possible with the help of chitosan as a core material, as it inhibits drug release under the alkaline condition of extracellular fluid. Numerous antitubercular agents are used to control the TB. Among all antitubercular agents, the first line antitubercular agents, i.e., rifampicin (RIF) and isoniazid (INH), are widely used for an effective treatment of TB. RIF inhibits the DNA dependent RNA polymerase whereas INH inhibits the biosynthesis of mycolic acid of MTb. Conventional RIF and INH formulations (oral route) cannot retain their effective minimum inhibitory concentration (MIC) for a prolonged period of time, and tubercle bacilli develop resistance against them under such environments.15 Dry powder inhaler (DPI) is an innovative formulation strategy to deliver inhaled therapeutics for the treatment of lung infections.16 However, the aerodynamic properties of dry powder play an important role in order to achieve high drug concentration in the lung. Nanoparticles, because of their high surface area to volume ratio, offer the distinct advantage of passive accumulation at the target site followed by pulmonary administration.17 Further, nanoembedded microparticles meet the specific requirement of sustained release drug delivery and offer excellent aerodynamic properties. Spray drying is a very rapid and commonly used technology to prepare dry powders. However, conventional spray drying suffers from certain shortcomings, particularly nonuniform distribution of drugs in the end products. Further, drugs with poor solubility in dispersion medium require appropriate carriers to increase residence drug contact in the final spray dried process. Most often mannitol and lactose have been commonly used, and they provide ample flexibility to operate in continuous mode with high throughput. Nekkanti et al. (2009) reported that a drug complex with cyclodextrin could help to produce reproducibility in drug content when processed under optimized spray drying conditions.18 The proposed strategies involving nanoembedded microparticles could help to overcome the aforementioned limitations and also increase the drug content by avoiding drug loss during spray drying. In this present study, we developed a chitosan, guar gum, mannan, and guar gum coated chitosan carrier system containing entrapped INH and RIF, to be administered through the pulmonary route. The particle size, morphology, entrapment efficiency, in vitro deposition behavior, in vitro drug release, pharmacokinetic, and biodistribution studies of spray dried powders were examined. Chemotherapeutic efficacy and toxicity were also investigated against primary experimental murine TB.

process yield (%) = weight of final powder /total weight of solids × 100

(1)

Characterization of Spray Dried Nanoembedded Microparticles. Morphological Studies. The particle size and polydispersity index (PDI) of the prepared spray dried nanoembedded microparticles were characterized by using a zeta sizer (DelsaNano C, Beckman Coulter Pvt. Ltd.). Briefly, B


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formulations at 37 ± 1 °C. Each formulation was separately added into the activated dialysis membrane (MWCO 12000− 14000 Da, Himedia, India) and suspended in a beaker containing 50 mL of phosphate buffer solutions (PBS, pH 7.4). The formulations were stirred by using a shaking incubator (LSB-1005RE, Daihan Labtech. Co. Ltd., Korea) at 50 rpm. Samples were withdrawn at predetermined time intervals, replaced with the same volume of PBS, and analyzed spectrophotometrically (UV-1601 Shimadzu, Japan) for INH and RIF content at 262 and 473 nm, respectively. Release studies were carried out in sink conditions. The studies were performed in triplicate.3 In Vitro Cell Line Studies. J-774 macrophage cells (obtained from National Centre for Cell Science, Pune) were used to determine cell viability and uptake potential of prepared optimized formulations. In the macrophage uptake study, J-774 cells (100 μL, 1.5 × 105) were transferred into a 24-well plate and incubated for 12 h in a CO2 incubator (Heracell VIOS 160i C, Thermo Scientific) at 37 °C. Fluorescein isothiocyanate (FITC) dye loaded optimized formulations (prepared the same as above except using FITC dye (20 μL) instead of ATDs) were separately suspended (2 mg/well) in cell containing wells and incubated for 2 h. After 2 h, macrophage uptake was measured by flow cytometer (BD FACSCALIBUR) using FACSDiva6.1.2 software (BD Biosciences).24 Simultaneously to determine cell viability, the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay was used. Briefly, J-774 macrophage cells (1.5 × 105 per well) were transferred into a 96-well plate and incubated for 12 h in a CO2 incubator at 37 °C. After 72 h of culture, the medium in the well was replaced with fresh medium containing blank as well as drug loaded optimized formulations at the concentrations corresponding to 1 mg/mL of drug. After 12 h, 50 μL of MTT solution (5 mg/ mL in PBS) was added to each well, and incubated at 37 ± 1 °C for 4 h for MTT formazan formation. Consequently the supernatant was carefully removed and formazan crystals were dissolved in DMSO. The absorbance values were measured by using a micro plate reader (Bio-Rad, model 550 Microplate Reader, USA) at wavelength 570 nm, blanked with DMSO solution. Three replicates were read for each sample, and the mean value was used as the final result.25 The relative cell viability (%) was calculated by the following equation:

