Synthesis of N1-(5-methyl-5H-indolo[2,3-b]quinolin-11-yl)benzene-1,4-diamine hydrochloride 4 (NIQBD)

To form the free amine of neocryptolepine derivative 3, The as prepared 11-chloroneocryptoleine 1, reacted with 1.4 phenylene diamine 2 in equimolar ratio with presence of dimethylformamide (DMF) as a solvent and three times excess of triethyl amine (TEA) as a base under reflux to afford the free amine 3in 87% yield. The free base 3 was dissolved in 2 mL of methanol and cooled in ice bath and acidified by addition of few drops of 1 M hydrochloric acid (HCl) till the complete precipitation of the hydrochloride salt. The salt was filtered off, washed with methanol and dried to afford pure 4 as depicted in Scheme 2 [22].

Scheme 2

Synthesis of hydrochloride salt of 4

The establishment of was elucidated by Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). The FTIR spectra showed υ(NH) at 3359 cm−1 whereas υ(CHAr) at 3044 cm−1 and υ(C=C) at1616 cm−1. Furthermore,1H NMR the (N-CH3) group of neocryptolepine core were reported at δ: 4.39 ppm while in 13C NMR the (N-CH3) group showed at δ: 36.27 ppm.

Characterization of NIQBD loaded soluble starch nanoparticles

The aim of our current work was designed to enhance the biomedical efficacy of NIQBD loaded StNPs. Via reacting soluble starch with sodium hydroxide (NaOH) at 80 °C, gelatinization of soluble starch was obtained. The use of absolute ethanol as a precipitating agent allowed NIQBD to be grafted into the gelatinized starch. The grafting of StNPs with NIQBD is triggered by ultrasonication waves. The encapsulation efficiency of NIQBD was found to be 93, 89,2 and 86,4%, respectively, for the prepared samples StNPs-1, StNPs-2, and StNPs-3, implying that NIQBD was encapsulated within StNPs.

The particle shape and homogeneity of NIQBD loaded StNPs were illustrated using TEM for the prepared samples; StNPs-1, StNPs-2 and StNPs-3 as can be seen in Fig. 1a, b, and c. All evaluated samples have nearly spherical shape. However, the particles tend to agglomerate together into clusters. As shown in Fig. 1, some NIQBD were deposited onto the surface of StNPs and tended to enlarge the particles of StNPs.

Fig. 1

Particle shape of NIQBD loaded StNPs using TEM where: a StNPs-1, b StNPs 2 and c StNPs-3

The particle size and polydispersity index (PDI) of the prepared NIQBD loaded StNPs ( StNPs-1, StNPs-2 and StNPs-3) were evaluated using dynamic light scattering (DLS). Figures 2 and 3 (a) illustrated the average size of NIQBD (different concentrations) loaded StNPs which recorded 246 nm for StNPs-1, 300 nm for StNPs-2, and 328 nm for StNPs-3.

Fig. 2

Average particle size of NIQBD loaded StNPs, a StNPs-1, b StNPs-2 and c StNPs-3

Fig. 3

a Particle size and b PDI of NIQBD loaded StNPs

Figure 3(b) displayed the polydispersity index (PDI) of the formed NIQBD loaded StNPs. As shown, all the prepared StNPs that loaded with different concentrations of NIQBD exhibited small PDI values. The PDI values of StNPs-1, StNPs-2 and StNPs-3 are 0.298, 0.177, and 0.262, respectively. All these PDI values are less than 0.5 which proved the homogeneity of the prepared NIQBD loaded StNPs.

Biochemical analysisEffect of MTX and different treatments on lung oxidants and anti-oxidants

Oxidative stress is a key indicator of cellular damage because it affects cell membrane permeabilization and impairs cellular functioning. To investigate the role of MTX as a reported oxidant, we estimated the levels of GSH, MDA, NO, and AOPP in lung tissues. Induction of MTX showed a marked decrease in the activity of lung GSH followed by a statistically substantial increase in the lung levels of MDA, NO, and AOPP in comparison to the control group (Table 1). In MTX group, the percent of change from the control group of GSH, MDA, NO, and AOPP were -55.4%, 525.8%, 288.7%, and 174.8% respectively as shown in Table 1.

Table 1 Lung oxidant and anti-oxidant levels in different studied groups

Treatment with StNPs alone or StNPs loaded with NIQBD in different concentrations; StNPs-1, StNPs-2, or StNPs-3 showed a statistically substantial increase in the activity of lung GSH with a percent of change 9.1%, 28.1%, 48.8%, and 74.7% respectively from the MTX group (Table 1).

Compared to the MTX group, treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 showed a significant decrease in the levels of lung MDA with a percent of change -14.4%, -27.7%, -51.8%, -66.8% respectively from the MTX group (Table 1). In addition, levels of lung NO showed significant decrease with percent of change -22.1%, -35.5%, -44.5%, -60.8% respectively from the MTX group (Table 1). Levels of lung AOPP showed significant decrease compared to the MTX group upon the treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 with a percent of change -30.7%, -39%, -44.7%, -51.4% respectively from the MTX group (Table 1).

