The principal compounds in [email protected] were identified using HPLC by comparing their characteristics absorbance peak with those of standards. The both chromatograms of total ion current (TIC) and the corresponding HPLC (258 nm) were displayed in Fig. 1.
3.2. Preparation and characterization of [email protected]
The desolvation method was followed for [email protected] NPs production. Here, ethanol and glutaraldehyde were used as desolvating and cross-linking agent respectively . As presented in schematic Fig. 2, this method validates the production of homogeneous distribution of least aggregated particles. Also, the particle remains stable both in water and culture medium. Glutaraldehyde is a water-soluble reagent and has the ability to form stable covalent bonds. It reacts with protein and forms Schiff bases between the amino groups and two carbonyl ends of glutaraldehyde. Thus, the BSA amino moieties are key in the formation of nanoparticles. The absorption maximum of [email protected] NPs was assessed from 300 to 600 nm (Fig. 3a). Albumin shows a characteristic absorption peak at 270 nm. However, bergenin recorded absorbance peak at 290nm. Interestingly, the bergenin encapsulated albumin NPs ([email protected] NPs) showed a characteristic peak at 376 nm. This change in spectra could be due to cross-linking of amino acids and the formation of protein–drug complex. The niclosamide drug encapsulated albumin NPs had shown a peak at 346 nm .
FTIR spectrum of BSA showed functional peaks at 1510.21, 1623.05, 3100, and 3356.28 cm− 1 assigned to the N–H and C–N vibrations, stretching vibration of –NH, C = O stretching of the peptides and stretching vibrate of –OH respectively (Fig. 3b). Bergenin alone had a major peak at 1084.04 to 1176.10, 1229.35 to 1247.90 and 1480.17 to 1501.05 indicating the involvement of the C–OH, C = O, NO2 groups of bergenin in the formation of a complex. Furthermore, the interaction between the nanoparticles have led to wide spectrum. A typical reduction in the stretching was observed in BG-BSA NPs (Fig. 3b). The peak of BSA shifted from 1623.05 to 3356.28 cm− 1 may be attributed to the cross-linking of amino groups of proteins and the interaction of bergenin. It was demonstrated that electrostatic interactions help in the binding process of protein with a drug. Likewise, niclosamide interacts with the tryptophan residues (Trp 212) of BSA [19, 20].
The XRD pattern of bergenin and [email protected] NPs is shown in Fig. 4(a). The synthesized NPs showed Bragg’s reflection peaks at 38.4, 42.5, 63.2 and 76.4 in the 2 h range between 30 and 80 which can be indexed to the (220), (311), (400), (422) and (511) planes respectively. The diffraction peaks match with the standard JCPDS No: 04-0784. The thermogravimetric (TG) analysis of bergnin, BSA and [email protected] NPs were performed (Fig. 4b). The degradation rate of NPs was slower indicating their improved stability compared to BSA and bergenin. At 300–600°C, a remarkable difference in weight loss was noticed. On the other hand, only 10% was remaining for bergenin alone which confirmed the slower degradation potential of BG-BSA NPs when compared to bergenin alone. A faster degradation of BG-BSA NPs was observed compared to BSA at > 500°C. This is because of the crystalline nature of NPs. The differential thermal analysis (DTA) of the bare bergenin showed an endothermic peak at 235°C. However, [email protected] NPs had greater stability compared to bergenin and albumin. This substantiates the amorphous nature of NPs as evident from Fig. 4(c). The increased amorphous nature of the therapeutic system contributes to the drug delivery efficiency of NPs .
The surface morphology of the [email protected] NPs was determined by SEM and TEM analysis. [email protected] NPS had a spherical shape with uniform size distribution as shown in Fig. 5(a). However, the TEM image reflected the hexagonal shaped particle as shown in Fig. 5(b). The size of [email protected] NPs was measured by AFM (Fig. 5c). The average grain size was 124.26 nm. The nanoparticles size was most important factor in drug delivery system. The particles less than 400 nm have increased permeability and retention (EPR) effect . The element composition of [email protected] NPs were measured using XPS analysis. The ratio of carbon was greater than that of oxygen, nitrogen, phosphorus and sulfur (Fig. 5d). The ratio of C/N was much lower than that of HNP, in CS-NP . [email protected] NPs exhibited good stability which was confirmed by zeta potential analysis. Figure 6a showed the zeta potential value of [email protected] NPs, it was found to be -24.2 mV while comparing to the values of BSA (-18.9 mV). This result confirmed that [email protected] NPs are highly stable. The electrostatic repulsive force of the negatively charged surface of NPs attributed to the higher stability of the colloidal solution . The particle size analysis of [email protected] NPs was shown in Fig. 6b. The mean size was found to be 124.26 nm. However, the BSA NPs and bergenin were found to be 288.24 nm and 267.02 nm respectively in size. The size of NPs plays a key role in drug delivery, as the nanoparticles up to 400 nm get can have “enhanced permeability and retention (EPR)” effect .
