4.1 Particle size, PDI, and zeta potential measurement
In our investigation, among the three different F1, F2, and F3 formulations, the F2 formulation was selected and proceeded for further characterization due to its desirable particle size, PDI, and zeta potential of 310 ± 5.5nm, 0.15 ± 0.1, and − 11.5 ± 0.4mV, respectively as shown in Table 1. Compared to the F2 formulation, particles size of both F1 and F3 formulations were beyond our limit ranges (200-400nm). Therefore, F2 formulation was regarded as an optimized one. The increased size of F1 nanoparticle in respect to F2 is due to the aggregation of PLGA particles, as the PVA concentration is lower. Moreover, the F3 formulation has a comparatively higher particles size compared to F2 which might be due to the increased concentration of PLGA. The effect of increased PLGA concentration with increased particle size was also been investigated by Manchanda et al [26]. Likewise, lower PDI of F2 formulation compared to F1 is due to optimum PVA concentration that makes the nanosuspension more stable. Although PDI of F3 is comparatively higher than F2 which might be the reason for larger particles size leading to decreased stability of nanosuspension. Furthermore, negative zetapotential is particularly due to the effect of PVA. Increased negative zeta potential of F3 formulation is due to higher PVA concentration. A similar effect of PVA concentration on particles size, PDI, and zeta potential was observed in various investigations [32,33]. However, the particle size, PDI, and zeta potential of reconstituted freeze-dried NAC-PLGA@Cs were found to be 320 ± 6.2nm, 0.20 ± 0.1, and − 11.8 ± 0.6mV respectively. The results were obtained with a slight changes, indicating that our developed formulation even after coating with lactose remains stable as depicted in particle size distribution graph of Fig. 1: 1(E) coated NAC-PLGA@Cs (Formulation), and 1(F) free NAC.
4.2 Drug loading (DL), Entrapment efficiency (EE), and yield recovery determination
As shown in Table 1, F2 formulation exhibiting desirable particle size, PDI and zeta potential with additional comparative higher drug loading (10%) and entrapment efficiency (82%) compared to F1 formulation with slight difference irrespective to F3 formulation further proceed for its selectivity. Although some studies suggested that increased PVA concentration does not alter the % DL and, % EE significantly, instead alter the particles size, PDI and zeta potential [27, 28]. Besides this, Drug: PLGA ratio significantly changes the DL and EE. Increasing the PLGA concentration by keeping the drug concentration constant, enhances the drug loading and entrapment efficiency as observed in the study. Chaisri et al and Halayqa et al also depicted the similar results [34,35]. In addition to this, % yield recovery of freeze-dried NAC-PLGA@Cs was found to be 90%, while freeze-dried NAC-PLGA particle was found to be 89%.
4.3 Morphology study by FESEM and HR-TEM
After morphological evaluation of uncoated as well as coated PLGA particles by FESEM, particles were observed as a spherical shape. Irrespective of uncoated NAC-PLGA NP, HR-TEM images confirmed that the lactose coating of PLGA particles indicates the formation of inhalable PLGA composites. The given below Fig. 1 indicates (A) uncoated NAC- PLGA particles by FESEM, (B) coated NAC-PLGA@Cs by FESEM, (C) coated NAC-PLGA@Cs by HR-TEM.
4.4 FTIR
FTIR spectrum of NAC, PLGA, physical mixture of NAC-PLGA-Lactose, and NAC-PLGA@Cs were studied,with finding compatibility between drug and excipients as shown in Fig. 2. FTIR spectra of NAC (A) observed at a different wavelength of 3360 cm− 1, 2550 cm− 1, 1700 cm− 1, 1650cm− 1, 1350 cm− 1, 1080–1190 cm− 1 is due to the presence of various functional groups characterized with stretching O-H, S-H group, C = O bond, COO bond, N-H group and C-N groups of NAC respectively. Moreover, ascribed peaks at a wavelength of 3400-3500cm− 1, 1750 cm− 1, 1450 cm− 1, 748 cm− 1 were observed in the FTIR spectrum of PLGA (B), which was due to the presence of the O-H group, COO group, CH3 group, CH2 group, respectively.