each sample (5 mg) was dispersed in acetone (5 mL) for 5 min and transferred into the small volume cell of the respective instrument. Scanning electron microscopy (SEM) (JSM 6510LV, JEOL) was used for imaging the shape and size of the optimized formulations. In this technique, samples were vacuum-dried, coated with gold palladium, and observed microscopically at different magnification levels.20 Angle of Repose Analysis. Angle of repose of the prepared spray dried microparticles was determined by a fixed funnel technique. Briefly, the sample powder materials were thrown down through a funnel tip, and a powder pile was prudently built up from a certain height (2 cm). The angle of repose was calculated by the following equation: angle of repose (θ) =

height of the pile of particles (h) distance from the center of the pile to the edge (r) (2)

Moisture Content Determination. The moisture content or water content in the prepared spray dried powders was determined by Karl Fisher volumetric titration technique using a Karl Fisher autotitrator (T- 50M, 51303566960, Mettler Toledo, Switzerland). In this technique, the moisture content in a sample (0.5 g) was monitored by measuring the consumption of iodine. One mole of I2 is consumed for each mole of H2O. The end point was detected by a bipotentiometric method. The water content was measured in triplicate.21 In Vitro Lung Deposition Studies. A cascade impactor (10709 Mark II 1-ACFM, Andersen Instrument Inc., Atlanta, USA) was used for determination of aerodynamic particle size distribution and deposition of DPI formulations in each region of lungs. Briefly, the instrument was linked to the opening of a jet mill collection vessel which was already connected to a vacuum pump. 2 g of powder sample was introduced into a cascade impactor with a flow rate of 5 L/min. Every plate of Anderson cascade impactor was then analyzed for the total drug deposited, and particle size was determined from the cumulative mass distribution.22 Drug Encapsulation Studies. Briefly, drug-loaded spray dried microparticles were separated from free drug by ultracentrifugation (3K-30, Sigma SV Instruments, New Delhi) at 10,000 rpm for 15 min at 4 °C (indirect method). In the clear supernatant, the free drug amounts of INH and RIF were measured using a UV spectrophotometer (UV 1700 Pharma Spec Lambda 35 Shimadzu, Japan) at 262 and 473 nm, respectively.23 To reconfirm, the loading efficiency of spray dried particle after separation was analyzed for drug content. Entrapment efficiency (EE %) and drug loading capacity (LC %) were calculated according to the following equations:

cell viability (%) = absorbance of the test sample /absorbance of control sample × 100 (5)

Microbial (MIC) Studies. The modified broth micro dilution MIC test was performed to determine drug susceptibility against microbial strain (Mycobacterium tuberculosis, an avirulent strain, purchased from Institute of Microbial Technology, Chandigarh, Punjab, India). First, the fresh culture of tested organism was developed in Lowenstein−Jensen medium. Aliquots of the culture were prepared in normal saline. The broth culture was incubated at 35 °C until the turbidity was developed. Suspension culture was analyzed spectroscopically to determine the colony forming unit (CFU) count using the calibration curve. All optimized formulations and drug solution in HPLC grade water were inoculated with Mycobacterium tuberculosis for 48 h. Each experiment was repeated at least three times.26 Confocal Microscopy Studies. Briefly, FITC dye incorporated spray dried microparticles (preparation technique was the

entrapment efficiency (EE%) = total drug concentration − drug concentration in supernatant/total drug concentration × 100

(3)

drug loading capacity (LC%) = total drug concentration − drug concentration in supernatant/weight of nanoparticles × 100

(4)

In Vitro Drug Release Studies. The dialysis tube diffusion method was used to determine the drug release from optimized C