Effect of MTX and different treatments on lung inflammatory markers

In order to verify the mechanisms of inflammatory processes induced by MTX, we estimated the levels of MMP-9, IL-1β, and NF-kB in lung tissues. Our results showed that MTX induced inflammation resulted in a statistically substantial increase in the levels of lung MMP-9, IL-1β, and NF-kB compared to the control group. The percent of change of MMP-9, IL-1β, and NF-kB were 307%, 280%, and 66.7% respectively from the control group (Table 2).

Table 2 Lung inflammatory markers in different studied groups

Treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 showed a significant decrease in the levels of lung MMP-9 with a percent of change -5.5%, -11%, -15%, -48% respectively from the MTX group (Table 2), levels of lung IL-1β showed significant decrease with percent of change -11.5%, -22.4%, -43.1%, -55.3% respectively from the MTX group (Table 2). Lung NF-kB showed significant decrease compared to the MTX group upon the treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 with a percent of change -1.84%, -4.46%, -8.28%, -18.13% respectively from the MTX group (Table 2).

Effect of MTX and different treatments on liver oxidants and anti-oxidants

The liver's pivotal role in drug metabolism and clearance renders it highly susceptible to the toxic effects of these pharmacological agents. The hepatotoxic manifestations of MTX have been attributed primarily to the induction of oxidative stress.

In comparison to the control group, MTX administration showed a significant decrease in the activity of GSH in the liver tissues accompanied with a statistically substantial increase in the levels of MDA, NO, and AOPP in liver tissues (Table 3). In MTX group, GSH, MDA, NO, and AOPP percent of change from the control group were -61.2%, 219%, 371.3%, and 199.3% respectively as shown in Table 3.

Table 3 Liver oxidant and anti-oxidant levels in different studied groups

Using StNPs alone, StNPs-1, StNPs-2, or StNPs-3 as a treatment showed a statistically substantial increase in the activity of liver GSH with a percent of change from the MTX group were 16.6%, 36.8%, 69.4%, and 115.7% respectively (Table 3).

Compared to the MTX group, treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 showed a significant decrease in the levels of liver MDA with a percent of change of the treated groups from the MTX group were -13.3%, -23.5%, -36.5%, -53.2% respectively (Table 3). In addition, levels of liver NO showed significant decrease with percent of change -27.3%, -38.5%, -58.9%, -63.1% respectively from the MTX group (Table 3).

Levels of liver AOPP showed significant decrease compared to the MTX group upon the treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 with a percent of change -24.7%, -37.1%, -46.5%, -56.7% respectively from the MTX group (Table 3).

Effect of MTX and different treatments on liver inflammatory markers

Obtained results revealed that MTX-induced inflammation showed a statistically substantial increase in the levels of liver MMP-9, IL-1β, and NF-kB compared to the control group. The percent of change of MMP-9, IL-1β, and NF-kB were 169%, 155.8%, and 157.4% respectively from the control group (Table 4).

Table 4 Liver inflammatory markers in different studied groups

Treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 showed a significant decrease in the levels of liver MMP-9 with a percent of change from the MTX group were -10.5%, -20.9%, -34.3%, -49.6% respectively (Table 4), levels of liver IL-1β showed significant decrease with percent of change from the MTX group were -16.3%, -34.5%, -44.5%, -52.2% respectively (Table 4).

Levels of NF-kB in liver tissues showed significant decrease compared to the MTX group upon the treatment with StNPs alone, StNPs-1, StNPs-2, or StNPs-3 with a percent of change –5.2%, -8.1%, -16.3%, -34.2% respectively from the MTX group (Table 4).

Effect of MTX and different treatments on serum Homocysteine

Serum homocystein levels showed a highly statistically substantial increase in MTX-induced inflammation group compared to the control group with a percent of change 1690% from the control group (Table 5).

Table 5 Serum homocystein levels in different studied groups

On the other hand, levels of serum homocystein showed a significant decrease by using StNPs alone, StNPs-1, StNPs-2, or StNPs-3 as treatments, with percent of change from the MTX group -22.5%, -43.2%, -55.6%, and -79.2% respectively (Table 5).

Histopathological resultsEffect of MTX and different treatments on lung tissue

Microscopic examination of lung sections from control rats revealed normal lung architecture with thin interalveolar septa, clear rounded or polygonal alveoli, and alveolar sacs. The interalveolar spaces were extremely narrow, and the interstitial tissue contained blood vessels (Fig. 4A.