Henceforth, after observing the FTIR spectrum of physical mixture (C), it was clearly demonstrated that there is compatibility between drug and excipients. Most importantly, appearance of broad band at 3300–3500 with characteristics loss of S-H and C-N group’s peak at wavelength of 2550 cm− 1 and 1080–1190 cm− 1 respectively in NAC-PLGA@Cs (D), suggested complete dispersion of NAC-PLGA nanoparticles inside lactose matrix. Our obtained results were compared with Du W et al, Mahumane et al, Ahmaditabar et al, Zu Y et al, where results were observed to be similar [36,37,38,39].
4.5 DSC
In the study, thermal properties of NAC, PLGA, physical mixture of NAC-PLGA-Lactose, and NAC-PLGA@Cs were studied throughout the analysis of DSC thermogram as shown in Fig. 3. With exhibiting crystalline behavior, the obtained DSC thermogram of NAC (A) illustrated a sharp endothermic peak with a melting temepearture (Tm) at 111.950C while the DSC thermogram of PLGA (B) showed glass transition temperature (Tg) at 500C due to its amorphous characteristics. However, the DSC thermogram of the physical mixture of NAC-PLGA-Lactose (C) exhibited the melting temperature (Tm) of NAC at 110.300C, Tg for PLGA at 55-600C, and Tm for lactose was observed at a range of 160-1750C. The obtained results were satisfactory with the results examined by Mahumane et al [37]. Finally, evaluating the DSC thermogram of NAC-PLGA@Cs (D), it was observed that the intense peak of NAC was diminished along with the peak of PLGA, indicating that the NAC-PLGA particle was significantly dispersed within the lactose dispersant matrix.
4.6 PXRD
PXRD pattern of NAC, PLGA, physical mixture of NAC-PLGA-Lactose and NAC-PLGA@Cs were depicted in Fig. 4. The sharp intense peaks of NAC (A) was visualized at 270C followed by 210C, 200C at 2θ, suggesting the crystalline nature of the drug. In addition to this, a very broad pattern with a low intense peak of PLGA (B) at 190C for 2θ, depicted for its amorphous property. Moreover, each diffraction pattern for the respective NAC, PLGA, and lactose in the given physical mixture sample (NAC-PLGA-Lactose) (C) was observed at its position in thegiven diffractogram, confirming compatibility between drug and excipients. Importantly, diffraction pattern of NAC was not been observed in the final formulation NAC-PLGA@Cs (D), although a sharp intense peak of lactose was seen at 200C followed by small two peaks at 120C and 16.50C at 2θ, which concluded that NAC-PLGA particles were purely encapsulated within lactose dispersant matrix with no interaction between them. However, the results obtained in our study were satisfactory as compared to the Muhamane et al findings [37].
4.7 Flow property of the powder
The angle of repose, hausenr ratio and car’s index are considerable indirect indicators to measure the powder flow property. The flowability of powder greatly influences the in vitro deposition [40]. In our study, the flowability of uncoated NAC-PLGA particles and coated freeze-dried NAC-PLGA@Cs were investigated. The obtained values as listed in Table 2, for the angle of repose, hausner ratio, and carr’ index for NAC-PLGA@Cs suggested improved flow property but NAC-PLGA particles demonstrated very poor flowability. The reason for the undesirable flowability of uncoated particles was due to particle-particle aggregation [41]. Herein, the lactose as a dispersant matrix prevents particles from aggregating hence enhanced the flowability. As a result, the values determined by each indicator were recorded as displayed in the table.
Table 2
Study for Powder flow property of both coated NAC-PLGA@Cs and uncoated NAC-PLGA particles.