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organs were isolated, weighed separately, minced into pieces, and homogenized by a tissue homogenizer (WiseMix HG-15D, Dathan Scientific, New Delhi) in phosphate buffer. The homogenized tissues were deproteinized with acetonitrile and vortexed for 5 min. Supernatant was filtered through a membrane filter, used for the analysis of INH and RIF upon suitable dilution with mobile phase by high-performance liquid chromatography technique, and compared with preformed calibration graphs.31 In Vivo Antitubercular Activity. Female BALB/c mice were infected with M. tuberculosis H37Rv strain (4.2 × 107 bacilli/ mL) through the pulmonary route using an aerosol chamber (Glascol-inhalation exposure system, USA). After 15 days, the Ziehl−Neelsen staining technique was used for confirmation of the presence of mycobacteria. Additionally, diluted (1:10, 1:50, 1:250, and 1:500) and undiluted homogenates were plated on Mycobacteria 7H11 agar medium supplemented Middlebrook OADC enrichment medium for the determination of basal colony forming units (CFU). Consequently, the mice were grouped as follows (n = 6 per group): group 1, untreated control; group 2, naive group; group 3, free drugs, aerosol; group 4, drug loaded CNPs, aerosol; group 5, drug loaded GNPs, aerosol; group 6, drug loaded MNPs, aerosol; and group 7, drug loaded GCNPs, aerosol. The animals were sacrificed after 2, 4, and 6 weeks of chemotherapy; lungs was removed and homogenized in sterile normal saline solution. The diluted and undiluted homogenates were plated on the above medium for cfu estimation on 14th, 28th, and 45th days postinoculation.32 Histopathology Assays. Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. The histopathology studies were performed on female mice following administration of the tested formulation. The isolated lung tissue was fixed in 5 mL of 10% neutral buffered formalin. Lungs were embedded, sectioned horizontally, stained with hematoxylin as well as eosin, and observed under an optical microscope for evaluation of epithelial cell disruption and inflammatory response.33,34 Statistical Analysis. Statistical tests were performed with Graph Pad Instat Software. The pharmacokinetic data were analyzed by Student’s unpaired t test and cfu data analyzed by analysis of variance (ANOVA) to compare the control and treated groups. Difference with p ≤ 0.05 was considered statistically significant.

same as mentioned in the cell uptake study) were administered to mouse lungs by using a dry powder insufflator (Penn Century, USA). The mice were grouped with three mice per group, and the study was repeated three times. The mice were sacrificed via cervical dislocation, and lungs were collected. The tissues were further analyzed via confocal microscopy (Olympus FV1000 MPE) at different magnifications to determine if the fluorescence observed was still in the particulate matter or had been leached out.27 In Vivo Lung Deposition Studies. Gamma scintigraphy was performed to evaluate in vivo lung deposition of optimized carrier systems in mouse lungs. The formulation for gamma scintigraphy was screened on the basis of in vitro performance. Briefly, MNPs and GCNPs were radiolabeled with technetium99 (99mTc) using a direct labeling method at 30 °C for 30 min. Radiolabeled formulations were inhaled through a nebulizer machine (Penn Century, USA) at the dose of 1 mg/kg body weight. The mice were placed on an imaging board, and imaging was performed using a gamma camera at different time intervals.28 In Vivo Studies. Female BALB/c mice (20−30 g) were used for in vivo studies of optimized spray dried formulations. Experiments were conducted as per Committee for Prevention, Control and Supervision of Experimental Animals/Institutional Animal Ethics Committee (IAEC), approval no. ISF/CPCSEA/ IAEC/2013/100 guidelines. Bioanalytical Method Development. A selective and sensitive optimized analytical method was developed for simultaneous determination of RIF and INH using RP-HPLC with diode array detector (DAD) (WATERS, USA). A 200 μL standard solution containing RIF and INH was spiked into BALB/c plasma (200 μL). The spiked sample was later mixed with acetonitrile (1.4 mL) and vortexed for 30 s at room temperature. After centrifugation at 4000 rpm for 15 min, the supernatant was separated and analyzed. A 20 μL aliquot from the final preparation was injected into the HPLC column. Calibration curves were prepared by linear regression analysis of the plot for peak area against simultaneous concentration of RIF and INH. The concentration of plasma samples was determined from the area of the chromatographic peak using the calibration curve.29 Pharmacokinetic and Biodistribution Study. Female BALB/c mice (20−30 g) were used for pharmacokinetic and organ distribution studies of optimized formulations. Various optimized spray dried formulations were administered through a suitable nebulizer device (Pan Century, USA). Throughout the study, the drugs were used at therapeutic dosage (INH, 10 mg/kg, and RIF, 12 mg/kg body weight). BALB/c mice were grouped as follows, with six animals in each group: group 1, free drugs, aerosol and oral (using cannula); group 2, drug loaded CNPs, aerosol; group 3, drug loaded GNPs, aerosol; group 4, drug loaded MNPs, aerosol; and group 5, drug loaded GCNPs, aerosol.30 In pharmacokinetic study, animals from each group were bled after 1, 2, 4, 8, 12, and 24 h through retroorbital plexuses. Collected blood samples (0.5 mL) were centrifuged at 1000 rpm for 20 min; plasma was separated and filtered through a membrane filter (0.45 μm). Drug estimation in plasma was done using a developed HPLC technique (discussed above), and a calibration curve was obtained by analyzing pooled blank plasma spiked with a known amount of drug. The other pharmacokinetic parameters were calculated by using Sigma Plot software (version 8.0). At the same time, animals were sacrificed via cervical dislocation and selected

