Particle size with suitable and uniform distribution of particle size is the utmost criteria for controlled drug delivery system. Along with high drug loading, there is also the requirement of delivering less amount of drug in pulmonary delivery. These parameters depend on many factors like polymer amount, surfactant concentration and organic phase volume. Polymer amount increases the particle size as well as entrapment efficiency, whereas surfactant concentration and organic phase volume influences the particle size and charging of particles. From the preliminary trials, a 3³ Box-Behnken Design was employed to study the effect of three independent variables, i.e. PLGA amount (X₁), PVA concentration in % w/v (X₂), and Dichloromethane volume in ml (X₃), on three dependent variables like particle size (Y₁), percentage drug entrapment (PDE) (Y₂), Polydispersibility index (Y₃). Effects of independent variables were studied at three different levels (Table 1). Nanoparticles were prepared as per the experiment design matrix generated by the software.

After centrifugation, un-entrapped PTH in the supernatant was estimated by UV-spectrophotometric method as reported previously [15Debnath SK, Saisivam S, Dash DK, Debnath M. Development and validation of UV-spectrophotometric methods for quantitative estimation of Prothionamide in pure and pharmaceutical dosage forms. Int Curr Pharm J 2015; 4(7): 402-4.
[http://dx.doi.org/10.3329/icpj.v4i7.23590] ]. Entrapped drug was calculated by subtracting un-entrapped drug from total drug. Further percentage drug entrapment was calculated from the entrapped drug [3Jain DS, Athawale RB, Bajaj AN, et al. Unraveling the cytotoxic potential of Temozolomide loaded into PLGA nanoparticles. DARU J Pharm Sci 2014; 22(1): 18.
[http://dx.doi.org/10.1186/2008-2231-22-18] [PMID: 24410831] , 4Pirooznia N, Hasannia S, Lotfi AS, Ghanei M. Encapsulation of alpha-1 antitrypsin in PLGA nanoparticles: in vitro characterization as an effective aerosol formulation in pulmonary diseases. J Nanobiotechnology 2012; 10(20): 1-15.
[http://dx.doi.org/10.1186/1477-3155-10-20] [PMID: 22607686] ]. Average particle size and Poly Dispersity Index (PDI) of the prepared nanoparticles were determined by laser dynamic light scattering using Zetatrac, Microtac Inc.USA. The PDI value indicates the particle size distribution of nanoparticles in a given sample. Higher value of PDI indicates low homogeneity due to wide distribution of NPs, which results in aggregation of particles. Zeta potential indicates the surface charge on the particles and was measured to determine the stability of nanoparticles in the suspension [16Sharma D, Maheshwari D, Philip G, et al. Formulation and optimization of polymeric nanoparticles for intranasal delivery of Lorazepam using Box-Behnken design: In vitro and in vivo evaluation. Biomed Res Int 2014; 1-14.].

The shape and surface morphology of the nanoparticles were examined for the optimized batch by Scanning Electron Microscopy (Karl Zesis with SE detector, EVO-18). The samples were sputter-coated with gold and observed for morphology at an acceleration voltage of 7.0 kV with 10.66KX and highest magnification of 19.99 KX.

Aerodynamic diameter of a particle controls its deposition in pulmonary tract. MMAD represents aerodynamic diameter below which 50% particles remain. MMAD of the optimized dry powder inhaler was determined using an eight stage cascade impactor. Firstly, DPI were passed through the cascade impactor with a flow rate of 28.3 l/min, thereafter calculated the Prothionamide content in each stage by direct measurement of Prothionamide using RP HPLC C18 Column, length-250mm. The mobile phase selected was water: Acetonitrile at 70:30 ratio with a flow rate 1.0ml/min. Absorbance was detected at 290nm. These values were inserted into the MMADCALCULATOR to obtain MMAD and geometric standard deviation (GSD) [17Sinha B, Mukherjee B. Development of an inhalation chamber and a dry powder inhaler device for administration of pulmonary medication in animal model. Drug Dev Ind Pharm 2012; 38(2): 171-9.
[http://dx.doi.org/10.3109/03639045.2011.592532] [PMID: 21721851] ].