3.3. Drug loading efficiency and release profile
The encapsulation efficiency of [email protected] NPs was 90.15% (Fig. 7a). Previously, it was reported that BSA provides the encapsulation efficiency of 92.36% . The in vitro drug release profiles of [email protected] NPs was studied at regular time intervals (Fig. 7b). Drug release is the diffusion and degradation of drug molecules into the external environment. The drug release was 36.25%. The initial burst release is due to the surface bound bergenin, while the controlled release is due to the encapsulated bergenin. Our results are in close proximity with earlier reports on niclosamide based NPs .
3.4. Effect of [email protected] NPs on cell viability and NO production
Briefly, NR8383 cells were treated with [email protected] NPs (1–100 mg mL− 1) for 24 h and 48 h and the cell viability were determined following CCK8 assay (Fig. 8a). The viability of cells before LPS stimulation and measured nitric oxide (NO production). Following LPS stimulation, NR8383 cells had a greater level of NO production compared to the control cells (Fig. 8b). [email protected] NPs slightly suppressed the NO releasing activity at a minimum concentration, but the inhibitory effect was strongly enhanced at 10 and 25 mg mL− 1. Cells treated with [email protected] NPs at 10 and 25 mg mL− 1 showed 44% and 62% decrease in NO production respectively (Fig. 8c). NR8383 macrophages cells treated with BM60 had reduced NO production .
3.5. Total cell count and lung wet to dry ratio
Neutrophils are involved in the innate immune system in the lung. These cells can be found at the injury site and release numerous cytotoxic products . In LPS induced group, the neutrophils modulated the expressions of TNF-a, IL-1b, and IL-6 and finally cause the pulmonary injury . The neutrophil count in this study was increased in the lung following LPS exposure. The effect of [email protected] NPs on the inflammatory cells in BALF was shown in Fig. 9a. The animal administrated with LPS alone showed a significantly increased level of total cells count and neutrophils. Interestingly, the induced group of animals treated with [email protected] NPs showed a decreased level of total cells count and neutrophils. This finding revealed that [email protected] NPs had significantly reduced the neutrophils level in the lungs.
Lung wet-to-dry ratio was used to evaluate the pulmonary edema. It is a typical symptom of inflammation . In this study, the lung wet/dry ratio was increased in LPS treatment (Fig. 9b). However, the lung wet/dry ratios were greatly decreased in animal treated with [email protected] NPs, thus confirming the protective effect of [email protected] NPs. BM60 decreased the lung wet-to-dry ratio, which suggested the protective effect on LPS-induced ALI .
3.6. Antioxidant activity
The measurement of MDA and SOD are useful in minimizing the damaging effects caused by ROS [29–31]. Currently, LPS treated mice showed an increased level of MDA in the lung (Fig. 10a). In contrast, the SOD was greatly decreased (Fig. 10b). Treatment with [email protected] NPs reduced MDA and increased SOD in the lung. Similar results have been documented using BM 60 .
3.7. MPO and inflammatory cytokine activity
MPO is important for acute and chronic inflammations . MPO measurement indicates neutrophil confiscation in tissue. In this study, the increased level of MPO was observed in lung tissues of LPS challenged animals. However, [email protected] NPs have the ability to attenuate the neutrophil infiltration in the lungs indicating their protective effects against inflammation (Fig. 11a). The inflammatory cytokines in BALF serve as a marker to evaluate the inflammatory responses [33,34]. [email protected] NPS may inhibit the expression of TNF-a, IL-1b, and IL-6. The TNF-a, IL-1b, and IL-6 in treated groups. Overall, the treatment of [email protected] NPS decreased pro-cytokines while compared to control (Fig. 11b-d). Similarly, the inflammatory cells and TNF-a, IL-1b, and IL-6 production were greatly inhibited by BM60 .
3.8. Histopathology analysis
Neutrophils, one of the most important components of the initial innate immune response in the lung against bacterial infections are the earliest immune cells to be recruited to the site of injury and express multiple cytotoxic products . In this study, histopathological examination was performed to reveal the protective effect of [email protected] NPs on acute lung infection (Fig. 12). In LPS induced acute lung injury (ALI), the neutrophils accumulated in the lungs, changed the expressions of pro-inflammatory cytokines, such as TNF-a, IL-1b, and IL-6 and finally led to the pulmonary injury. In this study, we found that the neutrophils clearly increased in lung tissues after LPS exposure and the effect of [email protected] NPs on the number of inflammatory cells in BALF was observed. The number of total cells and neutrophils increased significantly after LPS treatment. [email protected] NPs dose dependently inhibited the number of total cells and neutrophils in BALF induced by LPS. As expected, [email protected] NPs pre-treatment significantly decreased the neutrophils in lung tissues. It was evident that the histopathological damage and neutrophil infiltration was greatly inhibited by [email protected] NPs. This indicates that [email protected] NPs may be used to treat lung infections.