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RESULTS AND DISCUSSION Preparation and Characterization of Spray Dried Nanoembedded Microparticles. All spray dried nanoembedded microparticle formulations were successfully preTable 1. Particle Size and Polydispersity Index (PDI) of Optimized Spray Dried Formulations (Mean ± SD, n = 3) formulation code CNPs-RIF CNPs-INH GNPs-RIF GNPs-INH MNPs-RIF MNPs-INH GCNPs-RIF GCNPs-INH D

particle size (nm) 875 824 1425 1300 1125 900 1760 1575

± ± ± ± ± ± ± ±

5.81 3.42 5.42 8.43 9.44 6.45 7.26 5.57

polydispersity index (PDI) 0.210 0.195 0.315 0.341 0.110 0.190 0.260 0.185

± ± ± ± ± ± ± ±

0.019 0.025 0.009 0.120 0.111 0.009 0.021 0.015


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Figure 1. SEM micrographs of different optimized formulations which were considered in interpreting the surface morphology.

Table 2. In Vitro Characterization of Optimized Spray Dried Formulations (Mean ± SD, n = 3) formulation code CNPs-RIF CNPs-INH GNPs-RIF GNPs-INH MNPs-RIF MNPs-INH GCNPs-RIF GCNPs-INH

angle of repose 17.41 22.21 18.52 23.93 23.04 28.25 16.42 23.21

± ± ± ± ± ± ± ±

0.5 0.6 0.3 1.4 2.1 3.2 0.6 0.3

% yield 55.81 57.58 59.03 57.00 53.44 43.49 65.81 62.58

± ± ± ± ± ± ± ±

5.52 4.74 6.62 7.15 4.52 7.12 4.92 6.78

moisture content 3.64 3.51 3.91 3.75 4.91 4.33 4.14 4.51

± ± ± ± ± ± ± ±

aerodynamic diameter (μm)

0.34 0.21 0.11 0.66 0.55 1.21 0.14 1.01

1.17 1.21 1.57 1.43 1.92 1.76 1.58 1.64

pared and characterized. The particle size and polydispersity index (PDI) of all optimized formulations are summarized in Table 1. Figure 1 represents the SEM micrographs of different experimental formulations. Spray dried mannan particles exhibited irregular surface morphology with external voids, which could be related to low molecular weight of mannan, leading to agglomeration due to rapid drying and reduced turbulence of the atomized droplets. Further, the low viscosity of the feed causes the rapid expansion of solvent during spray drying. On the other hand, guar gum spray dried particles showed a smooth surface. The high molecular weight of guar