5 mg PTH equivalent DPIs were dispersed in 2ml of Simulated lungs fluid (SLF) of pH 7.4 [18Marques MR, Loebenberg R, Almukainzi M. Simulated biological fluids with possible application in dissolution testing. Dissolut Technol 2011; 18(3): 15-28.
[http://dx.doi.org/10.14227/DT180311P15] , 19Debnath SK, Saisivam S, Omri A. PLGA ethionamide nanoparticles for pulmonary delivery: Development and in vivo evaluation of dry powder inhaler. J Pharm Biomed Anal 2017; 145: 854-9. [Internet].
[http://dx.doi.org/10.1016/j.jpba.2017.07.051] [PMID: 28826144] ] and subsequently filled in dialysis bag (12,000 molecular cut off). To avoid floating, the drug filled bag was tied over the glass plate and kept at the bottom of the dissolution chamber. In-vitro release study was carried out in 1L SLF of pH 7.4 at 37 ±0.5ºC with a paddle speed of 100 rpm [20May S, Jensen B, Wolkenhauer M, Schneider M, Lehr CM. Dissolution techniques for in vitro testing of dry powders for inhalation. Pharm Res 2012; 29(8): 2157-66.
[http://dx.doi.org/10.1007/s11095-012-0744-2] [PMID: 22528980] ]. 10 mL of dissolution fluid was withdrawn each hour up to 24h and replaced with fresh SLF.

Accelerated stability study was carried out as per the ICH guideline Q1A (R2). Paraffin tape was used to seal cryoprotectant vials contained freshly prepared DPI of PTH nanoparticles. These vials were kept in stability chamber and maintained at 25±2°C & 60±5% RH. The nanoparticles were analyzed for the period of 6 month [15Debnath SK, Saisivam S, Dash DK, Debnath M. Development and validation of UV-spectrophotometric methods for quantitative estimation of Prothionamide in pure and pharmaceutical dosage forms. Int Curr Pharm J 2015; 4(7): 402-4.
[http://dx.doi.org/10.3329/icpj.v4i7.23590] , 21Fernandes CA, Vanbever R. Preclinical models for pulmonary drug delivery. Expert Opin Drug Deliv 2009; 6(11): 1231-45.
[http://dx.doi.org/10.1517/17425240903241788] [PMID: 19852680] , 22Chougule M, Padhi B, Misra A. Nano-liposomal dry powder inhaler of tacrolimus: preparation, characterization, and pulmonary pharmacokinetics. Int J Nanomedicine 2007; 2(4): 675-88.
[PMID: 18203434] ]. Zeta size, zeta potential, PDI, drug entrapment and drug release were carried out to check the stability of dry powder inhaler of PTH nanoparticles.

Animal experiments were performed according to the guidelines of institutional animal ethical committee of Bengal College of Pharmaceutical Sciences and Research, Durgapur, West Bengal with prior approval (Registration No: 1799/PO/ Ere/15/S/CPCSEA under CPCSEA, India). Wistar rats (either sex, 4-6 month old & average weight 200-250g) were used in this study to assess the pharmacokinetic parameters of PTH in the form of DPI. The rats were housed for 12-h light/dark cycle with free availability of food and water. Rats were divided into two groups: one was treated with DPI of Prothionamide nanoparticles; and the other group was treated with pure Prothionamide [23Guidance for industry. Estimating the maximun safe starting close in initial clinical trial for therapeutics in adult health volunteers.. 2005. Available at: http://www.fda.gov/downloads/Drugs/Guidance/UCM078932.pdf]. Dry powder inhaler delivery device was designed with little modification as described previously [24Okuda T, Suzuki Y, Kobayashi Y, et al. Development of biodegradable polycation-based inhalable dry gene powders by spray freeze drying. Pharmaceutics 2015; 7(3): 233-54.
[http://dx.doi.org/10.3390/pharmaceutics7030233] [PMID: 26343708] ]. Tongue was gently pulled outside and sprayed the DPI after placing the DPI device in the trachea region. After administration, three separate studies were performed. 1ml blood was withdrawn from the tail vain of rat to check the bio-distribution of PTH at 1, 2, 3, 6, 12 & 24h. After scarification in these specified time interval, lungs were isolated and filled it with 4 ml Phosphate buffer solution (PBS) by injected slowly in fractions to fill the lungs [22Chougule M, Padhi B, Misra A. Nano-liposomal dry powder inhaler of tacrolimus: preparation, characterization, and pulmonary pharmacokinetics. Int J Nanomedicine 2007; 2(4): 675-88.
[PMID: 18203434] ]. The fluid was withdrawn very slowly followed by one repetition. To estimate PTH remaining to be released, this fluid was vortexed for 5 min with 1ml of methanol. To check the tissue distribution, lungs and trachea portion were homogenized with 10 ml of PBS solution and 1 ml methanol followed by vortexed for 5min. 30% trichloroacetic acid was added to each sample for deprotenization. After neutralized with sodium bi carbonate, each sample was centrifuged for 10min at 8000rpm. Supernatant containing Prothionamide was estimated by HPLC as described previously. Maximum concentration attained by PTH (C_max) at specified time (T_max) and area under curve (AUC) were calculated thereafter.