Fig. 4

A micrograph of rat lung of: A control group showing normal architecture including alveolar sacs (AS), numerous normal regular alveoli (A) thin interalveolar septa (arrows). B MTX induced inflammation group showing disruption of normal lung architecture and distortion of the interalveolar septa with thickening (arrow), the pulmonary blood vessel showed dilatation and congestion (BV). Inflammatory cellular infiltration in perivascular (IF), and interstitial tissue with haemorrhage into alveolar spaces (arrowhead) were found. C MTX induced inflammation treated with StNPs showing moderated improvement with reduction of thickening of the interalveolar septa (arrow) and inflammatory cell infiltration (IF) and interstitial tissue with haemorrhage into alveolar spaces (arrowhead), blood vessel dilatation and congestion (BV). D MTX induced inflammation treated with StNPs-1group (low concentration) showing reduce thickening of the interalveolar septa (arrow) with minimal inflammatory infiltration (arrowhead), interstitial tissue with haemorrhage into alveolar spaces (arrowhead) and dilatation and congestion (BV). E MTX induced inflammation treated with StNPs-2 group (medium concentration) showing reduced thickening of the interalveolar septa (arrow) with minimal inflammatory infiltration (IF) and interstitial tissue with haemorrhage into alveolar spaces(arrowhead) and blood vessel dilatation and congestion (BV). F MTX induced inflammation treated with StNPs -3group (high concentration) showing marked reduced thickening of the interalveolar septa (arrow) with minimal inflammatory infiltration (IF) and blood vessel dilatation and congestion (BV). H&E stain, Scale bar: 60 μm

Histopathological evaluation of lung sections from MTX-treated rats showed disruption of the normal architecture. This disruption was characterized by apparent alveolar damage, including collapse and distortion of the interalveolar septa with thickening. Additionally, the pulmonary blood vessels exhibited dilatation and congestion, accompanied by extravasation of red blood cells into the alveolar lumen. Inflammatory cell infiltration was observed in the perivascular and interstitial tissues, with hemorrhage into the alveolar spaces (Fig. 4B).

In the group received MTX and treated with StNPs revealed moderated improvement with reduction of thickening of the interalveolar septa and inflammatory cell infiltration and congestion of interstitial vessels (Fig. 4C).

However, MTX and treated with StNPs-1 revealed ameliorated by administration of different doses of StNPs-1, StNPs-2, and StNPs-3 evidenced by a reduction in interalveolar septal thickening, a histological hallmark of improvement with minimal inflammatory infiltration and congestion of interstitial vessels in dose dependent manner however the group of StNPs-3 (high concentration) is better than StNPs-1 and StNPs-2 (low & medium concentrations) (Fig. 4D, E and F respectively).

Effect of MTX and different treatments on liver tissue

Microscopic examination of liver sections from the control group revealed normal morphology with regular arrangement of hepatocytes, distinct nuclei, and patent sinusoids (Fig. 5A.

Fig. 5

A micrograph of rat liver of: A control group showing hepatic architecture. Central vein (CV), hepatocytes (H), blood sinusoids (S) and nucleus (S) are shown. B & C MTX induced inflammation model group showing B degeneration changes with eosinophilic cytoplasm, necrotic areas (arrowhead), deeply stained pyknotic nuclei (P) with mild activation of Kupffer cells (K). C Showing congested central vein (CV) associated with moderate inflammatory cell infiltration (arrow). Deeply stained pyknotic nuclei (P) and mild activation of Kupffer cells (K) were seen. E MTX induced inflammation treated with StNPs group showing moderate improvement. Mild inflammatory cell infiltration (arrow) around the congested central vein (CV), mild activation of Kupffer cells (K) and pyknotic nuclei were noticed (P). F MTX induced inflammation treated with StNPs-1 (low concentration) group showing moderate improvement with inflammatory cell infiltration (arrow) around congested central vein (CV), mild haemorrhage in between blood sinusoids (S) and pyknotic nuclei (P). G MTX induced inflammation treated with StNPs-2 (medium concentration) group showing an improvement associated with small area of inflammatory cell infiltration (arrow) around congested central vein (CV). Mild activation of Kupffer cells (K) and pyknotic nuclei (P) were seen. H MTX induced inflammation treated with StNPs-3 (high concentration) group showing an improvement associated with mild congested central vein (CV), mild activation of Kupffer cells (K) and pyknotic nuclei (P). H&E stain, Scale bar: 30 μm

In the group that received MTX, hepatocytes exhibited vacuolation, disruption of the normal hepatic architecture, and increased evidence of necrosis (Fig. 5B). On the other hand, moderate accumulation of inflammatory cells around the congested central veins, most sinusoids showed numerous Kupffer cells and pyknotic nuclei (Fig. 5C).

Liver of the MTX group that treated with StNPs showed moderated improvement. Mild inflammatory cells infiltration around the congested central veins, mild activation of Kupffer cells and pyknotic nuclei were noticed (Fig. 5D).

The liver of MTX group that treated with StNPs-1 showed a moderated improvement of tissue. Mild inflammation around the congested central veins, mild haemorrhage in between blood sinusoids and pyknotic nuclei were seen (Fig. 5E).

MTX group that treated with StNPs-2 reversed the morphological changes caused by inflammation agent resulting in the restoration of normal liver architecture, reduced inflammatory cell infiltration, and preserve distinct lobular structure (Fig. 5F).

MTX group administration StNPs-3 attenuated congestion of central vein in liver tissues, reduce the number of the infiltration of inflammatory cells with hepatocyte arrangement closely resembled that of a normal liver (Fig. 5G).