± ± ± ± ± ± ± ±

0.02 0.02 0.01 0.01 0.02 0.03 0.03 0.02

entrapment efficiency (%) 70.81 68.82 52.43 63.44 45.44 58.43 62.42 60.81

± ± ± ± ± ± ± ±

6.62 7.02 5.02 4.04 5.03 6.09 5.02 5.12

loading capacity (%) 42.48 40.16 31.14 37.23 23.33 41.34 35.22 26.14

± ± ± ± ± ± ± ±

4.90 5.06 4.16 5.28 6.26 4.17 2.40 3.18

gum enables controlled turbulence with minimum aggregation under the defined spray drying condition. Chitosan formulations exhibited the best surface morphology among the experimental lots, which could be related to the best mechanical strength of preformed chitosan nanoparticles that resist particle squeezing during flash evaporation of the spray drying process. Further addition of guar gum offers an additional advantage to manage particle morphology at the time of atomization. In contrast to guar gum formulation, mannan batches, by virtue of their low viscosity, possess difficulty in maintaining a smooth surface texture during spray drying. The low PDI in the case of E


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Figure 2. In vitro drug release profile of experimental formulations in PBS (pH 7.4; mean ± SD, n = 3).

Figure 3. Cell uptake study of optimized formulations at different time intervals as analyzed through FACS (mean ± SD, n = 3).

Figure 4. Cell viability (%) at different time intervals of optimized formulations as studied by MTT analysis (mean ± SD, n = 3).

properties of all experimental formulations. Spray dried mannan particles show relatively poor flow properties among the tested formulations, which could be related to irregular particle morphology and higher moisture content of the resultant particle. When the micrometric properties of mannan particles were compared with similar size range particles, they exhibited reduced aerodynamic diameter owing to their high affinity for water and large surface discontinuation. Guar gum batches exhibited excellent flowability attributed to their optimum moisture content and uniform size distribution. Moisture content analysis indicated that the coated formulations show higher moisture content, which could be due to higher polymer

mannan particle could be attributed to its intrinsic density where only the larger particle of narrow size distribution was controlled in the cyclone under the applied spray dried condition, whereas smaller particles were removed by virtue of their low density. On the other hand, chitosan and guar gum particle due to their higher density were collected in the cyclone irrespective of their size with relatively higher PDI over mannan formulations. Drug Encapsulation and Micrometric Properties of Optimized Spray Dried Formulation. Table 2 represents micrometric properties of optimized formulations after spray drying. D-Mannitol and L-leucine improved the yield and flow F


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Figure 5. Confocal images of lung epithelium of treated animal showing the target efficacy of the optimized formulations: (A) CNPs, (B) GNPs, (C) MNPs, and (D) GCNPs.

Figure 6. Gamma scintigraphy images of mice showing the presence of radioactivity: MNPs (A) and GCNPs (B) (pulmonary).

content per unit mass. Further, the size of the final formulation plays an important role in the moisture content. CNPs possess smaller size compared to GCNP; as a result CNPs possess a higher surface area turning higher heat transfer than GCNPs. The aerodynamic diameter values for most of the particle

systems were within the range necessary (1−5 μm) for particles to deposit predominantly in the middle to deep regions and deposit by sedimentation due to gravitational settling.35 All experimental formulations exhibited excellent aerodynamic diameter ranging from 1.12 to 1.92 μm, ascertaining the G


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Molecular Pharmaceutics Table 3. Comparison of Pharmacokinetic Parameters Following Pulmonary Administration of Optimized Formulations into BALB/c Mice (Mean ± SD, n = 3)

Table 4. Chemotherapeutic Efficacy of Various Formulations against Experimental Tuberculosis Infected Mice (Mean ± SD, n = 6)

pharmacokinetic parameters optimized formulations free RIF free RIF (oral) free INH free INH (oral) CNPs- RIF CNPs-INH GNPs- RIF GNPs-INH MNPs-RIF MNPs-INH GCNPs- RIF GCNPs-INH

Cmax (μg/ mL) 6.65 5.25 5.55 4.85 4.23 4.67 3.21 3.70 4.76 5.27 3.82 4.13

± ± ± ± ± ± ± ± ± ± ± ±

1.18 1.14 1.23 1.11 1.93 2.23 1.23 1.11 0.91 1.89 1.03 1.20

Tmax (h) 2.89 3.12 2.03 2.47 16.24 18.32 28.23 31.20 7.72 9.21 20.22 24.02

± ± ± ± ± ± ± ± ± ± ± ±

0.31 0.52 0.61 0.23 4.03 3.22 2.23 3.33 2.21 3.01 3.43 3.49

log10 CFU (lungs)