Polymer amount is known to play an important role in controlling particle size along with release of drug from the matrix. Average particle size was found in the range of 224 to 510nm. The effect of average particle size was explained by the following quadratic equation:

(Y₁)= + 299.67 + 20.87X₁ - 22.50X₂ + 30.13X₃ + 13.25X₁X₂+ 92.50X₁X₃ - 27.25X₂X₃ + 20.17X₁² - 37.58X₂² + 48.17X₃².........1

X₁, X₂ and X₃ had the significant effect on the particle size, as the coefficient values were more. Positive effect was seen in X₁ and X₃ variable due to ‘+ve’ sign. Hence, the average particle size of nanoparticles was increasing with increasing the PLGA amount. Our results are in accordance with those observed by other authors [16Sharma D, Maheshwari D, Philip G, et al. Formulation and optimization of polymeric nanoparticles for intranasal delivery of Lorazepam using Box-Behnken design: In vitro and in vivo evaluation. Biomed Res Int 2014; 1-14., 25Tripathi A, Gupta R, Saraf SA. PLGA nanoparticles of anti tubercular drug: Drug loading and release studies of a water in soluble drug. Int J Pharmatech Res 2010; 2(3): 2116-23.]. Mainardes et al. (2005) reported particles size increased with rising the volume of Dichloromethane, but here particle size increased with increasing Dichloromethane volume [13Mainardes RM, Evangelista RC. PLGA nanoparticles containing praziquantel: Effect of formulation variables on size distribution. Int J Pharm 2005; 290(1-2): 137-44.
[http://dx.doi.org/10.1016/j.ijpharm.2004.11.027] [PMID: 15664139] ]. On the other side, negative value of factor X₂ signifies that the particle size is indirectly proportional with the increasing PVA concentration. With decrease in PVA concentration, the particle size of the NPs increased [16Sharma D, Maheshwari D, Philip G, et al. Formulation and optimization of polymeric nanoparticles for intranasal delivery of Lorazepam using Box-Behnken design: In vitro and in vivo evaluation. Biomed Res Int 2014; 1-14.].

Percentage drug entrapment (PDE) of prepared nanoparticles was found to be in the range of 55 to 82%. The effect of average particle size was explained by the following quadratic equation:

(Y₂)= +72.67 + 2.13X₁ - 6.63X₂- 3.00X₃ + 0.75X₁X₂- 4.00X₁X₃-6.00X₂X₃ - 0.71X₁²- 3.21X₂² + 1.04X₃² ........................2

Table 1
Adopted variables levels in Box-Behnken design.

Table 2
Box Behnken experimental design.

Table 3
Optimization of dry powder inhaler.

Table 4
Accelerated stability study.

Table 5
In-vivo analysis.

There was less influence of X₁, X₂ & X₃ on the PDE value due to low coefficient value. Influence of X₂ on PDE was more in comparison to X₃ and X₁. Here, percentage drug entrapment was increased by increasing the PLGA amount (X₁) and decreasing the PVA concentration (X₂) and organic phase volume (X₃).

Polydispersibility Index or PDI (Y₃) was found in the range of 0.607 to 2.248. Correlation tried to establish the relation between PDI with X₁, X₂ and X₃. The value of zeta potential of all the batches was found to be within 5mV. Particle aggregation is less likely to occur for charged particles (i.e. high zeta potential) [26Kharia AA, Singhai AK, Verma R. Formulation and evaluation of polymeric nanoparticles of an antiviral drug for gastroretention. Int J Pharm Sci Nanotechnol 2012; 4(4): 1557-62.]. In this range, particle has more tendency to aggregate resulting in wide distribution of particle size. Hence, no significant influences were established between independent variables with polydispersibiliy index.

All the measured responses that may affect the quality of the product were taken into consideration during the optimization [27Pandya VM, Patel JK, Patel DJ. Formulation and optimization of nanosuspensions for enhancing Simvastatin dissolution using central composite design. Dissolut Technol 2011; 18(3): 40-5.
[http://dx.doi.org/10.14227/DT180311P40] ]. Particle size and PDE were taken under consideration for the selection of optimized batch. Formulation was optimization with set criteria of maximum entrapment efficiency and minimum particle size, followed by higher disability factor. Design suggested that optimized formulation contained 125mg of PLGA, 0.36%w/v solution of PVA and 2ml of DCM. Nanoparticles prepared using this composition and found the particle size of 310 ± 12 nm, PDE 80.08 ± 0.26% and PDI of 0.386 ± 0.13.