Formulations | Angle of repose (θ) | Bulk density (g/cm3) | Tapped density (g/cm3) | Car’s index | Hausner ratio |
---|
NAC-PLGA@Cs | 34.58 ± 1.20 | 0.152 ± 0.008 | 0.186 ± 0.004 | 18.27 ± 3.87 | 1.22 ± 6.04 |
NAC-PLGA particle | 62.16 ± 4.06 | 0.092 ± 0.004 | 0.150 ± 0.08 | 38.66 ± 5.20 | 1.63 ± 0.44 |
4.7 In vitro drug release study
In vitro release study scrutinized that the release of drug from NAC-PLGA@Cs in phosphate buffer (at pH 7.4) was observed to be a biphasic pattern as depicted in Fig. 5(B).The initial burst release with 40% for 2hrs was visualized in NAC-PLGA@Cs due to rapid dissolution and release of adsorbed NAC at the surface of the particle. However, slow and sustained release profile was attained after 48 hrs with 90% drug release indicating slow drug diffusion and matrix erosion of PLGA nanoparticle after comes in contact with dissolution media. Additionally, drug dissolution profile of free NAC was estimated to be 100% within 3 mins as shown in Fig. 5(A), indicating hydrophilic nature (solubility: 100mg/ml) of NAC, as enlisted in BCS class I [42,43,44]. Although initial burst followed by sustained drug release from biodegradable and biocompatible PLGA particle varies from study to study. This is mainly straightforward towards the reasons that the drug has a very low molecular weight and is highly hydrophilic in nature, resulting in rapid leakage from polymeric particles [45,46,47].
4.8 In vitro pulmonary deposition study
However, for the efficient delivery of dry powder via inhalation route, aerodynamic properties should be taken into consideration. Some studies revealed that MMAD of 1–5µm could be optimal range particles size for pulmonary delivery [48]. In our study, obtained results suggested that developed PLGA composites showed an acceptable range of MMAD (2.50 ± 0.20 nm), GSD (1.49 ± 0.25), FPF (65.50 ± 5.50%), and ED (92.28 ± 0.2%) as shown in Table 3, signifying an efficient for drug delivery [49,50]. Moreover, the drug deposition study performed by Next Generation Impactor (NGI) showed the highest deposition for NAC-PLGA@Cs as compared to NAC-PLGA NPs at stage 3 followed by 4 and 5indicating the deep lung deposition of developed inhalable composites as shown in Fig. 6. Singh et al., also observed the similar deposition of inhalable liposphere at stage 3 followed by 4 in previous study [51].
Table 3
In vitro aerosolization study of freeze-dried NAC-PLGA@Cs and NAC-PLGA particles.
Formulation | MMAD (µm) | GSD | FPF (%) | ED (%) |
---|
NAC-PLG@Cs | 2.50 ± 0.20 | 1.49 ± 0.25 | 65.50 ± 5.50 | 92.28 ± 0.2 |
NAC-PLGA particle | 6.30 ± 0.40 | 1.68 ± 0.35 | 28.60 ± 5.50 | 68.43 ± 0.4 |
4.9 In vitro study for antimycobacterial activity
The potency for antimycobacterial activity of free NAC and developed NAC-PLGA@Cs were investigated against MTB H37Rv strain. Minimum inhibitory concentration (MIC) and Minimum bactericidal Concentration (MBC) values were determined with 2.69 mg/ml and 19.48 mg/ml for free NAC, while 0.73 mg/ml and 5.29 mg/ml for NAC-PLGA@Cs, respectively. Inhibitory potential of NAC against mycobacterial strain were studied by various researchers with finding significant potentiality to inhibit the growth of mycobacterium. In our study, NAC-PLGA@Cs exhibited 4-folds enhancement of antimycobacterial activity compared to free NAC [26,52]. we concluded that drug incorporated PLGA composites could be prominent strategy for treatment of pulmonary tuberculosis.