AUC0−24h (μg/mL/ h) 45.51 42.12 32.43 28.54 150.23 386.93 223.33 467.45 107.45 147.28 180.93 430.23

± ± ± ± ± ± ± ± ± ± ± ±

experimental groups disease control free ATDs (RIF-INH) CNPs- RIF-INH GNPs- RIF-INH MNPs- RIF-INH GCNPs- RIF-INH

3.75 4.45 5.25 3.98 6.08 8.08 8.93 9.15 6.09 8.05 3.09 8.02

after 28 days 5.40 4.99 2.23 1.10 1.82 0.20

± ± ± ± ± ±

0.15 0.34 0.19 0.07 0.09 0.01

after 45 days 5.81 5.23 1.72 0.67 2.62 0.05

± ± ± ± ± ±

0.30 0.28 0.71 0.03 0.05 0.01

In Vitro Drug Release Studies. Figure 2 shows the release behavior of both drugs from various optimized formulations in PBS (pH 7.4). RIF takes almost 2 h whereas INH takes 30 min to reach 95% cumulative release. Spray dried mannan because of its higher affinity for water exhibited high solubility in release medium and releases drug within the first few minutes. The difference in release profile of RIF and INH from mannan could be related to the intrinsic solubility of individual drugs. The kinetic studies revealed that the drug release from mannan particles follows first order kinetics where the amount of drug release is a function of drug concentration in spray dried particles. Controlled drug release behavior in chitosan batches is associated with its polyelectrolyte nature where chitosan remains nonprotonated at the alkaline pH of the selected release medium. Further, the cross-linking of chitosan restricts the flux of dissolution medium, thereby allowing controlled diffusion of encapsulated drugs. In addition to the above, guar gum acts as a rate limiting barrier, which potentially contributes in the delayed drug release in GCNP formulation. Additionally, the high molecular weight guar gum swells to form a rigid gel matrix. Further, the release exponent (n) values obtained were between 0.5 and 0.6, suggesting that the mechanism of drug release from the spray dried mannan depends on the drug concentration. Further, the release behavior from the experimental carrier was ascertained by the value of correlation coefficient (R2) values. The highest R2 value was considered to

deposition of experimental formulations in the posterior part of lungs. There was no significant difference observed in the aerodynamic diameter of different formulations. However, the minor difference in aerodynamic diameter could be a function of their density and particle size distribution. The higher drug encapsulation in chitosan nanoparticles could be attributed to the higher drug:polymer ratio as well as an excellent ionic gelation between TPP and chitosan. Relatively, lower drug entrapment in GCNP batches may be possibly due to the mannosylation reaction involving guar gum and chitosan which might cause leaching of ATDs in the reaction medium. The difference in encapsulation efficiency and loading capacity of INH and RIF in all experimental batches is owing to their intrinsic solubility in the respective dispersion medium, partition coefficient, and physicochemical properties of base components selected for experimental studies. To reconfirm, the loading efficiency of spray dried particle after separation was analyzed for drug content, which was found to be the same as the results obtained from the indirect method.

Figure 7. Antitubercular drug (ATD) lung distribution at different time intervals following pulmonary administration of optimized formulations (mean ± SD, n = 3). H


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Figure 8. Photographic images of experimental animal lungs represent the chemotherapeutic efficacy of various formulations against Mycobacterium tuberculosis infected mice.

be the best fit model to describe the drug release behavior. The R2 value (0.9728) obtained for fitting the drug release data to the Higuchi model inferred that the drug release behavior from chitosan batches was diffusion controlled, indicating that the rate of drug dissolution was much slower than the polymer erosion under the experimental condition. Further, the n values obtained were between 0.53 and 0.874 for all experimental formulations, suggesting more than one mechanism involved in release kinetics referring to combination of drug dissolution and erosion based drug release mechanism. In Vitro Cell Line Studies. Figure 3 demonstrated that chitosan formulations have relatively delayed cell uptake efficiency, which could be a function of low charge density of resultant chitosan particles. Chitosan by virtue of its cationic property facilitates the cell uptake process. However, charge density per unit volume was largely impaired by the ionic gelation process and particle size distribution. Low charge density and larger size of chitosan formulation allow relatively delayed cell uptake efficacy. Mannan batch has excellent macrophage uptake related to its mannose subunit, which serves as a specific agent for the glycosylated receptors located on the macrophage cells. Further, the geometric size also plays an important role in this process. A comparable macrophage uptake was observed in guar gum batches, which could be related to its intermittent mannose unit, exhibiting relatively poor affinity for its mannan counterpart. However, the intact nature of guar gum particles promotes the uptake mechanism via receptor mediated phagocytosis. The preferential uptake of GCNPs suggested the presence and selective uptake capability of mannose moiety present in guar gum to the specific cell surface of macrophages. In cell viability studies, a significant evidence of lesser cytotoxicity was observed for blank and drug