The size and shape of particles are the main parameters that determine their deposition in the different parts of lungs. Aerodynamic particle size in range of 0.5-5 µm can reach to the small airways and alveoli [28Fernández Tena A, Casan Clarà P. Deposition of inhaled particles in the lungs. Arch Bronconeumol 2012; 48(7): 240-6.
[http://dx.doi.org/10.1016/j.arbr.2012.02.006] [PMID: 22464044] ]. Prepared DPI of Prothionamide nanoparticles aerodynamic particle size was 1.69 µm and geometric standard deviation was 1.95. Maximum fine particle fraction, extra fine particle fraction and emitted dose were found as 76.88%, 23.88% and 91.46% respectively. This signifies that the prepared DPI can reach deeply to lungs.

The optimized Prothionamide nanoparticles (OP Batch) loaded DPI was subjected for in-vitro drug release behavior. SLF was used as dissolution medium for evaluating the release pattern of Prothionamide from PLGA nanoparticles. The optimized Prothionamide-PLGA nanoparticles showed initial burst release of 20% and then showed sustained drug release up to 43.52% in 24h (Fig. 3). The initial burst release will help to reach the desired plasma concentration in lungs, and the sustained release will help to maintain the PTH dose for prolong period of time. The in-vitro release data was analyzed through zero order, Higuchi, Peppas models and obtained correlation co-efficient (r²) were found to be 0.982, 0.966, and 0.933 respectively. The release best fitted with zero order kinetics.

Fig. (3) In-vitro release of optimized DPI in simulated lungs fluid at pH 7.4. Data represents mean ± SD (n=3).

Various literatures reported the instability of nanoparticles during storage. 6 month accelerated stability study revealed no clamping or aggregation of particles. Zeta size, PDI, and drug release demonstrated that the conservation of dry powder inhaler was stable during stress testing (Table 4).

The minimum inhibitory concentration (MIC) of Prothionamide (0.5µg/ml) was obtained for approximately 16h by a regimen of 250–375 mg twice daily [29Lee HW, Kim DW, Park JH, et al. Pharmacokinetics of prothionamide in patients with multidrug-resistant tuberculosis. Int J Tuberc Lung Dis 2009; 13(9): 1161-6.
[PMID: 19723408] ]. But this dose is never suitable for the pulmonary administration. So, pulmonary dose was calculated based on the human lungs volume (4.341 l) [21Fernandes CA, Vanbever R. Preclinical models for pulmonary drug delivery. Expert Opin Drug Deliv 2009; 6(11): 1231-45.
[http://dx.doi.org/10.1517/17425240903241788] [PMID: 19852680] ]. The prepared DPI of Prothionamide nanoparticles contained two parts: loading and maintenance dose. As both the dose release at a time, corrected dose was calculated as 78 mg for human administration. Dose calculation for rat was carried out with equivalent surface and weight with no observed adverse effect in 7.7mg/kg body weight [22Chougule M, Padhi B, Misra A. Nano-liposomal dry powder inhaler of tacrolimus: preparation, characterization, and pulmonary pharmacokinetics. Int J Nanomedicine 2007; 2(4): 675-88.
[PMID: 18203434] ]. Three different studies were carried out to determine percentage drug release in-vivo, lungs tissue distribution and bio-distribution of Prothionamide (Table 5). In contrast to pure PTH administration, PTH nanoparticles gave sustained release more than 24h, whereas pure PTH gave release 99.84±0.08 in 6h. AUC increased significantly when PTH was given in the form of nanoparticles. As PTH reached very slowly to plasma from PTH nanoparticles, AUC was relatively less 1.16±0.02 µgh/mL in comparison to pure PTH. C_max was 2.31±0.03 µg/mL for PTH DPI, whereas 4.567 ± 0.31 µg/mL for pure PTH reached at T_max 1h. DPI of PTH nanoparticles maintained plasma concentration above the MIC for the period of 6h, but pure PTH could be maintained for up to 3h. Concentration attained by PTH nanoparticles 0.22 ± 0.02 µg/mL, which is lower than the MIC. Hence, prepared formulation may be used once every day to maintain the plasma concentration above MIC for 24h. PTH was detected after 24h, when given in the form of nanoparticles, which signified prolong drug residency in lungs.