loaded optimized formulations as compared to free INH and RIF after treatment (Figure 4). Microbial Studies. The minimum inhibitory concentrations (MIC) of RIF and INH solution (135 ± 5.4 μg/mL) were approximately 15−16 times higher than those of drug loaded formulations (CNPs-RIF-INH 16.86 ± 0.9 μg/mL and GCNPs-RIF-INH 8.53 ± 1.8 μg/mL). A mannan and guar gum formulation shows a comparable antimicrobial efficacy to CGNPs-RIF-INH batches. However, the antimicrobial activity of guar gum formulation increase from 12 to 24 h could be related to dual release mechanism. Burst effect is beneficial as it helps to achieve the inhibitory concentration followed by a delayed release to ensure inhibitory drug concentration over a period of prolonged action, thereby decreasing the total therapeutic dose. Confocal Microscopy Studies. Figure 5 represents confocal micrographs of lung epithelium of treated animals. The confocal micrographs suggested that guar gum coated formulation and mannan particles show strong intracellular fluorescence in the cytosol of the macrophages. Both mannan and guar gum formulations exhibiting excellent fluorescence could function as excellent active targeting ligands which can be used selectively to achieve better targeting. The results are indicative of good targeted drug delivery systems. Confocal microscopic studies also supported the outcomes of cell uptake studies. Confocal microscopic studies also revealed that the alveolar macrophage cell internalization is predominantly a function of receptor mediated endocytosis where glactomannan content carries preferentially endocytosis. Fluorescent intensity was relatively poor in chitosan batches, reinforcing the hypothesis that the receptor mediated endocytosis process is a rate limiting step for the cell internalization process in alveolar I


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Figure 9. Histogram of lung tissues indicating the pathological changes observed in different experimental animal treated with different formulations: (A) naive group, (B) control group, (C) CNPs, (D) GNPs, (E) MNPs, and (F) GCNPs.

and particle morphology plays an important role in achieving the site directed targeted delivery. Results demonstrated the dark spot at the posterior apical part of the lungs which is generally regarded the highest prevalence site for pulmonary lesion during TB. Further, the posterior apical site of lungs is regarded as a primary center for extraversion of bacilli to extrapulmonary organs. Thus, the highest drug concentration at the posterior apical site helps to minimize the bioburden of TB to extrapulmonary organs. Pharmacokinetic and Biodistribution Studies. Pharmacokinetic study is an indicator for therapeutic utility and systemic toxicity of proposed strategies. Table 3 represents different pharmacokinetic parameters of the developed optimized formulation. It was observed that peak plasma drug concentrations (Cmax) were achieved rapidly in the case of free drug compared to the developed formulation due to the controlled release. Higher AUC values of developed formulations showed that drug concentrations were maintained

macrophage. Both the cell uptake and confocal microscopy studies suggested that the proposed therapeutic strategy comprising a dual targeting approach could serve as a potential therapeutic modality to treat lung infection. In Vivo Lung Deposition Studies. Figure 6 represents gamma scintigraphy images of mice to visualize lung uptake. A uniform distribution of radioactivity was observed in lungs after pulmonary administration of optimized formulations. The presence of a red dark spot showed the presence of carrier system in lungs. During the gamma scintigraphy study, the presence of carrier systems was detected in the lungs for 4−8 h. However, the percent radioactivity had significantly decreased (t1/2 of 99mTc(1/4) 4−5 h). The presence of carrier systems in the lungs could not be assessed after 12 h of administration due to negligible radioactivity. Gamma scintigraphy further confirms the specific localization of selected carrier system at the target tissue. In addition to the principle of active targeting, results of gamma scintigraphy revealed that the route of administration J


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containing galacto mannan subunits like xyloglucan, gum karaya, etc. that could serve as excellent drug vectors for lung infection by virtue of their mucoadhesiveness and biodegradable and ligand specific characteristics. From the Results and Discussion, we concluded that all optimized formulations showed controlled and sustained drug release for longer duration of period. The optimized formulations also showed lower cytotoxicity and enhanced lung uptake of drugs. Guar gum coated chitosan (GCNP) formulations additionally resulted in almost 5-fold reduction of the number of bacilli in the lungs as compared to free drug. Further, dual targeting strategy composed of DPI and site directed drug delivery could serve as an innovative approach to treat local pathological conditions of lung like cystic fibrosis, chronic bronchitis, lung cancer, etc. Although the preclinical results of the present study were quite interesting, however, it requires extensive clinical studies before being considered for clinical use.

within the pharmacologically effective range for longer period of time. Figure 7 represents the biodistribution studies of the developed optimized formulation. The results clearly indicate superiority of the ATD loaded optimized formulations in comparison to the free drug in increasing the accumulation of ATDs within the organs rich in macrophages, i.e., lungs. Pharmacokinetic study suggested that mannan formulations are preferentially taken up by the alveolar macrophage by virtue of their affinity for glycosylated receptor present on alveolar cells. However, due to their intrinsic solubility, they exhibit relatively lesser residence time compared to guar gum coated chitosan formulation. Hence further mannan formulation, irrespective of their site specific targeting or their inability to restrain or localize at the target site, significantly impaired their therapeutic efficacy. Further guar gum and chitosan formulation, by virtue of their mucoadhesiveness, was retained at the target site for a prolonged period. On the other hand, guar gum coated chitosan batches demonstrated prolonged residence at the target site, thereby improving the therapeutic utility of drug with a significant reduction in systemic toxicity. In addition to higher residence time, the guar gum coated chitosan formulation served to be a better therapeutic strategy against the experimental tuberculosis. In Vivo Antitubercular Activity. The antitubercular activity of the optimized formulations was implemented on the TB induced BALB/c mice. After 45 days treatment, microbial loads in the lung were significantly reduced as compared to control group (Table 4). Challenge study suggested that the poor antitubercular activity in mannan formulation could be related to poor residence time at the target site. Further, the rapid dissolution rate of mannan formulation could be largely contributed to its impaired antimicrobial activity. Guar gum coated formulation on the other hand shows significant antimicrobial activity by virtue of its targeting efficacy and sustained release profile. The optimized drug loaded GCNP formulation has resulted in almost 5-fold reduction of the number of bacilli as compared to control group. All experimental observations clearly indicated that GCNPs-RIF-INH is more effective against the mycobacterium than other formulations (Figure 8). Histopathology Assays. Figure 9 shows the microscopic images of histopathology samples. Control group tissues show necrotizing granuloma surrounded by a rim of epithelioid histiocytes with multinucleated giant cells. Mannan batches show relatively higher pathological consequences evident by the presence of inflammatory cells and parenchyma degeneration. This could be related to high burst release of ATDs, resulting in high local drug concentration causing accidental cell death. Higher evidence of toxicity in mannan formulation is further related to preferential accumulation of mannan carrier in phagocytic cells due to the presence of mannose receptor predominantly found on their cell surface. Other drug loaded formulations show a thin layer of connective tissue and numerous capillaries lined with simple squamous epithelium without any necrotizing granuloma.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 09878286888. Fax: 01636-236564. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors (under IYBA scheme; BT/01/IYBA/2009 dated 24/05/2010) are thankful to the Department of Biotechnology (DBT), New Delhi, India, for providing financial assistance to carry out research work.

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REFERENCES

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CONCLUSIONS AND FUTURE PROSPECTS Various natural (chitosan, alginate, and gelatin) and synthetic (PCL, PLGA, or PLG) polymers have been extensively used as drug delivery systems. On this basis, two natural polymers, chitosan and guar gum, were selected for delivery of antitubercular drugs (ATDs) against experimental tuberculosis. This study explored the potential of natural polysaccharides K


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